CHAPTER ONE

1.0 INTRODUCTION
1.1 WHEAT
Wheat (Triticum aestivum L. em Thell.) is the first important and strategic cereal crop for the
majority of world’s populations. It is the most important staple food of about two billion people
(36% of the world population). Worldwide, wheat provides nearly 55% of the carbohydrates and
20% of the food calories consumed globally (Breiman and Graur, 2005). It exceeds in acreage
and production every other grain crop (including rice, maize, etc.) and is therefore, the most
important cereal grain crop of the world, which is cultivated over a wide range of climatic
conditions and the understanding of genetics and genome organization using molecular markers
is of great value for genetic and plant breeding purposes.
The grass family Poaceae (Gramineae) includes major crop plants such as wheat (Triticum
aestivum L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), rye (Secale cereale L.), maize
(Zea mays L.) and rice (Oryza sativa L.). Triticeae is one of the tribes containing more than 15
genera and 300 species including wheat and barley.
Wheat belongs to the tribe Triticeae (Hordeae) in the grass family Poaceae (Gramineae)
(Briggle and Reitz, 1963) in which the one to several flowered spikelets are sessile and alternate
on opposite sides of the rachis forming a true spike. Wheats (Triticum) and ryes (Secale) together
with Aegilops, Agropyron, Eremopyron and Haynalidia form the s
ubtribe Triticineae (Simmonds, 2001).
Linnaeus in 1753 first classified wheat. In 1918, Sakamura reported the chromosome number
sets (genomes) for each commonly recognized type. This was a turning point in Triticum
classification. It separated wheat into three groups. Diploids had 14 (n=7), tetraploids had 28
(n=14) and the hexaploids had 42 (n=21) chromosomes. Bread wheat is Triticum aestivum. T.
durum and T. compactum are the other major species. All three are products of natural
hybridization among ancestrals no longer grown commercially (Briggle, 2001).
1.2 MUSHROOM
Mushrooms is called as a 'white vegetables' or 'boneless vegetarian meat' contain ample amounts
of proteins, vitamins and fibre apart from having certain medicinal properties. Mushroom
contains 20-35% protein (dry weight) which is higher than those of vegetables and fruits and is
of superior quality. Mushrooms are now getting significant importance due to their nutritional

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and medicinal value and today their cultivation is being done in about 100 countries. At present
world production is estimated to be around 5 million tonnes and increasingcontinually. Though
20 mushroom varieties are domesticated about half a dozen varieties viz; button, shitake, oyster,
wood ear and paddy straw mushrooms contribute 99% of the total world production. Mushroom
offers prospects for converting lignocellulosic residues from agricultural fields, forests into
protein rich biomass. Such processing of agro waste not only reduces environmental pollution
but the by-product of mushroom cultivation is also agood source of manure, animal feed and soil
conditioner.
Mushroom is a macro fungus with a distinctive fruiting body, which can be either epigeous or
hypogeous and large enough to be seen naked eye and to be picked by hand" (Chang and Miles,
2002).
Mushroom science is the discipline that is concerned with the principles and practices of
mushroom cultivation. Consistent production of successful mushroom crops will be built upon
scientific knowledge and practical experience (Chang and Miles, 2001). Edible mushrooms
provide high quality of protein that can be produced with greater biological efficiency than
animal protein. They are rich in fibre, minerals and vitamins, and have low crude fat content,
with a high proportion of polyunsaturated fatty acids (72 to 85 %) relative to total content of
fatty acids. These properties are major contributing factors to the traditional recognition of
mushrooms as "healthy" foods. A large number of mushroom species are not only edible and
nutritious but also possess tonic and medicinal qualities. However, some mushrooms are lethally
poisonous, and one should eat mushrooms only if one knows their names and their properties
with considerable precision. There are at least 12,000 species of fungi that can be considered as
mushrooms with at least 2,000 species showing various degrees of edibility (Chang,2004).
Furthermore, over 200 species of mushroom have been collected from the wild and utilized for
various traditional medical purposes mostly in the Far East. To date, about 35 mushroom species
have been cultivated commercially and of these, about 20 are cultivated on an industrial scale.
The majority of these cultivate species are both edible and possess medicinal properties.
However, two of the major medicinal mushrooms, viz. GanodermalucidumandTrametes
(Coriolus)spp. are distinctly inedible. Overall, the world production of cultivated edible and/or
medicinal mushrooms was recorded as 4,909.3 x 103 tons in 1994 increasing to 6,158.4 x 103 in
1997 with an estimated value in excess of 14 billion US dollars (Chang, 1999b).In percentage

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terms, output yield of the leading 10 species cultivated made up92% of total world production of
these six species, viz: Agaricusbisporus(31.8%); Lentinusedodes (25.4%), Pleurotus spp.
(14.2%), Auricularia auricular (7.9%), Flammulinavelutipes (4.6%), and Volvariellavolvaceae
(7.9%), made up 87% of the total production. Overall, world production of mushrooms is
increasingly being dominated by species that are both edible and have medicinal properties. It is
pertinent to note that world production of mushrooms is now dominated by China with over 64%
of total production. China has become a major producer and consumer of both edible and
medicinal mushrooms. Furthermore, China is also the major producer of the non-edible
medicinal mushrooms, e.g. Wolfiporia (Poria) cocos (10,000 tons) and Ganodermalucidum
(4,000 tons). The traditional acceptance of mushrooms in herbal medicine and in expanding
pharmaceutical industries, will ensure that China will continue to be a major exploiter of
medicinal mushroom technology (Yamanake, 2009). The cultivation of Agaricusbisporus is an
outstanding example of a biotechnological enterprise that challenges the combined skills of
industrial and biological technologies. A. bisporus cultivation in Western countries has achieved
its current pre-eminence in the mushroom industries because of a solid foundation in basic
scientific research in all aspects of Agaricus biology (genetics, physiology, biochemistry),
bioprocess technology and, above all, the use of modern management principles (Chang et al.,
2003). This foundation made possible a highly technical approach involving the creation and
utilization of specialized equipment and advanced engineering technology. However, much
fundamental knowledge has been acquired in recent years which will be of considerable value
for other cultivations.
1.3 Objectives of the study
 Pasting Characteristics of wheat flour supplemented with mushroom flour
 Proximate Analysis of wheat flour supplemented with mushroom flour

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 CHAPTER TWO

2.0 ORIGIN AND EVOLUTION OF WHEAT

The first cultivation of wheat occurred about 10000 UD years ago, as part of the Neolithic
Revolution, which saw a transition from hunting and gathering of food to settled Agriculture.
These earliest cultivated forms were diploid (genome AA) (einkorn) and tetraploid (genome
AABB) [Cmmer) wheats and their genetic relationships indicate that they originated from the
south-eastern part of Turkey (Heun et al, 2001, Neshiti, 2004, Dubcovsky and Dvorak 2007)
Cultivation spread to the Near East by about 9000 years ago when hexaploid bread wheat made
its first appearance (Feldman. 2001). The earliest cultivated forms of wheat were essentially
landraces selected by farmers from wild populations? Presumably because of their superior yield
and other characteristics, an early and clearly non-scientific form of plant breeding! However,
domestication was also associated with the selection of genetic traits that separated them from
their wild relatives. This domestication syndrome has been discovered in detail by others, but
two traits are of sufficient importance to mention here. The first is the loss of shattering of the
spike at maturity, which results in seed loss at harvesting this is clearly an important trait for
ensuring seed dispersal in natural populations and the non-shattering trait 15 determined by
mutations at the Br (brittle rachis) locus (Nalam et al, 2006), The second important trait is the
change from hulled forms in which the glumes adhere tightly to the grain. to free-threshing
naked forms. The free forms arose by a dominant mutant at the Q locus which modified the
effects of recessive mutations at the Tg (tenacrous glume) locus (Jantusuriyarat et al. 2004,
Simons et al, 2006, Dubkovsky and Dvorak. 2007). Cultivated forms of diploid, tetraploid, and
hexaploid wheat al have a tough rachis apart from the spelt form of bread wheat. Similarly, the
early domesticated forms of einkorn. emmer, and spelt are all hulled, whereas modern forms of
tetruploid and hexaplod wheat are free-threshing Whereas einkom and emmer clearly developed
from the domestication of natural populations. bread wheat has only existed in cultivation,
having arisen by hybridization of cultivated emmer with the unrelated wild grass Triticum
tauschii (also called Acgilops tauschii and Ae. squarosa) This hybridization probably occurred
several times independently with the novel hexaploid (genome AABBDD) being selected by
farmers for its superior properties. The genetic changes during domestication mean that modern
wheats are unable to survive wild in Competition with better adapted species. This was elegantly

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demonstrated by john bunnes lawes in the 1880s when he decided to allow part of the famous
long-term broad hulk experiment at Rothamsted to return to its natural state (Dyle, 2004) he
therefore left part of the wheat crop unharvested in 1882 and monitored the growth in successive
years after a good crop in 1883 the weeds dominated and in 1885 the few remaining wheat plants
which were spindly with small ears) were collected a photographed. The A genomes of tetraploid
and hexaploid wheats are clearly related to the A genomes of wild and cultivated einkorn, while
the D genome of hexaploid wheat is clearly derived from that of T. tauschii, ln fact, the
formation of hexaploid wheat occurred so recently that little divergence has occurred between
the D genomes present in the hexaplod and diploid species. By contrast, the B genome of
tetraploid and hexaploid wheats is probably derived from the S genome present in the
Sitopsissection of Aegilops, with Acspeltoides being the closest extant species. The S genome of
Acspeltoides is also closest to the G genome of T, timopheevi. A tetraploid species with the A
and G genomes (Feldnman, 2001). The spread of wheat from its site of origin across the world
has been elegantly described by Feldman (2001) and is only summarized here. The main route
into Europe was via Anatolia to Greece (8000 BP) and then both northwards through the Balkans
to the Danube (7000 BP) and across to Italy, France and Span (7000 BP) finally reaching the UK
and Scandinavia by about 5000 BP. Similarly, Wheat spread VIN Iran into central Asia. reaching
China by about 3000 BP and to Africa, initially via Egypt. It was introduced by the Spaniards to
Mexico in 1529 and to Australia in 1788.
2.1 CULTIVATION OF WHEAT CROP
Climate and Soil:
The ideal temperature requirement varies from plant type and stages of growth. The dwarf
varieties require the following temperature for their growth and development:

Growth stages Temperature requirement

Germination 20 to 25 0 C mean daily temperature
Tillering 16 to 20 0 C mean daily temperature
Accelerated growth 20 to 23 0 C mean daily temperature
Proper grain filling 23 to 25 0 C mean daily temperature.
Wheat plants are sensitive to very cold or frost injury at any stage of growth particularly at
reproductive growth if temperature is below 150

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Land Preparation:
The wheat crop requires a well-pulverized but compact seed bed for good and uniform
germination. Three or four ploughings in the summer, repeated harrowing in the rainy season,
followed by three or four cultivations and planking immediately before sowing produce a good,
firm seed bed for the dry crop on alluvial soils. For the irrigated crop, the land is given a pre-
sowing irrigation (palewa or raund) and the number of ploughings is reduced. Where white ants
or other pests are a problem, Aldrin 5% or BHC 10% dust at the rate of 25 kg/ha should be
applied to the soil after the last ploughing or before planking.

Sowing:
a) Sowing time:
Based on above temperature requirement it has been found that for indigenous wheat last week
of October, for long duration dwarf varieties like Kalyansona, Arjun, etc. first fortnight of
November and for short duration dwarf wheats like Sonalika, Raj 821 etc. second fortnight is the
best sowing time. Under exceptionally late sown condition it may be delayed to latest by 1st
week of December beyond which if area is very small transplanting may be practiced.

b) Seed rate:
Generally, a seed rate of 100 kg/ha has been found to be sufficient for most of the varieties like
Kalyan Sona, Arjun, Janak, etc.which have moderate tillering and medium sized grains. But a
higher seed rate of 125 kg/ha is desirable for late sown wheat and normal sown for varieties like
Sonalika, Raj 821 etc. which have bold grains and shy tillering habits.

c) Spacing:
For irrigated, timely sown wheat, a row spacing of 15 to 22.5 cm is followed, but 22.5 cm
between the rows is considered to be the optimum spacing. Under irrigated late-sown conditions,
a row spacing of 15-18 cm is the optimum. For dwarf wheats, the planting depth should be
between 5 and 6 cm. Planting beyond this depth results in a poor stand. In the case of
conventional tall varieties, the depth of sowing may be 8 or 9 cm.

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d) Seed treatment:
The seed of loose smut-susceptible varieties should be given solar or hot-water treatment. If the
wheat seed is used only for sowing, and not for human consumption or for feeding cattle, it can
be treated with Vitavax.

Application of Mannures and Fertilizer

It is desirable that 2 to 3 tonnes of farmyard manure per hectare or some other organic matter is
applied 5 or 6 weeks before sowing. The fertilizer requirement of the irrigated wheat crop are as
follows:
With assured fertilizer supply:
Nitrogen (N) @8- - 120 kg/ha
Phosphorus (P2O5) @ 40- 60 kg/ha
Potash (K2O) @ 40 kg/ha.

Under fertilizer constraints:

N @ 60-80 kg/ha
P2O5 @ 30-40 kg/ha
K2O @ 20-25 kg/ha.
Total quantity of Phosphorus and potash and half the quantity of nitrogen should be applied at
the time of sowing. Remaining quantity of Nitrogen should be applied at the time of crown root
initiation for the late sown irrigated wheat crop, the NPK fertilizer dose recommended is:
N – 60-80 kg/ha
P2O5 – 30-40 kg/ha
K2O – 20-25 kg/ha.
Interculture:
Generally weeding is done after 1 ½ to 2 months after sowing or weedicides like 2,4 D, Avadex
or Nitrofen (Tok E-25) for controling Chenopodium sp, Angallis sp. Asphodelus sp. Phalaris sp.
of weeds.
Irrigation:
The high yielding wheat varieties should be given five to six irrigations at their critical growth
stages viz. Crown root initiation, tillering, jointing, flowering, milk and dough which come at 21-

7
25 days after sowing (DAS), 45-60 DAS, 60-70 DAS, 90-95 DAS, 100-105 DAS and 120-125
DAS respectively. Off these irrigation at CRI stage is most important.
Harvesting and Storage:
a) Harvesting:
The rain-fed crop reaches the harvest stage much earlier than the irrigated crop. The crop is
harvested when the grains become hard and the straw becomes dry and brittle. The harvesting is
mostly done by sickle. The crop is threshed by treading with cattle on the threshing-flour or by
power driven thresher.
b) Yield:
The national average yield of wheat grain is about 12 to 13.8 quintals per hectare.

c) Storage:
The grains should be thoroughly dried before storage. The storage life of the grain is closely
related to its moisture content. Grains with less than 10 percent moisture store well. The storage
pits, bins or godowns should be moisture-proof and should be fumigated to keep down the stored
– grain pests including rats. Zinc phosphide is very effective against rats.
2.1.1 TYPES OF WHEAT CROP
Wheat flour is available in many varieties: the categorization is regional, and the same name may
have several different regional meanings.
CANADA: whole wheat flour in Canada may have up to 5% or the gram removed, most of the
germ is often removed to prevent the flour from going rancid
Whole grain flour contains the whole grain. including bran, germ, and endosperm, but not
the chaff.
FUI: sharp flour is produced in Fi and primarily used in INDIAN cuisine.
USA WHEAT: American flours are categorized by gluten/protein content, processing, and Use.
 All-purpose or plain flour is blended wheat with protein content lower than bread
flour, ranging between 9% and 12%. Depending on brand or the region where it is
purchased. it may be composed of all hard or soft wheat, but is usually a blend of the two.
And can range from low protein content to moderately high. It is marketed as an
inexpensive alternative to baker' s flour which is acceptable for most household baking
needs.

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  Bread flour or strong flour is always made from hard wheat, Usually hard spring wheat. It
has very high protein content, between 10% and 13%, making it Excellence for yeast
bread baking. It can be white or whole wheat or in between
 Cake flour is finely milled white flour made from soft wheat. It has very low protein
content, between 8% to 10%, making it suitable for soft-textured cakes and cookies The
higher protein content of other flours would make the cakes tough. Highly sifted caked
flours may require different volume amounts in recipes than all-purpose flour Using the
scoop und level method, well-sifted flour usually produces 125g per cup. American cake
flour is bleached; in countries where bleached flour is prohibited, plain flour can be
treated in a domestic miico-wave to improve he texture of the end product Related to
cake Flour are masa harinat (from maize), maida flour (from wheat or tapioca), and pure
starches (J.R fraser, 2001)

Durum flour: flour made of durum wheat which is suited for pasta making, traditional
pizza and flat bread for doner kebaba.
Graham flour is a special type of whole wheat flour. The endosperm is finely ground as in white
flour while the bran and germ are coarsely ground, Graham four is uncommon outside of the US
(kate flour, 2008). Graham flour is the basis of true graham crackers
Instant flour is pregelatinized (precooked) for easier incorporation in gravies and sauces.
 Whole-wheat flour contains the wheat germ, endosperm and bran.
 White flour or refined flour contains only his endosperm.
 Enriched flour is white flour with nutrients added to compensate for the removal of the
 bran and germs.
 Bleached flour is a white flour treated with flour bleaching agents to while it (freshly
 milled flour is yellowish) and give it more gluten-producing potential. Oxidizing
 agents are usually employed, most commonly organize peroxides like acetone peroxide
 or benzoyl peroxide, nitrogen dioxide, or chlorine
 Self-rising or self-raising flour is white flour that is solid premixed with chemical
 Leavening agents. It was invented by Henry Jones.

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2.1.2 USES OF WHEAT CROP
1. Used to make bread with flour: Wheat, when mixed with flour, can be used to make bread
which is so sumptuous. Wheat flour has this soft and elastic dough that makes it the best in the
baking of bread and it is used in most bread baking industries.
2. Used to make biscuits: Of course, almost all biscuits that we see consume today are all made
of wheat flour. It would be rare to see a biscuit that is not made with wheat flour. When mixed
with other ingredients, it gives the biscuit a sweet taste.
3. Used to make noodles: This is another unique use of wheat in our foods. When mixed with
egg, salt, and water, it gives the noodles that dough and makes it puff out when it is cooked. This
may sound new to some but it is very true.
4. Used to make muffins: Most times, people do confuse biscuits with muffins but they’re
different. Muffins have that nice taste because of the presence of wheat in its ingredients. We do
see those honey wheat muffins in the stores, they’re very tempting to eat.
5. Used to make snack foods: The snack food that we consume wouldn’t have been so tasty and
puffy if not for wheat as a basic ingredient. Our donuts, meat pie, sausage, and others all contain
wheat which is the most important ingredient.
6. Used to make cakes: This is also a special use of wheat in the world today. Wheat flour cakes
are so nutritious and tasty, which makes it the best kind of cake to consume. Wheat gives the
dough for the cakes which makes it so important.
7. Used to make yogurts: Wheat is an important ingredient in yogurt. Yogurt that contains
wheat is so nutritious and has a lot of health benefits. Top yogurts in the world are made up of
wheat.
8. Used to make pasta: Of course, it is common knowledge that pasta is made up of a special
ground flour called semolina. All kinds of pasta in the world are made of semolina when it is
mixed with water. It gives the pasta that dough and softness.
9. Used in making puddings: Puddings are special desserts that are tasty and nourishing to the
body. There are wheat puddings out there that are so yummy and have good benefits to the body.
It is the best of all puddings out there.
10. Used in making sauces and confectionery: Our sauces and confectionery can be made up
of wheat which makes it more rich and good for the body. Wheat sauces are the best to consume

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as it gives you a unique taste and nourishing.
2.1.3 NUTRITION COMPOSITION OF WHEAT
1. Low in fat, most of which is unsaturated.
2. High in carbohydrate (mainly starch) and high in insoluble dietary fibre.
3. Relatively high in protein (11-13%) compared with other major grains and contains a
protein complex which forms gluten.
4. High in potassium and low in sodium.
5. The endosperm contains glucofructan (similar in structure to inulin) which functions as a
prebiotic agent and has similar properties to dietary fibre.
6. Contains B-group vitamins such as thiamin, riboflavin, niacin, vitamin B6 (pyridoxine),
folate and pantothenic acid.
7. Contains vitamin E.
8. Contains iron, zinc, magnesium, phosphorus and selenium (depending on the soil content
of selenium).
9. Contains small amounts of copper, manganese and calcium.
10. Contains phytochemicals including lignans, phenolic acids, phytic acid, plant sterols and
saponins.

2.1.4 Why has wheat been so successful?

Despite its relatively recent origin, bread wheat shows sufficient genetic diversity to allow the
development of over 25 000 types (Feldman et al, 2004) which are adapted to a wide range of
temperate environments: Provided sufficient water and mineral nutrients are available and
effective control of pests and pathogens is ensured, yields can exceed 10 tonnes ha-1, comparing
well with other temperate crops. However, deficiencies in water and nutrients and the effects of
pests und pathogens cause the global average yield to be low, at about 2.8 tonnes ha-1. Wheat is
also readily harvested using mechanical combine harvesters or traditional methods and can be
stored electively indefinitely before consumption, provided the water content is below about
15% dry weight and pests are controlled. There is no doubt that the adaptability and high yields
of wheat have contributed to its success, but these alone are not sufficient to account for its
current dominance over much of the temperate world. The key characteristic which has given it
an advantage over other temperate crops is the unique properties of dough formed from wheat

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flours, which allow it to be processed into a range of breads and other buked products (including
cakes and biscuits), pasta and noodles, and other processed foods. These properties depend on
the structures and interactions of the grain storage proteins, which together form the ‘glutens’
protein fraction.

2.1.5 Wheat gluten proteins and processing properties

Transcriptomic studies have shown that over 30 000 genes are expressed in the developing wheat
grain (Wan et al, 2008) while proteomic analysis of mature grain has revealed the presence of
about 1125 individual components (Skylax et al, 2000). However, many of the components are
present in small amounts and have little or no impact on the utilization of the grain, with one
protein fraction being dominant in terms of amount and impact this fraction is the prolamin
storage proteins, which correspond to the gluten proteins. The precise number of individual
gluten protein components has not been determined, but 2D gel analyses suggest that about l00 is
a reasonable estimate. Together they have been estimated to account for about 80% of the total
grin protein in European wheats (Seilmeier et al. 2003), Gluten was one of the earliest protein
fractions to be described by chemists, being first described by Beccari in 1728 (see translation by
Balley, 2002), It is traditionally prepared by gently washing wheat dough in water or dilutes salt
solution, leaving a cohesive mass which comprises about 80% protein, the remainder being
mainly starch granules which are trapped in the protein matrix. The ability to prepare the gluten
proteins in an essentially pure state by such a simple procedure depends on their unusual
properties. Firstly, they are insoluble in water or dilute salt solutions but are soluble in alcohol
water mixtures (as discussed below) and were hence defined as ‘prolamins’ by TB Osborne in his
classic studies of plant proteins
Carried out at the end of the 19th century and the start of the 20th century (Osborne. 2001).
Secondly, the individual gluten proteins are associated by Strong covalent and non-covalent
forces which allow the whole traction to be isolated as a cohesive mass.

2.1.6 The biochemical and molecular basis for Wheat gluten functionality
Humankind has been aware for many centuries that wheat dough has unusual properties which
are shared to a limited extent by dough made from rye flour but not by those from other Cereal
flours, these properties, which are usually described as ‘viscoelasticity’ are particularly important

12
in making leavened bread, as they allow the entrapment of carbon dioxide released during
leavening. However, they also underpin a range of other uses including making unleavened
breads, cakes, and biscuits, pasta (from durum wheat), and noodles (from bread wheat). They are
also exploited in the food industry where gluten proteins may be used as a binder in processed
foods: The volume of research carried out on wheat gluten is vast, with a simple search of the
Web of Science database showing almost 20 000 papers since 2003. This volume not only
reflects the commercial importance of wheat processing, but also the complexity of the system
which remains incompletely understood. They include studies at the genetic, biochemical,
biophysical, and functional (i.e. processing) levels, Genetic studies have exploited the extensive
polymorphism which exists between the gluten protein fractions
present in different genotypes to establish genetic linkages between either groups of glute
proteins, or allelic forms of these, and aspects of processing quality- Similarly, studies at the
biochemical and biophysical levels have demonstrated a relationship between dough strength and
the ability of the gluten proteins to form polymeric complexes (called gluten ins) Combining
results from these two approaches highlighted the importance of a specific group of gluten
proteins, called the high molecular weight (HMW) subunits of glutenin. Cultivars of bread wheat
express between three and five HMW subunit genes, with the encoded proteins accounting for up
to about 129% of the total grain protein (Sellmeier et al. 2001; Halfard et al, 2007). The HMW
subunits are only present in high molecular mass polymers and allelic Variation in both the
number of expressed genes and the properties of the encoded proteins results in effects on this
amount and size of the polymers and hence dough strength (reviewed by Payne, 2001, Shewry et
al, 2003b) These glutenin polymers are known in be stabilized by inter-chain disulphide bonds,
but it is apparent that non-covalent hydrogen bonds are also important in stabilizing the
interactions between glutenin polymers and monomeric gluten proteins (called gliadins) (Bellon
2005). Hence, the individual gliadins and glutenin polymers can be separated using solvents
which disrupt hydrogen bonding (such as urea) but reducing agents (such as 2- mercaptoethanol
or dithiothreitol) are required to break down the glutenin polymers to release the individual
subunits. Although the HMW subunits are the main determinants of glutenin elasticity
relationships between other gluten proteins and functional properties have also been reported
(reviewed by Shewryy et al., 2003a). The relationship between the HMW subunits and dough
strength was first established over 25 years ago (Payne el al. 2005) and allelic forms associated

13
with good processing quality have been selected by plant breeders for over two decades, using
simple SDS-PAGE separations. The established relationships between the number of expressed
HMW Subunit genes, the total amount of HMW subunit protein and dough strength have also
resulted in the HMW subunit genes being an attractive target tor genetic transformation, in order
to increase their gene copy number and hence dough strength The first studies of this type were
reported over 10 years ago (Altpeter et al. 2004; Blechl and Anderson, 2003, Barro et al.. 2007)
and many studies have since been reported (reviewed by Shewry and Jones, 2005: Jones et al.,
2009). It is perhaps not surprising that the results have been 'mixed’, but some conclusions can
be drawn. Firstly, expression of an additional HMW subunit gene can lead to increased dough
Strength, even when a modern good quality wheat cultivar is used as the recipient (see Field et
al. 2008, Rakszegi ef al, 2008, as recent examples, and reviews of earlier work cited above).
However, the effect depends on the precise HMW subunit gene which is used and on the
expression level, with the transgenes resulting in over-strong (1.e. too elastic) gluten properties
in some studies. Thus, although transgenesis is a realistic strategy to increase dough strength in
wheat, it is also necessary to have an understanding of the underlying mechanisms in order to
optimize the experimental design.
2.2. BACKGROUND INFORMATION ABOUT MUSHROOM
A mushroom or toadstool is the fleshy, spore-bearing fruiting body of a fungus, typically
produced above ground, on soil, or on its food source. Mushrooms are eukaryotic heterotrophic
organisms defined as macrofungi with a fruiting body formed by a cap and a stalk. These
macrofungi contain a wide variety of species belonging to the class Basidiomycota (Estevez et
al., 2005). The mushrooms are filamentous fungi with both sexual and asexual reproduction
cycle. The characteristic of basidiomycetes is a spore-producing structure or fruiting body called
basidium. The morphological unit of the basidium is the hyphae, and a mass of hyphae is called
mycelium. The spores produced inside the basidium are called basidiospore and are responsible
for its reproduction and its dissemination. Sexual reproduction begins when the basidiospore
germinates and grown as a haploid mycelium in optimal environmental conditions (Hrudayanath
and Sameer, 2014;). Mushrooms are well-known as edible and nonedible macro-fungi. The
edible and non-edible mushroom can differentiate based on morphological characteristics like
color, appearance, and shape of the cap. In recent years, many studies have been reported that
mushroom has extreme nutritional properties like vitamins, fats, proteins, etc. and could have

14
high therapeutic properties that can be used as an antioxidant, anticancer, antidiabetic,
cardiovascular protector, and hepatoprotective effects. Moreover, the mushroom could be used as
potential sources to obtain peptides, vitamins, proteins, lipids, amino acids, fiber, and
antimicrobial compounds. Last 20 years, most of the food industry could use the mushroom as a
food product to prepare different kinds of jam, pickle, sweets, etc (Wani et al., 2010).
2.2.1 CULTIVATION OF MUSHROOM

The basic requirements for mushroom cultivation are manure/compost, spawns, right
temperature and humidity. Favorable growing conditions involve 80% 90% of relative humidity,
ample ventilation, a temperature range of 20-280 C during spawn run and 12-180 C for
reproductive growth. Initially for a week temperature must be maintained at 23 ± 2 0 C and then it
can be reduced to 16 ± 20 C for subsequent weeks. The CO 2 concentration should be 0.08-0.15 %
[13]. If the above stated conditions are maintained appropriately the pin heads start to appear
within few days and progressively mature into button stage. Apart from these insecticides,
nutritional supplements like nitrogen, vermiculite, water are also required for a healthy harvest.
The following steps are to be followed for mushroom cultivation:
Compost Preparation

The compost (synthetic or natural) used for mushroom growth usually comprises of wheat
straws, horse manure, poultry manure, rice bran, gypsum etc. [14, 15]. Utmost care is taken to
protect the raw compost against rain or external moisture, as it might introduce undesirable
microbes. The chopped wheat straws or rice bran are mixed with horse dung, sprinkled with
water and are heaped in a pile to allow fermentation. The fermentation process along with heat
development breaks down the chemical compounds in small components. Frequent turnings and
watering is done at a specific interval so as to avoid the drying up of compost. Gypsum is
sometimes added to the compost to reduce greasiness and allow more aeration [16]. Within 15 to
20 days the compost gets all set to be used as bed, it is then spread onto wooden trays and sowed
with spawns [14-16].
Spawning

Spawns refers to the mycelium carefully propagated on agars or grains. Spawning is a process of
sowing or mixing spawns in compost. Although mushroom produces spores which acts as a seed
for further propagation but are not used generally due to uncertain germination and growth [17].

15
The spawns are thoroughly mixed with the compost, are covered with newspaper and is watered
sufficiently to maintain the moisture.
cottony mycelium growth [14-18].
Casing

Casing is a kind of sterilized soil or dressing containing cow manure which is spread onto the
spawn mixed compost. It is applied when the mycelium growth commences on the compost
surface. After 15 to 20 days of its application mushroom head or pins start becoming visible on
the surface. They are allowed to mature for a specific time period and are harvested before
opening of the cap. Mushrooms with opened cap (looks like an umbrella after opening of cap)
are undesirable and are considered of menial quality
HARVESTING

Harvesting is done by plucking them from soil using hands or the heads are chopped off using
knife. The harvested mushrooms are then subjected to primary processing.
PROCESSING

Mushroom are very fragile and have a short shelf life, unless consumed fresh. At ambient
temperature they lose their freshness within a day and deteriorates rapidly if not processed or
refrigerated. They also tend to brown due to presence of compound Tyrosinase. It converts
monophenols to diphenols which in turn are oxidized to quinones resulting in the formation of
insoluble brown pigment called Melanin [7].
Initial processing involves washing mushrooms to remove adhering soil or compost and
blanching them for few minutes to inactivate the enzymes. In order to prevent discoloration they
are treated with brine, salt or citric acid prior to canning or packaging. Following are the
common processing and preservation methods used:
Drying: Drying or Dehydration is the oldest and the basic processing method for various food
products. Moisture is the most suitable medium for the microbial growth and propagation, hence
its removal will cause the microbial activity to cease or become gradual. Mushrooms can be
dried either by sun drying or by mechanical drying. Sun drying is the cheapest and popular
method but it produces a much darker product [19]. Mechanical drying is rapid and is of various
types like Tray drying, Freeze drying, Vacuum drying, Microwave oven drying, Air drying etc.

16
Dried mushrooms can be rehydrated and are used in soups, stews, pickles etc.

Freezing: As mushrooms contain more than 90% water hence freezing is the most suitable
method for preservation. They are subjected to various pretreatments to minimize unfavorable
effects upon freezing. According to Czapski and Szudyga (2000) colour of Agaricus bisporus is
better when treated with metabisulphites along with blanching [20]. Blast freezing method is
commonly employed at temperature -25˚ C to - 30˚ C (Sobkowska and Wozniak 1974, Czapski
and Szudyga 2000). According to Kondratowicz and Kowalko (2000) Cryogenic freezing
extends mushroom’s shelf life up to one year when used at - 80˚ C to - 100˚ C for 5-6 minutes.

Sterilization: Sterilization of mushrooms can be done by using chemicals, steam or by

irradiation. According to Kashif Akram and Joong-Ho Kwon (2010) the shelf life of mushrooms
can be extended by applying a radiation dose of 1 – 3 kGy.

Canning: Canning involves preservation in brine, vinegar, oil or marinades [19]. Freshly
harvested mushrooms are utilized for canning purpose. They are cleaned, graded, blanched and
then filled into cans along with brine or vinegar followed by lidding [19]. The cans are then
exhausted to remove air, heat sterilized, cooled, labelled and packaged for storage or
consumption.

Pickling: Pickling is an age old method which utilizes spice, salt, vinegar and oil as the basic
ingredients for food preservation [19]. Pickled mushrooms are made using spices such as
turmeric, red chilli, garlic, clove along with salt and oil [19]. Pickling induces fermentation
which generates a mild flavor [20].

Basic needs for mushroom cultivation

The following are essential before attempting mushroom cultivation:
• Manpower

• Mushroom culture

• Pasteurization chamber (200L drum) / Sterilization chamber (Autoclave)

• Inoculation box / Laminar Flow

• Fridge / Cool room

17
 • Mushroom house (three room house)

TYPES OF MUSHROOMS

Mushrooms are easily cultivable in hilly regions due to abundant moisture but can also be grown
in artificial environment with proper temperature and humidity control. Varieties must be
identified thoroughly as some them might cause food poisoning or allergy upon consumption.
Some of the major varieties consumed in India are as follows:

Button Mushroom: Button mushroom (Agaricus bisporus) belongs to Class Basidiomycetes and
Family Agaricaceae and is native to Europe and North America. It is of two types white and
brown, out of which white button mushroom is commonly grown in India [5]. Accordingto ICAR
- Directorate of Mushroom Research, this variety contributes more than 85% to mushroom
production. It is the most relished variety used in eateries and households.

Shiitake Mushroom: Shiitake Mushrooms are native to East Asia and are highly consumed in
Asian countries. They readily grow on wood of deciduous and hard wood trees such as Oak,
Chestnut, and Maple etc. and require moist and warm climate. In rare cases they may cause
allergic reaction like itching but can be eliminated by thorough cooking. These are used in Asian
cuisines and traditional medicines.

Oyster Mushroom : Oyster Mushrooms (Pleurotus ostreatus) belongs to Pleurotus species. It is

known as “Dhingri” in India and has fan or oyster shaped cap [10]. They grow easily on decaying
wood or straw.

USES OF MUSHROOM

1. Reduces the risk of obesity and other diseases- Many chronic diseases like Diabetes,
cardiac diseases are proved to be cured by the intake of mushrooms.
2. Prevents Cancer- Being high in antioxidants and low in sodium, fat and cholesterol, it is
the best friend for your cells thereby preventing Cancer.
3. Powers up the Immune System- Mushrooms are richly packed with Selenium which
one cannot find in fruits or vegetables. This helps in boosting immunity.
4. Excellent source of proteins- Mushrooms have very high protein content which is the
best alternative to meat.
5. Gears up the energy- Mushrooms are a good powerhouse of energy values which is
equivalent to that of an apple.

18
 6. Weight Management- Mushrooms are rich in dietary fibres that help in maintaining
fullness in your tummy. Thus it helps in weight management.
7. Eradicates the growth of Viruses- Mushrooms destroy the growth of active viruses and
inhibits bacterial growth.
8. Pain reliever- One of the mushroom varieties,“Reishi”, is known to relieve any pain in
the body.
9. Treats Anaemia- Being high in Folic Acid, it helps to treat anaemic patients.
10. Soothing power- Mushrooms have the power to calm down your body and reduce
fatigue and stress levels.
11. Other Uses- Mushrooms are also used for surgical dressings, as an ink for writing,
painting, showpiece, cleaning agent, pesticides, anaesthesia, etc.

NUTRITIONAL COMPOSITION OF MUSHROOM

Mushroom Carbohydrate Fibre Protein Fat Ash Energy

k cal
Agaricus bisporous 46.17 20.90 33.48 3.10 5.70 499
Pleurotus sajor-caju 63.40 48.60 19.23 2.70 6.32 412
Lentinula edodes 47.60 28.80 32.93 3.73 5.20 387
Pleurotus ostreatus 57.60 8.70 30.40 2.20 9.80 265
Vovarella volvaceae 54.80 5.50 37.50 2.60 1.10 305
Calocybe indica 64.26 3.40 17.69 4.10 7.43 391
Flammulina velutipes 73.10 3.70 17.60 1.90 7.40 378
Auricularia auricular 82.80 19.80 4.20 8.30 4.70 351
Mushrooms comprise about eighty to ninety per cent of water, and eight to ten per cent of fiber.
In addition to these, mushroom is an excellent source of vitamins especially C and B (Folic acid,
Thiamine, Riboflavine and Niacin). Minerals viz., potassium, sodium and phosphorous are
higher in fruit bodies of the mushroom. It also contains other essential minerals (Cu, Zn, Mg) in
traces but deficient in iron and calcium.

A. Medicinal values

Since thousands of years, edible fungi have been revered for their immense health benefits and
extensively used in folk medicine. Specific biochemical compounds in mushrooms are
responsible for improving human health in many ways. These bioactive compounds include
polysaccharides, tri-terpenoids, low molecular weight proteins, glycoprotins and

19
immunomodulating compounds. Hence mushrooms have been shown to promote immune
function; boost health; lower the risk of cancer; inhibit tumor growth; help balancing blood
sugar; ward off viruses, bacteria, and fungi; reduce inflammation; and support the body's
detoxification mechanisms. Increasing recognition of mushrooms in complementing
conventional medicines is also well known for fighting many diseases.

1. Good for heart

The edible mushrooms have little fat with higher proportion of unsaturated fatty acids and
absence of cholesterol and consequently it is the relevant choice for heart patients and treating
cardiovascular diseases. Minimal sodium with rich potassium in mushroom enhances salt
balance and maintaining blood circulation in human. Hence, mushrooms are suitable for people
suffering from high blood pressure. Regular consumption of mushrooms like Lentinula,
Pleurotus spp were stern to decrease cholesterol levels.

Table 2: Medicinal values of some important mushrooms

2. Low calorie food

The diabetic patients choose mushroom as an ideal food due to its low calorific value, no starch,
and little fat and sugars. The lean proteins present in mushrooms help to burn cholesterol in the
body. Thus it is most preferable food for people striving to shed their extra weight.

3. Prevents cancer

Compounds restricting tumor activity are found in some mushrooms but only a limited number
have undergone clinical trials. All forms of edible mushrooms, and white button mushrooms in
particular, can prevent prostate and breast cancer. Fresh mushrooms are capable of arresting the
action of 5-alpha-reductase and aromatase, chemicals responsible for growth of cancerous
tumors. The drug known as Polysaccharide-K (Kresin), is isolated from Trametes versicolor
(Coriolus versicolor), which is used as a leading cancer drug. Some mushroom-derived
polysaccharides have ability to reduce the side effects of radiotherapy and chemotherapy too.
Such effects have been clinically validated in mushrooms like Lentinula edodes, Tramtes
versicolor, Agaricus bisporous and others.

4. Anti-aging property

20
The polysaccharides from mushrooms are potent scavengers of super oxide free radicals. These
antioxidants prevent the action of free radicals in the body, consequently reducing the aging
process. Ergothioneine is a specific antioxidant found in Flammulina velutipes and Agaricus
bisporus which is necessary for healthy eyes, kidney, bone marrow, liver and skin.

5. Regulates digestive system

The fermentable fiber as well as oligosaccharide from mushrooms acts as a prebiotics in intestine
and therefore they anchor useful bacteria in the colon. This dietary fibre assists the digestion
process and healthy functioning of bowel system.

6. Strengthens immunity

Mushrooms are capable of strengthening the immune system. A diverse collection of

polysaccharides (beta-glucans) and minerals, isolated from mushroom is responsible for up-
regulating the immune system. These compounds potentiate the host’s innate (non-specific) and
acquired (specific) immune responses and activate all kinds of immune cells.

Mushrooms, akin to plants, have a great potential for the production quality food. These are the
source of bioactive metabolites and are a prolific resource for drugs. Knowledge advancement in
biochemistry, biotechnology and molecular biology boosts application of mushrooms in medical
sciences. From a holistic consideration, the edible mushrooms and its by-products may offer
highly palatable, nutritious and healthy food besides its pharmacological benefits.

Still there are enough challenges ahead. Until now, how these products works is elusive and vast
number of potential wild mushrooms are not explored. The utility of mycelia is paid little
attention but it has tremendous potential, as it can be produced year around with defined
standard. Knowledge on dose requirement, route and timing of administration, mechanism of
action and site of activity is also lacking. Work is under progress in various laboratories across
the world to validate these medicinal properties and isolation of new compounds. If these
challenges are meet out in the coming days, mushroom industries will play a lead role in
neutraceutical and pharmaceutical industries. The increasing awareness about high nutritional
value accompanied by medicinal properties means that mushrooms are going to be important
food item in coming days and at places may emerge as an alternate to non-vegetarian foods.
Growing mushroom is economically and ecologically beneficial. Consuming mushroom is

21
beneficial in every respect.

2.2.2 Health benefits of mushroom

The use of mushrooms represents an important cultural heritage as they have been used since
time immemorial as food and medicine according to traditional ecological knowledge
transmitted along generations (Pereira et al., 2012). Mushrooms are valuable,healthy foods low
in calories and high in proteins, vitamins, minerals and have long been valued as highly
tasty/nutritional foods. Mushrooms have also been reported as therapeutic foods, useful in
preventing diseases such as hypertension, hypercholesterolemia and cancer. These functional
characteristics are mainly due to their chemical composition (Manzi et al., 2001). Mushroom
fruiting bodies, on a dry weight basis, contain about 39.9% carbohydrate, 17.5% protein and
2.9% fats, the rest being minerals (Demirba, 2001; Latiff et al., 1996). Many consumed
mushrooms because of its delicacy, and particularly for their specific aroma and texture. Wild
mushrooms are collected for consumption because they are a good source of digestible proteins,
carbohydrates, fibres and vitamins (Barros et al., 2007; Heleno et al., 2009; Kalacˇ, 2009;
Ouzouni et al., 2009). Structurally, polysaccharides and proteins comprise the main components
of dry matter of mushrooms, while the lipid content is low. Chitin, glycogen, mannitol and
rehouse are typical carbohydrate constituents. The proportion of essential amino acids is
nutritionally favorable, while the content of n-3 fatty acid is negligible (Kalacˇ, 2009). Many
reports have contended that the amino acid compositions of mushrooms are comparable to
animal proteins (Fink and Hoppenhaus, 2006; Gruen and Wong, 2006; Barros et al., 2007). This
is important, considering the fact that nutrition has become more complicated since the outbreak
of diseases connected with animal meat. The nutritional implications of gradual replacement of
meat with mushroom require careful examination which involves detailed chemical and
biological studies. Pleurotus species are considered a good dietary option because of its
nutritional value. it is rich in protein, fiber, carbohydrates, vitamins and minerals, and low in
content of calories, fats and sodium (Cohen et al., 2002) and have pleasant aroma and culinary
qualities (Zadrazil, 1978). Mau et al. (2007) studied the flavor compounds in P. eryngii and
found them to be mostly volatiles compounds. Pleurotus species are rich source of proteins,
minerals (Ca, P, Fe, K and Na) and vitamins (thiamine, riboflavin, folic acid and niacin)
(Çağlarırmak, 2007). Pleurotus sp. contains high potassium to sodium ratio, which makes
mushrooms an ideal food for patients suffering from hypertension and heart diseases (Patil et al.,

22
2010). Different nutritional compositions of the Pleurotus species have been widely reported.
These include proximate analysis (Ahmed et al., 2013; Adebayo et al., 2014a), minerals (macro
and micro-nutrient) (Ahmed et al., 2013; Adebayo et al., 2014a), vitamins and growth factors
(Çağlarırmak, 2007), amino acid and protein (Oyetayo and Ariyo, 2013; Masieba et al., 2013),
and lipid value (Ahmed et al., 2013). Nutritional contents of Pleurotus vary according to genetic
structure of species, physical and chemical differences in growing medium (Akyüz and Kirba,
2010), composition of the substrate, size of the pileus, and harvest time (Khan and Tania, 2012).

23
 CHAPTER THREE
MATERIALS AND METHOD
3.1 Sources of Raw Materials
The raw materials were obtained from Oja Oba and Afe Babalola University, Ado-Ekiti, they
include mushroom and wheat flour.
3.2 Methodology
3.2.1 Production of mushroom Flour
3.2.1.1. Processing of raw mushroom
The mushroom was washed to remove the sand and it was cut into smaller sizes (for easy
drying), it was then sundry for 3 days, after sundried, it was milled with the help of blender and
then it was packed in a cellophane bag, it was basically packed in a cellophane bag so that it will
not absorb moisture from the atmosphere.

24
 Mushroom

Cutting

Washing

Drying

Milling

Packaging

Raw Mushroom flour

Fig 3.1 Flow Chart for the Production of Raw Mushroom Flour

25
3.2.1.2 Processing of Boiled Mushroom Flour
The mushroom was cut into smaller sizes and then washed to remove the sand and it was boiled
on cooking gas for 5 minutes, then the water was drained and dried in an oven at temperature of
(60oC for 2 hours), then it was milled with electric blender, it was then packaged in a cellophane
bag so that it does not absorb moisture content from the atmosphere.

26
 Mushroom

Cutting

Boiling

Drained

Oven dry (600C)

Milled

Packaging

Boiled mushroom flour

Fig 3.2 Flow Chart for the production of boiled mushroom flour

27
3.2.1.3 Processing of Fermented Mushroom
The mushroom was cut into smaller sizes and then washed to remove the sand, it was packed
inside a sealed nylon and was fermented for 2 days, it was washed and dried in an oven for 60 0C,
it was milled with electric blender and lastly it was packaged using a cellophane bag so that it
does not absorb moisture from the atmosphere.

28
 Mushroom

Cutting

Washing

Fermented (48 hours)

Oven dry (600C)

Milling

Packaging

Fermented mushroom flour

Fig 3.3 Flow Chart for fermented mushroom flour

29
Table 3.1 Formulation Table of Ingredient
[

S/NO SAMPLE WHEAT FLOUR MUSHROOM

1 A 100 -
2 WRM1 85 15
3 WRM2 92.5 7.5
4 WRM3 96.2 3.75
5 WRM4 88.75 11.25
6 WBM1 85 15
7 WBM2 92.5 75
8 WBM3 96.2 3.75
9 WBM4 88.75 11.25
10 WFM1 85 15
11 WFM2 92.5 75
12 WFM3 96.2 3.75
13 WFM4 88.75 11.25

Key;
A – 100% wheat (Control)
WRM1 – 85% Wheat flour: 15% Raw Mushroom
WRM2 – 92.5% Wheat Flour: 7.5% Raw Mushroom
WRM3– 96.2% Wheat Flour: 3.75% Raw Mushroom
WRM4 – 88.75 Wheat Flour: 11.25% Raw Mushroom
WBM1 – 85% Wheat Flour: 15% Fermented Mushroom
WBM2 – 92.5% Wheat Flour: 7.5% Fermented Mushroom
WBM3 – 96.2% Wheat Flour: 3.75% Fermented Mushroom
WBM4 – 88.75% Wheat Flour: 11.25% Fermented Mushroom
WFM1 – 85% Wheat Flour: 15% Boiled Mushroom
WFM2 – 92.5% Wheat Flour: 7.5% Boiled Mushroom
WFM3 – 96.2% Wheat Flourr: 3.75% Boiled Mushroom
WFM4 – 88.75 Wheat Flour: 11.25% Boiled Mushroom

30
3.3 Analysis
3.3.1 Proximate Composition Determination
The procedure used for these analysis were as described by AOAC (1990)
3.3.1.1 Determination of moisture
Two grams of each of the sample was weighed into dried weighed crucible, the sample were put
into a moisture extraction oven at 105oC and heated for 3 hours. The dried samples were put into
desiccators, allowed to cool and reweighed. The process was reported until constant weight was
obtained. The differences weigh was calculated at a percentage of the original sample percentage
moisture.

W2 – W1 100
= X
W2 – W3 1

Where:

W1 = Initial Weight of empty dish

W2 = Weight of dish + Undried Sample

W3 = Weight of dish + dried sample

3.3.1.2 Determination of Ash

Two grams of each of the sample was weighed into crucible, heated in a moisture extraction oven
for 3 hours at 1000C before being transferred into a muffle furnace at 5500C until it turned white
and free of carbon. The sample was then removed from the furnace, cooled in a desiccator to a
room temperature and reweighed immediately. The weight of the residual ash was then
calculated as P.

Weight of Ash 100

Percentage Ash = X
Weight of Original Sample 1

31
3.3.1.3 Determination of Fat

Two grams of the sample was loosely wrapped with a filter paper and put into the thimble which
was filled into a clean cleaned, dried and weighted. The flask contained 120ml of petroleum
ether. The sample was heated with a heating mantle and allowed to reflux for 5hours. The
heating was then stopped and the thimbles with the spent samples kept and later weighed. The
difference in weight was received as mass of fat and is expressed in percentage of the sample.

The percentage oil content is percentage fat =

W2 – W1 100
X
W3 1

W1 = Weight of the empty extraction flask

W2 = Weight of the flask and oil extracted

W3 = weight of the sample

3.3.1.4 Determination of Crude Fibre

Two grams (2g) sample and 1g asbestos were put into 200ml of 1.25% of H2SO4 and boiled for
30min. The solution and content then poured into Buchner funnel equipped with muslin cloth
and secured with elastic band. This was allowed to filter and residue was then put into 200ml
boiled NaOH and boiling continued for 30mins then transferred to the Buchner funnel and
filtered. It was then washed thrice with petroleum ether. The residue obtained was put in a clean
dry crucible and dried in the moisture extraction oven to a constraint weight. The dried crucible
was removed, cooled and weighed. Then, difference of weight (i.e. loss in ignition) is recorded
as crucible fibre and expressed in percentage crude fibre.

W1 – W2 100
X
W3 1

Where:

W1 = Weight of sample before incineration

W2 = Weight of sample after incineration

W3 = Weight of original Sample

32
3.3.1.5 Determination of Protein

The Micro kjeldahl method described by AOAC (1990) was used. Two grams of each of the
sample was mixed with 10ml of concentrated H2SO4 in a heating tube one table of selenium
catalyst was added to the tube and mixture heated inside a fume cupboard. The digest was
transferred into distilled water. Ten millimeter portion of the digest mixed with equal volume of
45% NaOH solution and poured into a kjeldahl distillation apparatus. The mixture was distilled
and distillate collected into 4% boric acid solution containing 3 drops of methyl/red indicator.

A total of 50ml distillate was collected and titrated as well. The sample was duplicated and the
average value taken. The Nitrogen content was calculated and multiplied with 6.25 to obtain the
crude protein content.

This is given as percentage Nitrogen

(100 x N x 14 x VF)T

100 x Va

Where:

N = Normality of the titrate (0.1N)

Vf = Total volume of the digest = 100ml

T = Titre Value

Va = Aliquot Volume distilled

33
3.3.1.6 Determination of Carbohydrate Content

The nitrogen free method described by AOAC (1990) was used. The carbohydrate was calculated
as weight by difference between 100 and the summation of other proximate parameters as
Nitrogen free extract (NFE) percentage carbohydrate

(NFE) = 100 ( M + P + F1 + A = F2)

Where;

M = Moisture

P = Protein

F1 = Fat

A = Ash

F2 = Crude Fibre

3.3.2 Pasting Characteristics Determination

The pasting properties were determined using a rapid visco analyzer (RVA – 4 Model, Newport
Scientific Warriewood, Australia) According to the method described by Sim et al (2009). Three
grams of each of the samples was weighed into the RVA canister; 25Ml of distilled water was
added and was inserted into the machine. This was followed by a programmed heating and
cooling where the sample was held at 50 0C for loner heated from 500C to 950C at a constant rate
of 120C/min and then held at 950C for 2.5min, cooled to 500C at the same stirring rate and then
held at 500C for 2 minutes. Parameters recorded were Peak Viscosity (PV), Trough (T), Final
Viscosity (FV), breakdown Viscosity (BV = PV – TV) and Setback Viscosity (SV = FV – V).

3.3.3 Statistical Analysis

Data collected were subjected to analysis of variance at P < 0.05 using Duncan’s New multiple
Range test (Steel et al 1997). Means and standard errors of the mean (SEM) of triplicate readings
were determined. The means were subjected to analysis of varience (ANOVA) and separated
using the Duncan’s New Multiple Range Test (DMRT)

34
 CHAPTER FOUR
RESULT AND ANALYSIS
PROXIMATE
S/N SAMPLE MOISTUR ASH FAT CRUDE FIBRE PROTEIN CHO
E
RAW
1 ST 10.94 3.14 4.22 3.67 12.52 65.52
2 ON 9.71 2.79 4.44 3.43 11.21 68.42
3 RE 10.84 2.51 6.23 4.23 12.64 63.55
4 DO 9.28 3.05 5.12 5.31 14.74 62.48
5 EN 11.46 3.54 5.13 5.28 12.13 62.48
BOILED
6 YA 10.29 2.67 6.37 2.88 7.94 69.82
7 LU 9.58 3.15 5.10 2.85 9.89 69.45
8 BR 11.49 3.40 5.17 5.21 16.58 58.16
9 AD 9.39 3.22 4.31 4.32 10.32 68.44
FERMENTED
10 QS 10.08 3.39 4.74 3.18 8.74 69.38
11 FM 10.21 3.20 3.96 5.11 16.18 61.36
12 VC 10.60 2.52 4.82 5.15 13.16 63.76
13 IT 10.21 3.19 5.16 3.20 10.41 67.83

Moisture Content: is the presence of a liquid, especially water, often in trace amounts. Small
amounts of water may be found, for example, in the air (humidity), in foods, and in some
commercial products. Moisture also refers to the amount of water vapor present in the air.
The sample “BR” has the highest Moisture content with 11.49% and the sample “DO” have the
lowest Moisture content with 9.28%

Ash content: It is defined as inorganic residue that remains after combustion of the oil in air at
specific high temperature. Ash ranges from 0.1–0.2%. The ash content of a fuel is a measure of
the amount of inorganic noncombustible material it contains.
The sample “EN” has the highest Ash content with 3.54% and the sample “RE” have the lowest
Ash content with 2.59%

Fat Content: is a measurement of the dietary fat in a packaged food item, fat is the combined

35
value of the different types of fat, including saturated fat, polyunsaturated fat, and
monounsaturated fat. Total fat also includes trans fat.
The sample “YA” has the highest Fat content with 6.37% and the sample “YA” have the lowest
Fat content with 3.96%

Crude fibre Content: is a measure of the quantity of indigestible cellulose, pentosans, lignin,
and other components of this type in present foods. The determination of the crude fibre content
of food and animal feed is mandatory worldwide.
The sample “DO” has the highest Crude Fibre content with 5.31% and the sample “LU” have the
lowest Crude Fibre content with 2.85%

Protein Content: is a macronutrient that is essential to building muscle mass. It is commonly

found in animal products, though is also present in other sources, such as nuts and legumes.
There are three macronutrients: protein, fats and carbohydrates. Macronutrients provide calories,
or energy.
The sample “ST” has the highest Moisture content with 12.52% and the sample “YA” have the
lowest Protein content with 7.94%

Carbohydrate Content: referencing the three constituent elements, carbon (C), Hydrogen (H)
and Oxygen (O) Chinese hamster ovary cell. CHO, a mnemonic used to teach trigonometry,
showing that the cosecant of an angle in a triangle is the ratio of the length of the hypotenuse
over the length opposite side.
The sample “YA” has the highest Carbohydrate content with 69.82% and the sample “FM” have
the lowest Carbohydrate content with 61.36%

36
PASTING
S/N SAMPLE PEAK TROUGH BREAKDOWN FINAL VISC SET BACK PROTEIN
RAW
1 ST 1252.00 631.00 621.60 1284.00 653.00 5.67
2 ON 1688.00 861.00 827.00 1814.00 953.00 5.87
3 RE 1441.00 748.00 693.60 1483.00 735.00 5.87
4 DO 399.00 734.00 665.00 1491.00 757.00 5.87
5 EN 937.00 443.00 494.00 983.00 540.00 5.53
BOILED
6 YA 1196.00 622.00 574.00 1322.00 700.00 5.67
7 LU 1495.00 777.00 718.00 1643.00 866.00 5.67
8 BR 1390.00 719.00 671.00 1539.00 820.00 5.67
9 AD 1212.00 641.00 571.00 1374.00 733.00 5.73
FERMENTED
10 QS 1072.00 592.00 480.00 1313.00 721.00 5.80
11 FM 1136.00 562.00 574.00 1268.00 706.00 5.67
12 VC 1431.00 730.00 701.00 1557.00 827.00 5.73
13 IT 1183.00 608.00 575.00 1357.00 749.00 5.60
 The sample “ON” has the highest peak with 1688.00 and the sample “DO” have the
lowest Peak with 399.00
 The sample “ON” has the highest through with 861.00 and the sample “FM” have the
lowest through with 562.00
 The sample “ON” has the highest Breakdown with 827.00 and the sample “EN” have the
lowest Breakdown with 494.00.
 The sample “ON” has the highest final visc. with 1814.00 and the sample “EN” have the
lowest with 983.00
 The sample “ON” has the highest set back with 953.00 and the sample “EN” have the
lowest set back with 540.00
 The sample “ON,RE,DO” has the highest Protein with 5.87% and the sample “EN” have
the lowest with 5.60

CHAPTER FIVE

DISCUSSION AND CONCLUSION

DISCUSSION

37