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ACTIVATION OF OXIDOREDUCTASES IN MILLET AND
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ACTIVATION OF OXIDOREDUCTASES IN MILLET AND COWPEA GRAINS
IMPROVES PROTEIN UTILIZATION FOR GROWTH.

By

Lovisa Hinandyooteti Kambonde

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE
Department of Food Science and Human Nutrition

2006

ABSTRACT

ACTIVATION OF OXIDOREDUCTASES IN MILLET AND COWPEA GRAINS
IMPROVES PROTEIN UTILIZATION FOR GROWTH.

By

Lovisa Hinandyooteti Kambonde

Cowpea (Vigna unquiculata L.) is a legume that provides an affordable source of protein
for many regions in Africa where animal sources of protein are not affordable. Cowpea is
especially important for providing protein for weaning foods. Millet (Panicum miliaceum
L.) is used in Africa, India and in some parts of Asia as a source of energy. During
germination, oxidoreductases reduce disulfide bonds of less digestible proteins in grains.
Germination has also been used to decrease the anti-nutrient content, enhance
consumption, and in some cases, increase the bioavailability of proteins. The aim of this
study was to evaluate protein quality of germinated and extruded millet and COWpea
foods. It was hypothesized that germination will increase the bioavailability of cysteine,
particularly in cowpea, thus increasing growth potential. Millet and COWpea were
processed by extrusion and conventional cooking and evaluated for growth potential and
protein digestibility. The study showed that a blend of millet plus cowpea diet yielded the
greatest growth in weanling rats. Germination did not affect protein digestion, however
germination yielded higher food intake (37.9g) in comparison to non-germinated feed
(35.2g). Extrusion processing retained higher protein digestibility of millet than cooking.
Germinated feeds also resulted in more growth (51.1g) than non-germinated feeds

(43.1 g), strongly suggesting that germination increased the bioavailability of cysteine.

ACKNOWLEDGEMENT

Grateful acknowledgement is extended to my major professor, Dr, Maurice R Bennink
for his excellent guidance and support throughout the completion of my program. His
providing of research resources and time are greatly appreciated. I also would like to
extend sincere appreciation to Dr. Perry Ng and Dr. Mark Uebersax for serving on my

committee and for offering their ideas and technical expertise.

I want to thank George and my lab mate Katie for their friendly help and all my friends at
Michigan State University for providing moral support and making my stay at MSU
exciting and memorable. I am also grateful to the Ministry of Education, Namibia and

Africa America Institute for granting me the scholarship to undertake graduate program.
My love and gratitude to my mother and family members, for inspiring and stimulating
my interest in learning, but most importantly for making me realize and believing in my

abilities.

Above all my, my deepest gratitude goes to God almighty whose grace has sustained me

during the duration of my studies and throughout my life.

iii

TABLE OF CONTENTS

Chapter Page
LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
I INTRODUCTION ........................................................................................................... 1
II LITERATURE REVIEW ................................................................................................ 3
2.1 MILLET (PANICUM MILIACEUML.) ........................................................................... 3
2.1.1 Global importance of millet ......................................................... 3

2.1.2 Uses of millet .......................................................................... 3
2.1.3 Millet porridge - common in Africa ................................................ 4

2.1.4 Millet varieties .......................................................................... 5

2.1.5 Description of millet plant and seed ................................................ 6

2.1.6 Nutritional profile of millet ......................................................... 7

2.1.7 Goiterogenic substances in millet ................................................... 8

2.2 COWPEA (VIGNA UNGUICULA TA) .............................................................................. 9
2.2.1 Global importance of cowpea ...................................................... 9

2.2.2 Description of cowpea plant and seed ........................................... 10

2.2.3 Cowpea varieties 11

2.2.4 Utilization ............................................................................ 12

2.2.5 Growing practices- African perspective .......................................... 12

2.3 ANT INUTRITIONAL FACTORS 1N MILLET AND COWPEA ........................ 13
2.3.1 Protease inhibitor .................................................................... 13
2.3.2 Phytohemagglutinin activity ........................................................ 14

2.3.3 Polyphenols, tannins and phytic acids ............................................ 15

2.4 GERMINATION ........................................................................... 16
2.5 EXTRUSION .......................................................................................................... 17
2.5.1 Extrusion cooking ................................................................... 17

2.5.2 Extrudate's starch viscosity ........................................................ 18

2.6 HUMAN REQUIREMENT FOR PROTEIN AND AMINO ACIDS ............... 19

iv

2.7 PROTEIN QUALITY ............................................................................................. 20

2.8 RATIONALE OF THE RESEARCH ..................................................................... 22
III MATERIALS AND METHODS ................................................................................. 23
3.1 Materials ............................................................................... 23

3.2 Methods ................................................................................ 23

3.2.1 Germination ......................................................................... 23

3.2.2 Non - germination .................................................................. 24

3.2.3 Extrusion ............................................................................. 24

3.2.4 Open kettle cooking and Drying .................................................. 25

3.2.5 Grinding .............................................................................. 25

3.2.6 Protein Extraction .................................................................. 26

3.2.7 Protein Determination ............................................................... 26

3.2.8 Diet Formulation and Preparation ................................................ 28

3.2.9 Protein Digestibility ................................................................. 29

3.2.10 Chemical Assays .................................................................. 34

3.2.11 Phytohemagglutinins Assay ...................................................... 35

3.2.12 Statistical Analysis ................................................................ 36

IV RESULTS AND DISCUSSION .................................................................................. 37
4.2 Total soluble proteins .................................................................... 37

4.2 Hemagglutinin activities .............................................................. 37

4.3 Amino Acid Score ..................................................................... 39

4.4 Protein Digestibility ................................................................... 42

4.5 Food Intake ............................................................................ 47

4.6 Growth ................................................................................. 49

4.7 Applications to Humans ............................................................... 53

V CONCLUSIONS ........................................................................................................... 55
VI SUGGESTIONS FOR FURTHER RESEARCH ........................................................ 56
VII APPENDICES ............................................................................................................ 57
Appendix I: The hemagglutinin levels of different diets as read at absorbance
740nm ....................................................................................... 58
Appendix 11: Protein Digestibility (%) for rats fed millet, cowpea and millet +
cowpea diets. ................................................................................ 59
Appendix III: Three way factorial AN OVA on protein digestibility ............... 60
Appendix IV: Food Intake for rats fed varying millet, cowpea and millet +
cowpea diets ................................................................................... 61
Appendix V: Three way factorial ANOVA on food intake ........................... 62

Appendix VI: Growth for rats fed varying millet, cowpea and millet + cowpea

diets ........................................................................................... 63
Appendix VH: Three way factorial AN OVA on grth .............................. 64
Appendix VIII:. Growth pattern for rats fed millet, cowpea and millet + cowpea
diets ........................................................................................... 65
VIII REFERENCES .......................................................................................................... 66

vi

LIST OF TABLES

Table Page
Table 3.1 Experimental design — a 2X2X3 factorial ............................................ 28
Table 3.2 Composition of positive control, metabolic proteins and millet diets. . . . . . . . ....31
Table 3.3 Composition of cowpea diets ........................................................... 32
Table 3.4 Composition of millet plus cowpea diets .............................................. 33
Table 3.5 Composition of basal mix used in all diets ........................................... 34
Table 4.1 Cowpea feed preparation and hemagglutinin % reduction ........................ 38

Table 4.2 Amino acid score for millet and cowpea in reference to the laboratory rat amino
acid requirement pattern ............................................................................ 40

Table 4.3 Amino acid score and limiting amino acids for millet and cowpea in reference
to the laboratory rat amino acids pattern requirements ......................................... 41

Table 4.4 Overall true digestibility (%) of protein as affected by germination, processing

method and grain type ............................................................................... 43
Table 4.5 True digestibility (%) for proteins in the various diets ........................... 43

Table 4.6 The Protein Di gestibility Corrected Amino Acids Score (PDCAAS) of

diets .................................................................................................... 46
Table 4.7 Overall treatment effect on food intake (weights are in g) ......................... 48
Table 4.8 Food intake for 3-day period (weights are in g) ..................................... 48
Table 4.9 Overall effect on grain weight (g) ..................................................... 49
Table 4.10 Average weight gain for rats fed varying sources of protein (g). . . . . . . . . ..50

vii

LIST OF FIGURES

Figure Page
Figure 2.1 Structure of a typical millet plant with seeds growing on panicles. .............. 6
Figure 2.2 Diagram illustrating the primary structures of the Cowpea plant .................... 11
Figure 2.3 Photomicro graph of raw starch with no moisture .................................. 18

Figure 3.1: Flow chart diagram showing the processing of foods used in the in vivo
study ................................................................................................ 25
Figure 4.1: Growth (g) in weanling rats as affected by the protein digestibility corrected

amino acid score ( %) of the diet consumed and food intake (g) ........................... 52

viii

ACRONYMS

AAS - Amino Acid Score
BV - Biological Value
FAO - Food and Agriculture Organization of the United Nations

F AOSTAT - Food and Agriculture Organization Statistic Database

HTA - International Institute of Tropical Agriculture
NDB - Nutrient Database
NPU - Net Protein Utilization

PDCAAS - Protein Digestibility Corrected Amino Acid Scores

PER - Protein Efficiency Ratio

RVU - Rapid Visco Units

TPD - True Protein Digestibility

USDA - United State Department of Agriculture

WHO - World Health Organization

ix

I INTRODUCTION

Cowpea is one of the important legume crops consumed in many parts of the world.
Because of its high protein content, this crop has a potential to influence the nutritional
profile of foods in countries where animal protein is unaffordable and/or not readily
available. However, the potential benefits can be limited by the presence of anti-
nutritional factors such as hemagglutinins (Oyeleke et al, 1985). Millet has been used for
centuries in India, parts of Afiica and Asia as a staple food to provide energy. Millet is
hardy and grows in hot and dry areas, adapting to local environments. Millet is more
efficient in utilization of soil moisture, has a higher level of heat tolerance and grows

better in these conditions than sorghum and maize ((FAO, 1991).

Cowpea is relatively high in protein (20-35%) and has therefore been used to improve
weaning foods in developing countries (Jirapa et a1, 2001; Oyeleke et al, 1985). Methods
have been developed (e.g. germination) to increase the bioavailability of legume and
cereal nutrients. During germination, a complex metabolic process takes places, where
lipids, carbohydrates and storage proteins in the seeds are broken down to energy and
amino acids (Urbano et a1, 2005). This improvement is usually as a result of breakdown
of complex carbohydrates and protein into smaller and more digestible molecules.
Oxidoreductases are enzymes that participate in redox reactions during germination, and
they reduce the disulfide bonds of storage proteins and proteins in the starchy endosperrn.
This reduction leads to the increased susceptibility of proteins to proteolysis.
Oxidoreductases also cause a reduction of anti-nutrients by inactivating specific enzyme

inhibitors (e. g. protease and amylase, Wong et al, 2004).

The preliminary work of the research focused on determining the optimum time for
germination and inactivation of phytohemaglutinin activities in both millet and cowpea.
The main focus of the research concentrated on evaluating the effect of germination,
processing methods (extrusion and traditional cooking) on protein digestibility of millet

and COWpea.

Millet and cowpea were allowed to either germinate or not, and extruded or cooked to
produce ingredients for an in vivo study. These products were formulated into 12 diets.
The 12 diet formulations were fed to male weanling rats to evaluate protein digestibility,
food intake and growth potential. Therefore the overall objective of these studies was to
investigate the effect of germination and processing methods on protein utilization of

millet and cowpea.

Thus the following hypothesis were tested:

1. Germination reduces disulfide bonds in some proteins to increase the “soluble protein”
fiaction and, therefore, growth.

2. Protein utilization is equivalent in extruded and traditionally cooked millet and

cowpea.

II LITERATURE REVIEW

2.1 MILLET (Panicum miliaceum L.)

2.1.1 Global importance of millet

Millet is one of the oldest foods known to humans and it has been used in Afiica and
India as a staple food for thousands of years. It is a summer cereal grass with large stems,
leaves and heads. Millet is an important food and forage cereal because it is a drought
tolerant crop that can be grown on poor, sandy soils in dry areas. It is more efficient in its
utilization of moisture than sorghum or maize. Millet grows well on poorly fertilized and
dry soils and fits well in hot climates with short rainfall periods and cool climates with
brief warm summers. The plants need good drainage, have a low moisture requirement
and do not grow well in waterlogged soils. Millet is unique due to its short growing
season. It can develop from a planted seed to a mature, ready to harvest plant in as little

as 65 days (FAO,1991).

2.1.2 Uses of Millet

In Afiica, millet is used to make bread, baby food, thick or stiff porridge and
ontaku/oshikundu, (a traditional non alcoholic beverage). It is also used as a stuffing
ingredient for cabbage rolls in some countries. Millet is a gluten free grain and lack of
gluten makes it a safe food for individuals who suffer from wheat allergies. Millet can be

used in pilafs, casseroles or most oriental dishes.

2.1.3 Millet porridge — common in Africa

Millet dishes vary according to the customs, culture and cultivars of each region. Millet
flour is used to make thin porridge, made by adding 10% flour to 90% water and boiling
for 10 to 15 minutes. This product is mainly consumed by children, because it has thin
consistency. Adults, consume thin porridges made from millet at breakfast, with fresh or
sour milk, butter, groundnut flour, sugar, honey, or limejuice as additives (FAO, 1991).
Thick porridges, soured or not, are made by adding approximately 40% (wzv) flour into
boiling water and stirring until the desired consistency is attained. The porridge is then
cooled and eaten with meat, vegetables, legumes or sour milk to enhance its palatability

as well as its nutritional value.

Germination of small grains has been shown to have beneficial effects in reducing the
bulk density of food (FAO, 1991) and thereby increasing its energy density. Souring the
porridge by the addition of a small quantity of starter that has been fermented for up to 48
hours is widely reported for Botswana, Tanzania, Zimbabwe, Zambia and Swaziland

(F A0, 1991). Soured porridges can be either of thin or thick consistency.

Thick ponidges are a staple food in many Afiican countries. Most meals feature stiff
porridges as a central element. According to the United Nations Food and Agriculture
Organization, "Cereals account for as much as 77% of total caloric consumption in
African countries, and contribute substantially to dietary protein intake (FAO, 1995).
The majority of traditional cereal-based foods consumed in Africa are processed by

natural fermentation.

Millet thick/stiff porridge is called oshithima, in Namibia, garri in parts of West Afiica
and ugali in East Africa. In Africa, porridges are generally served thick, with a solid
consistency that can be shaped and eaten with fingers, and are often served with saucy

stews and vegetables. It can also be served with fermented milk.

2.1.4 Millet varieties

There are many varieties of millet, but the four major types are Pearl, which comprises
40% of the world production, Foxtail, Proso, and Finger Millet. Pearl Millet produces the
largest seeds and is the variety most commonly used for human consumption. Pearl millet
is the most widely grown of all millets. It is a traditional crop grown in Western Africa,
particularly in the Sahel, however it is also grown in Central, Eastern and Southern
Africa. It is also known as bulrush millet, babala, bajra, cumbu, dukhn, gero, sajje, sanio

or souna (common names given in different countries).

Proso millet has been recently introduced as a grain crop in the South Eastern coastal
plain of the United States, and is grown in Colorado, North Dakota, and Nebraska. Most
of the crop is used for livestock, poultry, and bird feed with very little use for human
consumption. It is planted in spring and harvested in the fall and is often grown in
rotation to wheat, using the same planting and harvesting equipment (USDA, 2003).
Finger millet was once widely grown in Southern Africa, but its uses have been
overtaken by pearl millet. Finger millet is currently used as principle cereal grain in
Uganda. It is also used in India as popped grain and in Ethiopia to make arake, a strong

distilled liquor (Van Wyk & Gericke, 2000). Foxtail millet is one of the oldest cultivated

crops. It was used in India, China and Egypt before written records existed. This type of
millet is still used in Eastern Europe for porridge and bread and for making alcoholic

beverages (FAO, 1996).

2.1.5 Description of Millet plant and seed.

 

Figure 2.1: Structure of a typical millet plant with seeds growing on condensed panicles.

Source: www.fao.org/docrep/wl0808e/w1808e00.htm

Millet is a tall erect annual grass with an appearance strikingly similar to maize. The
plants will vary in appearance and size, depending on variety, and can grow anywhere
from one to 3 m tall. Generally the plants have coarse stems, growing in dense clumps
and the leaves are grass-like, numerous and slender, measuring about 2.54 cm wide and
up to 1.8 m long. The seeds are enclosed in colored hulls, with color depending on the

variety (FAO, 1996). The seed heads themselves are held above the grassy plant on a

spike like panicle 15 to 35 cm long. Because of a remarkably hard, indigestible hull, this
grain is generally de-hulled before it is used for human consumption. De-hulling does not
markedly lower the nutrient value, as the germ stays intact through the de-hulling
process. The de-hulled millet grains look like small yellow spheres with a hilum on one
side where it was attached to the stem. This gives the seeds an appearance similar to tiny,

pale yellow beads (FAO, 1996).

2.1.6 Nutritional profile of Millet

The nutritional quality of millet grain is directly related to its chemical composition. As a

cereal grain, millet grain is an important source of energy in the form of starch, but it can

also contribute a significant amount of fiber, mineral and other nutrients to the diet.

The limiting amino acid is lysine, although under better cultivation conditions, millet may
contain higher concentrations of lysine and other essential amino acids than sorghum,
maize, wheat or rice (Akingbala et a1, 2002). It is also a good source of B-complex
vitamins (niacin, thiamin, and riboflavin), lecithin, and vitamin E. It is particularly high
in the minerals iron, magnesium, phosphorous, and potassium. Protein quality is higher in
the outer layers of the endosperm and in the germ, because that is where the lysine-rich
albumins (water soluble proteins) and globulins (salt soluble proteins) are located. The
lysine poor prolamins (alcohol-soluble proteins) and the glutenins (acid or base — soluble
proteins) are located mainly in the inner endosperm of the seeds. In general as the
protein content of the cereal increases, its protein quality and digestibility decrease,
because the protein increase is mainly the lysine—poor, less digestible, prolamin protein

fraction (Kulp and Ponte, 2000).

2.1.7 Goiterogenic substances in millet

Millet has an interesting characteristic in the bulls and seeds contain small amounts of
goiterogenic substances (C — Glucosylflavones and their metabolites ) that limit uptake of
iodine by the thyroid (McDonough et al, 2000). Cooking destroys the enzyme that
converts thiocynates into active thyroid function inhibitors, and as such, cooked millet
should not be goiterogenic. However, some researchers feel that these "thyroid function
inhibitors" cause goiter (Akingbala et al, 2002) and they suggest that the correlation
between millet consumption and goiter incidence in some of the developing countries
where millet constitutes a significant part of the diet is due to the goiterogenic substances
in millet. But, in many of the developing countries where millet is consumed6, another

contributing factor may be a lack of sufficient dietary iodine.

2.2 COWPEA (Vigna unguiculata)

2.2.1 Global importance of cowpea

Cowpea (Vigna unguiculata), an annual legume, is also commonly referred to as southern
pea, black eye pea, crowder pea, lubia, niebe, coupe or frijole. This crop originated in
West Afiica where it was closely associated with the cultivation of sorghum and pearl
millet. It is widely grown in Africa, Latin America, and Southeast Asia. In the United
State the black-eyed cowpea type is grown primarily in California and is marketed as

California black-eyed peas (Davis et al, 1991).

Cowpea plant can be used at all stages of grth as a vegetable crop. The tender green
leaves are a significant food source in Africa and are prepared in a same way as spinach.
Immature snapped pods are used in the same way as snapbeans, often being mixed with
other foods. Green cowpea seeds may be boiled as a fresh vegetable, canned or fi'ozen.

Dry mature seeds are canned or eaten after boiling (Davis et al, 1991).

Cowpea seed can provide valuable nutrient component in the human diet. It is rich in
protein (18 — 35 %) and carbohydrates (55- 63%). The protein in cowpea seed is richer in
lysine and tryptophan compared to cereal grains. Therefore, cowpea seeds are often eaten
with cereals to improve the overall protein quantity and quality of the diet. Because of the
complex carbohydrates and dietary fiber, cowpea has a relatively low Glycemic Index
(G.I.) and is a good source of fiber (Helland et al, 2002). However, due to the long
cooking time and presence of the anti nutritional factors, its consumption has been
reduced (Leucona — Villanueva et al, 2006). The cowpea seed contain indigestible sugars

such as starchyose, raffinose and verbascose, which may cause flatulence in some

 

individuals, and as such make it an unpopular part of the human diet. Taste is also one of

the factors that limit the consumption of cowpea.

2.2.2 Description of cowpea plant and seed

The history of cowpea dates to ancient West Africa cereal farming, about 5 thousand
years ago and it was closely associated to sorghum and pearl millet. Cowpea is an annual
warm season crop and it adapts very well to many areas of the humid tropics and
temperate zones. It can withstand heat and dry conditions, but is intolerant of frost.
Cowpea plants are usually as erect and bushy, but other types are “climbers”. These
growth habits lead to differences from one species to another (Davis et al, 1991). The
cowpea seed can develop from a planted seed to a mature, ready to harvest plant in about
90 days. The seed coat can be smooth or wrinkled and of various colors including white,
cream, green, buff, red, brown, and black. Many are also referred to as blackeye or

pinkeye where the white colored hilum is bordered by another color.

10

 

 

 

Figure 2.2: Diagram illustrating the primary structure of the cowpea plant.

Source: International Institute of Tropical Agriculture (UTA)

2.2.3 Cowpea varieties

Cowpea plants are usually classified according to their seed type and coat color. Black
eye and purple eye—The immature pods shell easily, because the hull is easily bent and
the seeds come out of the pod clean and free. The shelled peas are suitable for processing.

The white hilum is surrounded by black, pink, or light-red color.

Brown eye-—-Pods vary in color fiom green to lavender and have a wide range of lengths.
The immature seeds, turn to a medium dark color upon cooking and they became very
tender with less natural flavor. Crowder—Seeds are closely packed in the pods and have
a globular shape. Cream—Seeds have a cream color and have no noticeable hilum.
White acre—This type is kidney shaped with a rounded end and is a semi-crowder,
generally tan in color and somewhat small with a hard pod. Each type produces a seed

with a distinctive appearance and flavor ( IITA, 2006).

11

 

2.2.4 Utilization

The dried black eye or purple eye cowpea types are mainly used for food products. The
dried beans are commonly sold directly to the consumer after cleaning and bagging
(common practice in Afiica). Another common use is the canned product. Cowpea is

also incorporated in various soups and bean mixes.

2.2.5 Growing practices — African Perspective

Traditionally, cowpea is grown on small farms, usually intercropped with cereals such as
millet and sorghum. The use of pesticides is not common because they are expensive and
often not available. The cowpea and cereal are usually planted in alternating rows. Millet
or sorghum is planted before the cowpea. The fast growth and spreading habit of
traditional cowpea varieties suppress weeds, and soil nitrogen is increased which
improves cereal growth. The two crops are harvested at different times, making food
available for a long period. West Africa is one of the biggest cowpea-growing regions in
the world with Nigeria producing 2,317,000 Mt in 2005 (FAOSTAT, 2006). Cowpea is
particularly important in West Afiica because of its adaptability to the tropical

environment.

12

2.3 ANT INUTRITIONAL FACTORS IN MILLET AND COWPEA

Cereal and legume grains contain compounds called anti-nutrients that reduce the
availability of nutrients. The anti-nutritional factors that are common to both millet and
cowpea are inhibitors of digestive enzymes (trypsin, chymotrypsin and amylase

inhibitors), phenolic compounds, tannins and phytic acids. Lectins are predominantly in

legumes (e.g. cowpea).

2.3.1 Protease inhibitor

Proteases inhibitors (trypsin and chymotrypsin inhibitors) bind with trypsin and
chymotrypsin and prevent protein hydrolysis. Amylase inhibitors will bind to amylase
and prevent starch hydrolysis. Consumption of these inhibitors will cause indigested
proteins and starch to pass to the colon where bacteria will use the protein and starch to
grow and will produce short chain fatty acids and gas as by—products. Extensive microbial

action will cause abdominal swelling (bloating), flatulence, and diarrhea.

If these inhibitors are consumed habitually, the pancreas will secrete more trypsin,
chymotrypsin and amylase to compensate or replace enzymes that are bound by the
inhibitors. Digestion will return to normal and abnormal discomfort will disappear.
Growth depressed by the trypsin/chymotrypsin inhibitor is due to poor digestibility of
dietary and endogenous proteins loss of amino acids in the form of enzyme being
secreted by hyperactive pancreas. Since pancreatic enzyme are rich in sulfur containing

amino acid, the effect of a hyperactive pancreas is to divert the amino acids from the

13

synthesis of the body tissue proteins to the synthesis of these enzyme that are

subsequently lost in the feces (Lajolo F M and Genovese M I, 2002).

2.3.2 Phflohemagglutinin activig

Consumption of lectins is more serious. Lectins bind to glycoproteins on cells that line
the digestive tract. As the lectins binds to the glycoprotein on cell membranes, the
membrane brakes and the cell dies. Destruction of cells lining the villi of the small
intestine causes sloughing of the villi tips, mal-absorption and bleeding. Large, acute
doses of lectins result in vomiting, abdominal pain, diarrhea and dehydration, may

require hospitalization.

Chronic consumption, such as forcing animals to eat lectins in raw legumes, will result in
weight loss, gastro intestinal bleeding and death (Bau et al, 1997). Since lectins bind to
glycoproteins of the red blood cells causing clumping of the cells, lectins are also known
as hemagglutinis. This property of causing red blood cell clumping is the basis for

detecting and quantification of active lectins in foods.

Other nutritional and physiological effects of phytohemagglutinins are lowering insulin
levels in blood, inhibition of disaccharides and proteases in the intestines, degenerative
changes in the liver and kidneys and intereference with absorption of non- heme iron and

lipid from the diet (Linier, 1994).

14

Lectins, protease inhibitors and amylase inhibitors are heat labile (Owen,1996).
Therefore, cooked or roasted foods are safe to consume as long as the foods receive
sufficient heat to inactivate these inhibitors. Other common food processing methods
such as, steaming, microwave cooking, drum processing and extrusion are efficient in
inactivating most or even all of the enzymes inhibitors. The inactivation is dependent on

the cooking time, temperature, particle size, and moisture conditions.

2.3.3 Polyphenols, tannins and phytic acids

Polyphenols, condensed tannins and phytic acids all interact with proteins to form
insoluble complexes. The resultant complexes reduce access of trypsin and chymotrypsin
to peptide bonds, which reduces protein digestibility. Since most of the phenols and
condensed tannins are in the hulls, de-hulling millet grain will increase protein

digestibility ( McDonough et al, 2000).

The brown red and black colors in the bulls of the millet and cowpea are mostly due to
the phenolic compounds and condensed tannins. Therefore dark colored varieties have

lower protein digestibility than light colored varieties unless bulls are removed.

15

2.4 GERMINATION

Germination technology is among the simple, inexpensive, easy to carry out and easily
adaptable technologies (Ariahu et al, 1999). Germination activates thioredoxins and
similar oxidoreductases, which reduce disulfide to produce sulflrydryl form. One purpose
of the oxidoreductases during germination is the inactivation of several inhibitors such as
amylase, trypsin, and chymotrypsin inhibitors (Wong et al, 2004). When the inhibitors
are inactivated, hydrolysis of protein and starch components occurs to provide seedlings

with raw materials to grow.

Germination is a simple technology that is sometimes used to hydrolyze starch and
reduce bulkiness of cereal foods to stimulate greater consumption of energy (Mbithi-
Mwikya et al, 2002). This is very important to infants and small children existing on
predominantly cereal diets. Germination may also, decrease cooking time, improve
sensory properties, increase vitamin C and of certain B- group vitamin content (Helland
et al, 2002) and decrease phytates (Trugo et al, 2000) and reduce the amounts of
oligosaccharides. However, germination is also a very active and complex metabolic
process that may alter the chemical, structural, and organoleptic properties of the seed,
and potentially decrease its nutritive value (Urbano et al, 2005). Reduction of disulfide
bonds has the general effect of increasing the solubility of proteins in aqueous solution.
Increased protein solubility may enhance digestibility of proteins and specifically
increase the bioavailability of cysteine - a sulfur containing amino acid. Sulfur containing
amino acids are often the first or second most limiting amino acids in hmnan diets that

contain predominantly cereals and legumes.

16

2.5 EXTRUSION

2.5.1 Extrusion cooking

Transformations that occur during processing is one of the most important factors that
distinguishes one food process and food type from another. Extrusion cooking, involves
heating to high temperatures for a short time, the application of mechanical mixing and
shear force, and formation of a structured product. Extrudates expand during the high
temperature, short time extrusion because the dough moisture cannot escape due to high

pressure, but instead flashes off as steam at the die exit (Onyango et al, 2004a).

The use of high temperatures and pressure reduce the processing time and allows a full
conversion of raw material to its functional form in periods as little as 30 to 120 seconds.
It is a relatively low moisture process compared with the conventional cooking (Guy R,
2001). Extrusion process produces a wide variety of foods and ingredients. Some
characteristics of extrusion are partial gelatinization of starch and denaturation of protein.
However, the degree of protein transformation is influenced by the pre and post
processing operations, and their interactions. Other characteristic includes inactivation of
many native enzymes and anti nutritional factors, reduction of microbial count, and
improvement in digestibility and biological value of proteins (Alarcon — Valdez et al,
2005). The complex physio — chemical changes that occur in carbohydrate rich foods
during high temperature short time extrusion are influenced by the operating conditions
of the extruder and the rheological property of the food. Extrusion thermo-mechanically
denatures and re—orients proteins in starchy foods resulting to changes in digestibility and

bioavailability of amino acids (Onyango et al, 2004b).

17

2.5.2 Extrudate’s Starch Viscosity

Unmodified starch granules are generally insoluble in water below 50°C. When Starch
granules are dispersed in water above 50°C, the granules absorb large amounts of water
and can swell to many times their original size. Above a critical temperature, the starch
granules undergo an irreversible process known as gelatinization. Starch begins to
gelatinize between 60 and 70°C, the exact temperature is dependent on the specific
starch. For example, different starches display different granular densities, which affect
the ease with which these granules can absorb water (Onyango, 2004b). The large
granules, which are usually less packed together, begin to swell first. The gelatinization
range is the temperature ranges over which all the granules are fully swollen. This range
is different for different starches. However, gelatinization can be observed visually,
because of increased translucency and viscosity. This is due to water being absorbed

away from the liquid phase into the starch granule (Guy, 2001).

 

Figure 2.3: Photomicrograph of raw starch with no moisture.

Source: Food Resource Nutrition and Food Management, Oregon State University

2.6 HUMAN REQUIREMENT FOR PROTEIN AND AMINO ACIDS

Proteins are made up of essential amino acids and non — essential amino acids. The
former cannot be synthesized in the body, therefore this type of amino acids must be
included in the diet and the latter group can be synthesized within the body and therefore
do not absolutely need to be in the diet. The essential amino acids are classified as such
because of the specific physiological roles they play in promoting growth in children,
preventing diseases and maintaining a positive nitrogen balance in children and adults.
The essential amino acid that worth mentioning is cysteine that is useful for pre-term
infants with metabolic disorder and malnourished patients suffering fiom compromised
liver functions such as cirrhosis. Proteins in the body are continually breaking down and
re-synthesized. A large proportion of the amino acids released during tissue protein
breakdown are reutilized for the synthesis of new proteins. Amino acids that are not
reutilized and those that are consumed in excess of the amounts needed for tissue
synthesis are degraded completely and their nitrogen is incorporated into urea, and

excreted quantitatively in the urine (Harper and Yoshimura, 1993).

Amino acids need to be consumed to replace the amino acids catabolized by the body.
For infant to children below 18, there is a need to consume more protein per kg of body
weight because new protein is slowly being deposited in the body (e.g., growth is
occurring). Synthesis of new protein requires a greater proportion of essential amino

acids and therefore children require better quality proteins than adults.

19

 

2.7 PROTEIN QUALITY

Apart from a favorable essential amino acid profile, easy digestibility is an important
attribute of a good quality protein. Chemical score does not take into account the
digestibility of protein or availability of amino acid. Biological methods based on grth
measurement and nitrogen retention assess the overall quality of the protein. These
methods include determination of protein efficiency ratio (PER), net protein utilization

(NPU), biological value (BV), and true protein digestibility (TPD) (FAO. 1995).

Protein digestibility corrected amino acid score (PDCAAS) is currently the most
appropriate method and its use is recommended by FAO. PDCAAS estimates protein
nutritional quality by combining information from calculations that determine the most
limiting amino acid in a given food protein based on the reference protein and an in vivo
assay that measures the true protein digestibility of the food protein. The PDCAAS is a
better estimate of protein quality for humans than PER that uses rat growth data. Rat
grth does not compare very well with the synthesis of protein in human adults, but is

more comparable to the human infant growth (Nielsen, 2003).

However protein digestibility by rats, is comparable to digestion of proteins by humans,
therefore the PDCAAS method most often estimates true digestibility of protein as
determined by the rats. One of the limitations of PDCAAS is that it uses the amino acid

score of only one amino acid and ignores the other essential amino acids.

20

 

The poor protein digestibility of cereals and legumes is caused by the polyphenols that
bind to the enzyme in the digestive tract and thus inhibiting the utilization of proteins and

carbohydrates (Onyango et a1, 2004 a).

The reaction of the phenolic compounds with the sulflrydryl and amino groups of protein
also results in decreased digestibility and availability of protein bound lysine and cysteine
(Owen, 1996). Other factors such as the interaction of proteins with the polysaccharides
and the interaction between proteins and dietary fiber also reduces the rate and the

completeness of protein hydrolysis.

21

2.8 RATIONALE OF THE RESEARCH

Germination is known to activate thioredoxin and other similar oxidoreductases that in
turn enhance protein in aqueous solutions. Preliminary work demonstrated that aqueous
soluble proteins were increased when millet and cowpea were germinated for 48 hours.
Existing literature suggest that germination has no effect or it slightly increases protein
digestibility perhaps by reducing di-sulfide bonds to the sulflrydryl form. The protein
digestibility data, however, are derived primarily from in vitro studies rather than in vivo
studies. Based on these findings, further research was designed to determine if increased
protein solubility would result in greater in viva protein digestibility. It was rationalized
that even if overall protein digestibility was not increased, the bioavailability of cysteine
could be increased as a result of germination. Moreover, open kettle cooking or boiling
of millet, the usual method to prepare millet-based foods in Afi'ica significantly reduces
protein digestibility (Kurien et al, 1961). It has been observed that boiling or open kettle
cooking sorghum also reduces the protein digestibility but extrusion retained high degree
of sorghum protein digestibility (Mosha T C E, 2004). Since most of the previous studies
that measured protein digestibility following germination was done in vitro, the effect of
germination on millet and cowpea protein digestibility is not well known. Also no one
has specifically examined if germination enhances bioavailability of cysteine. Therefore,
this study was designed to investigate the effect of germination and extrusion on the

millet and cowpea protein and cysteine utilization using an in vivo study.

22

 

III MATERIALS AND METHODS

3.1 Materials

Chemicals and reagents — the following items were purchased from Sigma Chemical Co
(St. Louis, MO): Tween 20 — P1379, bovine serum albumin — A4503, alkaline
phosphatate conjugate monoclonal anti- rabbit IgG- A2556, and p-nitrophenyl phosphate-
P7998; Vector Laboratories (Burlingame, CA): rabbit anti — Phytohemagglutinin IgG —
AS 2300; Bio Rad Laboratories (Hercules, CA): Bio-Rad DC protein assay kit 500-0111;
and Coming (New York, USA): 96 well EIA/RIA micro titer plate- 3369. All other
chemicals and reagents were of analytical quality and were purchased from local vendors.
Califomia black eye cowpea and millet grains were purchased from local retailers and

were stored in a cooler at 4°C.

3.2 Methods

3.2.1Germination

Germination of millet and cowpea followed the procedure described by Mubarak (2005)
with slight modifications. Extraneous materials were removed from both millet and
cowpea prior to soaking the grains in 95 % ethanol for 1 minute to provide surface
infection. Both millet and cowpea were rinsed three times with distilled water and
soaked in distilled water using the ratio of the grain to water of (l: 5 w/v) for 3 hours and
1 hour respectively. After soaking, both millet and cowpeas were germinated at room
temperature in trays for 48 hours with 12 hrs of alternating durations of light and

darkness. Water was sprayed on the trays at 12 hours intervals to wet the surface of the

23

 

grains. Millet had 93% germination and COWpea had 88%. Germinated millet and

cowpeas were then extruded or cooked.

3.2.2 Non-germination

Non-germinated millet and cowpea were manually sorted to remove the extraneous
material and soaked in ethanol for 1 minute. Both millet and cowpea grain were rinsed
three times with distilled water and soaked in distilled water using the ratio of the grain to
the water (1: 5 w/v) for 3 hours and 1 hour, respectively. After soaking, they were
divided into two equal parts, one half to be extruded and the other half to be cooked in
boiling water. The extruded portion was allowed to drain for 3 hours before extrusion,

and the cooked half was cooked immediately.

3.2.3 Extrusion

Extrusion of germinated and non-germinated millet and cowpea was carried out using a
JS3OA twin-screw extruder (screw diameter 30 mm and length of 420mm manufactured
by Quitong Chemical Industry Equipments Co, Ltd. Yantei, China). The barrel is divided
into three zones: the feeding zone was unheated, the middle zone was heated to 115°C,
and the exit end zone was heated to 130°C. The screw speed was 255 rpm and the die
used during extrusion was 7mm in diameter. The extruded products were air dried prior

to grinding.

24

3.2.4 Open Kettle Cooking and Dfling

The germinated millet and cowpea were cooked separately in steam-jacketed kettles in
distilled water. They were cooked for 45 minutes at 95°C. Cooked millet and cowpea
were then dried at 50°C in a large forced air oven (Model K12395, Proctor & Schwartz

Company, Philadelphia, PA, USA).

3.2.5 Grinding

After the drying, the cooked and extruded millet and cowpea were ground to pass through
a 1.6 mm screen (Model D hammer mill manufactured by Fitzpatrick Company Chicago,
IL, USA). These products were used to prepare diets that were subsequently used for the

in viva protein digestibility study.

Germination for 48 h or non germinated millet and cowpea

£1 E

Open kettle cooking Extrusion 130 °C exit end and
45 min/95 °C 105 °C middle zone
Oven 50°C Air-drying
Ground to pass though

1.6mm screen

Figure 3.1: Flow chart diagram showing the processing of foods used in the in viva study.

25

 

3.2.6 Protein Extraction

Proteins were extracted from the extruded and cooked samples using the extraction
method described by Wong et al, (2004) with few modifications. The extracted proteins
were separated into three fractions based on solubility in KCl and methanol. One g of
fine flour was suspended in 4 mL of cold (4°C) KCl buffer (SOmM Tris — HCl, lOOmM
KCl, 5 mM EDTA -— pH 7.8). The samples were incubated on ice for 5 minutes with
intermittent mixing (Vortex Genie 2, Scientific Industries, Inc, Bohemia, NY, USA) and
centrifuged at 4 °C for 15 minutes at 14, 000 rpm (Sorvall RC-5B refrigerated super
speed centrifuge, Sorvall Instrument, Newton, CT, USA). The supematants were
collected and the pellets were discarded. Five volumes of 0.1M-ammonium acetate in
methanol was added to the supernatant and the mixture was incubated overnight at ~20
°C. The samples were then centrifuged at 14, 000 rpm for 15 minutes at 4 °C to separate
the methanol insoluble fraction (pellets) from the methanol soluble fraction (supernatant).

The methanol insoluble fraction was resolubilized in the KC] buffer.

3.2.7 Protein Determination

The proteins in the methanol soluble and methanol insoluble fractions were determined
by a modified Lowry method using reagents and instructions provided by Bio-Rad. The
standard was prepared with bovine serum albumin. The modified Lowry method was
used instead of the Kj eldahl method because of the ammonium that was used during the
protein extraction. For total soluble protein, a modified Kjeldahl method was used
(Yasuhara and Nokihara, 2001). One hundred uL of the total protein fraction described in

section 3.2.6 was placed in the decomposing flask and 2 mL of concentrated sulfuric acid

26

(H2804) was added. The decomposing flask was placed on the heating unit (digesdahl
digestion apparatus model 23130 — 20, by Hach Company , Loveland, CO, USA) that
was preheated to 440°C. After the H2804 and protein extract boiled for 4 minutes, 4 mL
of 30% Hydrogen Peroxide (11202) was added. This mixture was boiled for an additional
10 minutes to boil off excess hydrogen peroxide. The flask was removed fi'om the heating
unit and cooled to room temperature before the digest was diluted with 10 ml of polished
water. Polished water is ultra pure water that has been passed through a C18 column-
(#6544 500mg —- Alltech Associate Inc, Dearfield, IL, USA). The digest was neutralized
with approximately 15 mL of 2 M Sodium Carbonate (NazCo3) and brought to 100mL
with polished water. The digested samples were further diluted 2, 25, and 50 times with
polished water for the colorimetric assay. One mL of n- hexane was pipetted into a test
tube. Two mL of sample and lml of reagent 1 (Phenol — 1 g and Sodium
Pentacyanonitrosyl Ferrate dihydrate — 5 mg, dissolved 100mL of polished water) was
added. The test tube was shaken for a few seconds, and lmL of reagent 2 (lmL of
sodium hypo — chloride and 1.5 g sodium hydroxide diluted in water to 100 mL) was

added. The tube was gently mixed and heated at 50°C for 40 minutes. Absorbance was
measured at 640 nm. A 2mM ammonium chloride (NH4C12) solution was used as a stock
standard. The stock standard was diluted to 1:20 and the following amounts of

ammonium chloride were concentrations were placed in series of test tubes: 0.04, 0.08,

0.12, 0.16 and 0.20umoles.

27

3.2.8 Diet Formulation and Preparation

A 2X2X3 factorial study was designed to determine the effects of germination, extrusion
and protein source on true protein digestibility and rat growth. The statistical design is
shown in Table 3.1.

Table 3.1 Experimental design — a 2X2X3 factorial

 

 

 

DIET EXTRUDED COOKED
Germinated Non- Germinated Non-
germinated germinated
Millet MGE MNGE MGC MNGC
Cowpea CGE CNGE CGC CNGC
Millet MCGE MCNGE MCGC MCNGC
+ Cowpea

Positive control — (Soy concentrate as a source of 10% protein)
Metabolic protein — (Lactalbumin that provided 2.5% protein)

Raw millet — (millet as a source of 10 % protein)

 

Twelve diets were formulated according to the AIN 93G guidelines (Reeves, 1993) —
Tables 3.2 — 3.6. The dietary products were as follows: Millet—non-germinated and
cooked (MN GC), cowpea—non-germinated and cooked (CNGC), millet + cowpea— non
germinated and cooked (MCNGC), millet—non—germinated and extruded (MNGE),

cowpea—non-germinated and extruded (CNGE), millet + cowpea—non-germinated and
extruded (MCNGE), millet—germinated and cooked (MGC), cowpea—germinated and

cooked (CGC), millet + cowpea—germinated and extruded (MCGE), millet-germinated

28

and extruded (MGE), cowpea—germinated and extruded (CGE), millet + cowpea—

germinated and extruded (MCGE).

The diets were formulated and processed in the laboratories of the Department of Food
Science and Human Nutrition. The millet plus cowpea diets were blended to provide the
maximum amino acid score for weanling rats. Three additional diets were included in the
study: a positive control (Table 3.2) that used soy concentrate as a source of protein, a
low protein diet used to estimate metabolic protein excretion, and a raw millet diet. All
diets (except the metabolic protein diet) were formulated to contain 10 % protein. All
other nutrients were provided at recommended levels and meet or exceeded known
requirements for rat growth (Reeves et al, 1993). All diets were stored at refrigerated

temperatures throughout the research period.

3.2.9 Protein Digestibility

Twenty one day old Male Sprague — Dawley rats were purchased from Harlan Sprague-
Dawley, (Indianapolis, IN) and were housed individually in suspended stainless steel
cages with wire bottoms. The temperature of the animal room was 22 i 2°C, the relative
humidity was 35 - 60% with alternating 12-hour periods of light and darkness. The rats
were fed the A1N- 93 G diet (Reeves et al, 1993) for an acclirnatization period of 4 days.
The rats were weighed and those rats at the extreme end of the distribution curve were
excluded from the study. The remaining animals were distributed into 15 groups of nine

animals each. The differences in mean weight between any two groups did not exceed 0.6

g. Twelve groups were assigned to millet and cowpea experimental diets as shown in

29

table 3.1. The 13th group was a control group fed a modified AIN- 93 diet (Table 3.2,
Reeves et al, 1993). The 14th group was fed a diet containing 2.5 % lactalbumin to
estimate metabolic protein. The 15th group was fed raw millet. Food and water were
provided ad libitum. The data collection period started after the fourth day of
acclimatization period, and lasted for 21 days. Animal weights were recorded every third
day during the 21 days period. Feces were collected for each rat on days 14 -17 and food
intake was measured during the same time period. The following protein quality indices
(apparent protein digestibility and true protein digestibility) were calculated fiom the data

collected from the in viva study:

Apparent (N ) digestibility (%) = t_o_t§l N consumed- total fec2_rl N * 100. (Eq. 1)
Total N consumed
True (N) digestibility (%) = tgfl N conpumed -—- (totaJMl N- metabolic N) *100 (Eq. 2)
Total N consumed
Amino acid score = lminfld content of feed type (test protein) (Eq. 3)
Amino acid content in the reference protein

The amino acid score of the feed type corresponds to the lowest ratio of essential amino
acids.

Protein digestibility corrected amino acid score (PDCAAS) = amino acid score * true

protein digestibility. (Eq. 4)

30

Table 3.2: Composition of positive control, metabolic protein and millet diets (g) used in

 

 

 

the rat feeding study.

INGREDIENTS POSITIVE METABOLIC MILLET

CONTROL PROTEIN NG-CI NG- E2 G-c3 G-E“
Millet 0.0 0.0 2760.0 2760.0 2760.0 2760.0
Cowpea 0.0 0.0 0.0 0.0 0.0 0.0
Cornstarch 2803.9 2330.4 0.0 0.0 0.0 0.0
Lactalburnin 0.0 75.0 0.0 0.0 0.0 0.0
Soy concentrate 504.0 0.0 0.0 0.0 0.0 0.0
Soy bean oil 280.0 210.0 90.0 90.0 90.0 90.0
Fiber 212.0 231.6 0.0 0.0 0.0 0.0
Basal (table 3.5) 200.1 150.0 150.0 150.0 150.0 150.0
Totals 4000.0 3000.0 3000.0 3000.0 3000.0 3000.0

 

1NG-C, non germinated and cooked
2 NG-E, non germinated and extruded
3 G-C, germinated and cooked

4 GE, germinated and extruded

31

Table 3.3: Composition of cowpea diets (g).

 

 

 

INGREDIENTS COWPEA

NG-C‘ NG- 132 G-C3 G-E‘
Cowpea 1160.4 1160.4 1160.4 1160.4
Corn starch 1661.3 1661.3 1661.3 1661.3
Lactalbumin 0.0 0.0 0.0 0.0
Soy concentrate 0.0 0.0 0.0 0.0
Soy bean oil 19.2 19.2 19.2 19.2
Fiber 9.0 9.0 9.0 9.0
Basal (see table 3.5) 150.0 150.0 150.0 150.0
Totals 3000.0 3000.0 3000.0 3000.0

 

1NG-C, non germinated and cooked

2 NG-E, non germinated and extruded

3 G-C, germinated and cooked

4 G-E, germinated and extruded

32

Table 3.4: Composition of millet plus cowpea diets (g).

 

 

 

INGREDIENTS MILLET + COWPEA

NG-c‘ NG- E2 G-c3 G-E‘
Millet 2167.6 2167.6 2167.6 2167.6
Cowpea 635.6 635.6 635.6 635.6
Cornstarch 976.5 976.5 976.5 976.5
Lactalbumin 0.0 0.0 0.0 0.0
Soy Concentrate 0.0 0.0 0.0 0.0
Soy bean oil 17.0 17.0 17.0 17.0
Fiber 3.2 3.2 3.2 3.2
Basal (table 3.5) 200.1 200.1 200.1 200.1
Totals 4000.0 4000.0 4000.0 4000.0

 

lNG-C, non germinated and cooked
2 NG-E, non germinated and extruded
3 G-C, germinated and cooked

4 G-E, germinated and extruded

33

Table 3.5: Composition of basal mix used in all diets.

 

 

BASAL MIX AMOUNT IN GRAMS
Mineral mix - minus calcium (AIN 93G) 1645.0

Vitamin mix (AIN 93G) 470.0

Calcium Carbonate (CaC03) 117.5

Choline Bitartrate 1 17.5

Butylated Hydroxy — Toluene (BHT) 0.7

 

3.2.10 Chemical Assays

Feces were dried for 3 days at room temperature, separated from the spilled food, and
dried to a constant weight in an oven at 100°C. The feces were finely ground using a
Science Ware-Micro Mill (Bel-Art products, Pequannock, NJ, USA). Total nitrogen in
the diets and the feces was determined by Dumas method using a F P 528 Nitrogen

Analyzer (LECO Corporation, St Joseph, MI, USA) and the crude protein content was

calculated using the factor N (6.25).

34

3.2.11 Phflohemagglutinins Assay

Active phytohemagglutinins in the processed food samples were determined by a
sandwiched enzyme - linked immuno sorbent assay (ELISA) as described by Boniglia et
a1 2003. One gram (1 g) of dried and finely ground food sample was extracted in 20 mL of
phosphate buffer solution (10mM phosphate buffer containing 150mM NaCl, pH7.2) by
stirring overnight at room temperature. A 96 well easy wash micro- titer plate, no lid high
binding certified, polystyrene plate was coated with 0.1 mL of porcine thyroglobulin
(8ng/ttL in 50 mM carbonate-bicarbonate buffer, pH 9.8) and incubated at 4°C overnight.
The treated microtiter wells were washed twice with PBT (phosphate buffer 10 mM, pH
7.2 containing 0.05% of Tween 20) and once with PBS. The coated micro titer wells were
treated with 0.1 mL of PBS containing 0.05 % bovine serum albumin and incubated at 37
0C for 60 minutes. After incubation the plate was washed as previously described. The
sample extracts (0.1mL) were loaded into the wells and incubated at 37°C for 60 minutes
and washed as described above. The micro titer plate was subsequently incubated with
0.1 mL of the following: 1) rabbit anti — Phytohemagglutinin IgG (diluted 1: 5000 in PBS
containing 0.25% BSA); 2) alkaline phosphatate conjugate monoclonal anti- rabbit IgG
(diluted 1: 10,000 in PBS containing 0.25%) and 3) p-nitrophenyl phosphate color
development solution. Between treatments 1 and 2, the micro titer plate was incubated for
60 min at 37°C, rinsed twice with PBT and once with PBS. After the third incubation, 50
uL of 3M NaOH was added to each well to stop the reaction. Absorbance of the final
solution was determined at 405 nm using a Microplate reader (Bio Rad, model 550,

Hercules, CA, USA). Samples of millet and cowpea flour that had not received any

35

thermal treatments were also analyzed and were considered to contain 100% of active

lectins for their respective grain.

3.2.12 Statistical analysis

Results are presented as mean i standard deviation or standard error of the mean (SEM)
as indicated. Data were subjected to a three-way analysis of variance (3 way - ANOVA)
using the Proth software, version 3.01 (Poly Software International Inc, Pearl River,
NY, USA). Treatment differences were considered significant at p S 0.05. Past hac

analysis was done by the Fisher‘s LSD test.

36

IV RESULTS AND DISCUSSION

4.1 Total Soluble Proteins

Germination increased protein solubility for both millet and cowpea as expected. Wong
et al, (2004) suggested that germination activates disulfide oxidoreductase to convert
disulfide bonds to the sulfhydryl form, which in turn alters certain proteins so that they
became more soluble in aqueous solutions. Germination increased the amount of soluble
protein by 30 % and 22 % for millet and cowpea, respectively. Germination was expected
to inactivate protease inhibitors by reducing disulfide bonds and in turn increase the

availability of the sulfur containing amino acid cysteine.

4.2 Hemagglutinin Activities

Intake of active phytohemagglutinins interfere with the intestinal mucosal membrane
brush border function by causing atrophy of the microvilli, and decreasing the absorption
of most of the nutrients (Owen, 1996). Moreover, consumption of active
phytohemagglutinin (for example insufficiently cooked beans) can be sufficiently serious
to require hospitalization. Since hemagglutinin activity is so potentially toxic, it is
important to determine hemagglutinin activity in any new process. Hemagglutinin
activity was reduced to negligible levels by both open kettle cooking and extrusion
(Table 4.1). There was no significant difference (p 20.05) between extruded and cooked
samples, suggesting that extruded legumes are as safe to eat as the cooked or boiled ones.
Since the residual phytohemagglutinin activities were very low, it suggests that extrusion

cooks the food sufficiently to render the food safe for human consumption.

37

Table 4.1:Cowpea feed preparation and hemagglutinin % reduction.

 

 

FEED TYPE % REDUCTION IN
HEMAGGLUTININ ACTIVITY‘

Cowpea non- germinated and extruded 99 i 2

Cowpea non-germinated and cooked 100 i 0

Cowpea germinated and extruded 96 2t 7

Cowpeas germinated and cooked 98 :t 3.4

For millet and millet + cowpea diet, the hemagglutinin activities were reduced to

undetectable levels

 

1 Mean :1: Standard deviation, % reduction is based on raw cowpea, and raw millet for

cowpea and millet diets respectively.

38

4.3 Amino Acid Score

Proteins that are deficient in one or more essential amino acid are considered as poor
quality and this deficient is often reflected in the amino acid scores. Lysine is limiting in
millet as in other cereal, and methionine is limiting in COWpea and other legumes. The
Amino Acid Score (AAS) compares the amino acid profile of the protein in a food to the
amino acid pattern required by a particular species at a particular age. For humans, the
amino acid pattern required by 2 — 5 year olds (FAO, 1991) is used because it surpasses
the amino acid requirement patterns of older children and adults. The AAS is calculated
as a percentage or alternatively the AAS can be expressed in decimal 0 — 1.0 format. Any
score above 100% is rounded to 100%. There is no benefit in consuming an amino acid
above the amount required, because the body can only utilize up to 100%. Foods that
have a score of less than 100% are considered to be limiting in one or more essential
amino acids and may not satisfy the amino acids need for normal growth of 2 — 5 years
old. The amino acid score for cowpeas alone (Table 4.2) was higher (0.51) than the
amino acid score for millet (0.33) when using amino acid pattern for the laboratory rat as
a standard (Table 4.2). Amino Acids listed in Tables 4.2 are from the USDA’s National
Nutrient Database for Standard Reference (www.nal.usdggov/fnic/foodcomb - millet
NDB No: 20031 and black eye pea NDB No: 16062). The most limiting amino acid in
millet is lysine as with many cereals. Cowpea is limiting in sulfur containing amino acids

as presented in table 4.2 and 4.3 for the laboratory rat requirement.

39

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40

4.3: Amino acid score and limiting amino acids for millet and cowpea in reference to the

laboratory rat amino acid pattern requirements.

 

 

 

 

Amino Acid MILLET COWPEA MILLET + COWPEA]

Score 0.33 0.51 0.67

Rank 2 Limiting Amino Acid

First Lysine Methionine + Cysteine Lysine

Second Threonine Threonine Methionine + Cysteine

Third Methionine + Valine Threonine
Cysteine

Fourth Valine + Histidine Isoleucine Tyrosine

 

T Sixty percent of the protein was derived from millet and 40% from cowpea.
2 The amino acid listed in each row is the amino acid that is most deficient, second most

deficient etc., for each column. The amino acid score is calculated using equation 3.

41

4.4 Protein Digestibiligy

Variable results have been reported for utilization of cereal and legume protein following
cooking and extrusion. For example germination of legumes and cereals was reported to
either have no effect, or to slightly increase protein utilization (Donangelo et al, 1995;
Mbithi-Mwikya et a1, 2002; Trugo et al, 2002 and Urbano et al, 2005). However it should
be noted that published data on utilization of millet and cowpea protein are primarily
limited to in vitro protein digestibility, and may not accurately reflect protein digestion by

mammals.

In this study there was a small overall difference in protein digestion between germinated
and non-germinated grains (Table 4.4), but the difference was not statistically significant
(p>0.05). The lack of significant difference in overall protein digestibility between
germinated and non-germinated grains is in part due to a significant interaction between
open kettle cooking vs extrusion and germination vs non-germination. When the grains
were cooked, germination improved protein digestion. However, when the grains were

extruded, germination lowered protein digestibility (Table 4.5).

42

Table 4.4: Overall true digestibility (%) of protein as affected by germination, processing

method and grain type.

 

TREATMENT

 

Germinated (75.4 a) vs. non-germinated (73.9 a)
Cooked (65.8 a ) vs. extruded ( 83.6 b)

Millet (70.9 a) vs. cowpea (80.9 b) vs. millet + cowpea (72.2 a)

 

1 Values are means. Values in the same row not sharing the same superscripts are
statistically different (p < 0.05). The true digestibility (%) for the positive control was

90.2 (0.5)h and 87.1 (1.0)gh for raw millet protein. The complete results for the 3-way

factorial ANOVA are shown in Appendix HI.

Table 4.5: True digestibility (%) for protein in the various dietsl.

 

FEED TYPE COOKED EXTRUDED

 

Non-germinated Germinated Non-germinated Germinated

 

Millet 52.6 (1 .5)a 57.3 (2.4)b 89.9 (0.9)h 83.6 (0.9)erg
Cowpea 78.6 (2.4)d 81.0(1.3)def 79.7 (1.7)“ 84.4 (2.1)fg
Millet + Cowpea 60.4 (1.0)b 64.6(1.1)c 82.3 (0.7)def 81.6 (1.1)def

 

1 Values are means 1 (SEM), N = 9, Values not sharing the same superscripts are
statistically different (p < 0.05), The true digestibility (%) for the positive control was

90.2 (0.5)h and 87.1 (1.0)gh for raw millet protein. The true protein digestibility was

calculated using equation 2.

43

There was an overall difference in protein digestibility depending on whether the grains
were cooked vs extruded (Table 4.4). Moreover, there was a significant interaction
between processing methods and grain type. It is evident from Table 4.5 that the most
pronounced differences due to processing were found in the diet that contained millet,
and that processing method had little effect on the protein digestibility of cowpea protein.
Digestion of raw millet protein was high (87.1%) and similar to soy concentrate protein

(90.2%).

Digestion of millet protein following extrusion was not different from raw millet.
However cooked millet had a low true protein digestibility (55%, Table 4.4) compared to
the extruded millet (87.1%). These results corresponded to the 52.9% protein digestibility
that Kurien et a1 (1961) reported for cooked pearl millet. Both processing methods
denature protein, but extrusion has limited moisture and higher heat for a short time
compared to boiling in an excess of water. It is not known why boiling reduces millet
protein digestion, but Eggum et al (1983) observed a similar decrease in protein

digestibility by cooking sorghum.

Comparing the cooked cowpea to cooked millet, cooked millet had a lower (p<0.05)
digestibility than cowpea, whereas raw and extruded millet yielded higher protein
digestibilities than cowpea (Table 4.5). The millet + cowpea diet had higher protein than
cowpea if the millet and cowpea were extruded whereas protein digestion was lower if
millet and cowpea were cooked. These differences were most likely due to the millet

rather than the COWpea.

44

The proportions of millet and cowpea in the millet plus cowpea diets were based on the
maximization of amino acid scores. This approach produced the amino acid scores listed
in table 4.3 and clearly show that a “complementary effect” was produced by blending
millet with cowpea. Since not all amino acids in a food are available (e. g. not all protein
is digested), multiplying the amino acid score of a particular protein by its protein
digestibility coefficient produces the protein digestibility corrected amino acid score
(PDCAAS). The PDCAAS is the best predictor of protein quality available today and the

PDCAAS for the diets used in this study are shown in Table 4.6.

The millet-based diets have low PDCAAS because of the very low amount of lysine. The
difference between cooked and extruded millet diets is due to the poor protein
digestibility when millet is cooked. Moreover, there is little differences between the
PDCAAS for cowpea and the cooked millet plus cowpea due to the poor digestion of
millet protein when millet is cooked. The safe levels of protein intake for 3-4 years old
children is 1.09 g/kg/day and decreases as the human body grow, and 0.75 g/kg/day for
young adult of 19 and older. According to FAO/WHO/UNU, the PDCAAS at 65% will
only be able to maintain the body and no growth support; this is specialty true for

growing children.

45

Table 4.6: The Protein Digestibility Corrected Amino Acid Score (PDCAAS) of diets1

 

 

 

DIETS ' PDCAAS (%) in reference to:
Weanling rat 2-5 yrs old

Millet non- germinated and cooked 17.49 17.49
Millet non- germinated and extruded 29.70 29.70
Millet germinated and cooked 18.81 18.81
Millet germinated and extruded 27.72 27.72
Cowpea non- germinated and cooked 40.29 79.79
Cowpea non- germinated and extruded 40.80 80.8
Cowpea germinated and cooked 41.31 81.81
Cowpea germinated and extruded 42.84 84.84
Millet + Cowpea non- germinated and cooked 40.47 40.2
Millet + Cowpea non- germinated and extruded 55.14 54.94
Millet + Cowpea germinated and cooked 43.28 43.55
Millet + Cowpea germinated and extruded 54.67 54.94

 

lThe PDCAAS was calculated using equation 4

46

4.5 Food Intake

Feed intake has a major impact on grth because it affects the amount of energy and
protein available for growth. The rat ate slightly more of the diet containing cooked foods
to the diets containing extruded foods, but this difference was not significant (Table 4.7).
Germination resulted in a significant increase in food intake (37.9g vs. 35.2g Table 4.7).
The rats ate about 60% more of the millet + cowpea diet than either the diets containing

millet or COWpea alone (Table 4.7).

There was a significant interaction between grain source and the type of processing. For
millet + cowpea, open kettle cooking or extruding had no effect on food intake, but for
millet or cowpea alone, rats ate 9% more of the cooked food than the extruded food

(Table 4.8).

Both sensory and metabolic effects are known to influence food intake. The sensory
attributes include the taste, physical characteristics while metabolic factor includes the
plasma amino acid homeostasis and satiety. There is no significant difference in food
intake regarding millet and cowpea individually. However, there is a significant

difference in food intake (p< 0.05) regarding the combination of millet and cowpea.

It is well known that the type and amount of protein influences the food intake, and foods

containing low and/ or imbalanced protein suppress the food intake, as reported by

Mosha T C E, (2004). Reversible, food containing too high protein suppress food intake.

47

Table 4.7: Overall treatment effect on food intake (weights are in g)1

 

TREATMENT

 

Germinated (37.9°) vs. non-germinated (35.2 2‘)
Cooked (37.4“) vs. extruded (35.7 8)

Millet (29.95 a )vs. cowpea (31.8 a ) vs. millet + cowpea (47.8 b )

 

r Values are means. Values in the same row not sharing the same superscripts are
statistically different (p < 0.05). The complete results for the 3-way factorial ANOVA are

shown in Appendix V.

Table 4.8: Food intake for 3-day period (weights are in g)1

 

 

 

FEED TYPES COOKED EXTRUDED

Non- germinated Germinated Non- germinated Germinated
Millet 30.3(1.8)abc 36.4 (2.0)“ 25.6(0.8) “ 27.5 (1.3) 3"
Cowpea 29.7 (2.1)“ 32.4 (1.6)°°°‘ 30.3 (1.4)“be 34.8 (3.0)cdc
Millet + Cowpea 47.1 (1 .3)f 48.2 (2.4) f 47.8 (1.9) f 48.1 (2.0) ‘

 

1 Values are means i (SEM), N = 9, Values in the same row not sharing the same
superscripts are statistically different (p < 0.05). The food intake was 38.0g (1 .1)° for the

positive control and 27.6g (1 .4)°° for raw millet.

48

4.6 Growth

All factors had a significant impact on grth but there were no significant interactions
between the main factors - grain type, processing, or germination. The most marked
effect was due to grain type. Rats fed the millet plus cowpea diet gained 1.7 times more
weight than rats fed the cowpea diets and 3.8 times more weight than rats fed millet diets
(Table 4.9). Likewise, rats fed cowpea grew twice as much as rats fed millet. Overall, rats
fed germinated millet and cowpea grew 19% more than rats fed the non-germinated
grains and rats fed cooked grains gained 9% more weight than rats fed the extruded
grains. It also noteworthy that rats fed the millet plus cowpea diets, regardless of
processing method or germination, all gained more weight than rat fed the positive
control (soy concentration protein, Table 4.10). Growths for individual diets are

presented in Table 4.10.

Table 4.9: Overall effect of germination, extrusion, and grain type on weight gain (g)l

 

TREATMENT

 

Germinated (5 1 . 1°) vs. non-germinated (43.1“)
Cooked (49.1 °) vs. extruded (45.2“)

Millet (20.3“) vs. cowpea (435°) vs. millet + cowpea (77 .5°)

 

I Values are means. Values in the same row not sharing the same superscripts are
statistically different (p < 0.05). The complete results for the 3-way factorial ANOVA are

shown in Appendix VH.

49

Table 4.10: Average weight gain for rats fed varying sources of protein (g)l

 

FEED TYPE COOKED EXTRUDED

Non- germinated Germinated Non- germinated Germinated

 

Millet 19.3 (1.2)a 28.9(1.7)° 12.7 (1.2)a 20.3 (1.18
Cowpea 40.2 (3.6)° 47.3 (2.9)c 39.5 (2.3)° 47.1 (3.1)c
Millet + Cowpea 74.3(1.8)e 84.4 (4.8)f 72.7 (4.1)c 78.8 (1.7cf

 

r Values are means i: (SEM), N = 9. Values in the same row not sharing the same
superscripts are statistically different (p < 0.05). Weight gain for the positive control was

57.5g (1.7)° and 16.0g (1.1)a for raw millet .

Growth is dependent upon consuming sufficient energy and all nutrients required for
growth. This research was designed so that the only limiting nutrient was protein.
Moreover, the sulfur amino acids were most limiting in the cowpea and the millet plus
cowpea diets. Therefore any small increase or decrease in available sulfur containing

amino acids was expected to cause detectable differences in growth.

A complicating factor in this type of research is food consumption. Ideally, food
consumption would be equal for all groups so that differences in growth would reflect
only differences in bioavailability of the sulfur containing amino acids. As the PDCAAS
becomes low, food intake is markedly depressed. The only small discrepancy in the data
to this generalization is that the rats ate more cooked millet than extruded millet even

though the PDCAAS’s for the extruded millet were greater than the cooked millet.

50

Multiple linear regression of growth as the dependent variable and PDCAAS and food
intake as the independent variables showed that 95% of the variation in growth is due to
these variables. Figure 4.1 illustrate this relationship. Rats that ate the germinated
cowpea and millet plus cowpea diets gained 13.6% more weight while only eating 5.6%

more than rats eating the corresponding cooked grains.

Alternatively, rats eating the germinated cowpea and millet plus cowpea diets require less
food (p<0.05) to gain 1 g of body weight than rats eating the cooked grains. Likewise, rats
eating the cooked diets gained more weight (p<0.05) per gram of protein consumed than

rats eating the cooked cowpea and the millet plus cowpea diets.

These measures of efficiency strongly suggest that germination increased the

bioavailability of cysteine, which in turn enhanced growth. Rats would have to be pair-

fed to conclusively demonstrate that germination enhanced utilization of cysteine.

51

   

3
c:
BS

50

 

 

 

 

40 Growth (g)

30
20
10

52

 

 

 

intake(g)

 

25

PDCAAS (%)

Figure 4.1 Growth (g) in weanling rats as affected by the protein digestibility corrected amino acid score %) of the diet consumed and

food intake (g).

4.7 Applications to humans

Dietary protein is needed to maintain existing protein in the human body and to support
normal growth. Adequacy of the protein component of the human diet depends upon the

total amount of protein consumed, the pattern of essential amino acid and digestibility.

The PDCAAS (Table 4.6) index reflects the estimated ability of the test protein to meet
the amino acid needs of an individual. The FAO/WHO/UNU recommended the use of the
arrrino acid requirement pattern of 2-5 year old child as a reference for food meant for
pre-school age children or older. The PDCAAS of the diets used in this study ranged
from 17 to 85 % when the amino acid profile for 2—5 year old was used as a reference.
The cowpea, regardless of germination or processing had the highest values ranging from
80-85%, while millet had the lowest scores. As for millet diets and the blend of millet
plus cowpea diets, lysine was the most limiting amino acid. The millet and cowpea blend
had high PDCAAS values (55%) compared to millet alone. This is due to the cowpea

protein, which improved the amino acid profile.

When the laboratory rat amino acid profile was used as reference pattern, all products had
serious deficiencies in essential amino acids, and the PDCAAS values ranged from 17 to
55 % for all foods. The requirement for sulfur containing amino acid requirement for rat
are greater than for pre-school children, and growth of weanling rats is much
proportionally faster than the growth of pre-school children. This difference explains the
low PDCAAS values for foods when the rat’s amino acid score was used. Since the

requirements for methionine + cysteine and lysine for growing rats are much higher than

53

the requirement for preschool age children, the growth observed for the growing rats in
this study are lower than what would have been if the diets has met all of the amino acids
requirements for rats. This suggests that a combination of millet plus cowpea would not

produce the complete amino acid requirement for growing rats.

A product that derived 66% of the protein from cowpea and 34% of the protein from
millet would be a complete protein with 100% PDCAAS and would meet the amino acid
needs for all people one year and older. This same product would also so be important in
rehabilitating malnourished children. Addition of minerals, vitamins and fat source would

make food nutritionally complete.

54

V CONCLUSIONS

This study showed that both the extruded and cooked blend of germinated millet and
cowpea diets were of high quality and displayed great potential to support growth. The
true protein digestibility values and growth response found in this study can be directly
translated to humans. Germination had no apparent effect on protein digestion, however
rats eating germinated grains ate more food (p<0.05) than they did if the grains were not
germinated. Overall germinated grains produced significantly higher growth (51.1 g) than
non-germinated grains (43.1 g). Extrusion processing yielded higher protein digestibility
(83.6%) than if the grains were not germinated (75.4%). This indicates that the type of
thermal processing is more important than germination when it comes to protein
digestibility. Activation of oxidoreductases in millet and cowpea grains improves protein
utilization for growth probably by increasing the bioavailability of the cysteine.
Appropriate combination of 52% millet and 48% cowpea offers great potential for
growth, because a high quality protein, especially for children and those who are HIV+,
is needed. On the other hand extrusion processing can be used to produce more versatile
and more digestible (83.6%) foods than open kettle cooking method (65.9%). Extrusion
cooking has the potential to replace the traditional labor intensive, energy and time
consuming cooking methods used in the production of many traditional foods. Extrusion
presents a valuable opportunity for overcoming food insecurity in developing world,
because extruded foods maintain extended shelf life and are ready-to-eat foods or they
can be reconstituted in hot water. Use of this extrusion technology will save time and

energy, which is a major constrain in traditional method of cooking food in boiling water.

55

VI SUGGESTIONS FOR FURTHER RESEARCH

In this study, bioavailability of cysteine was measured indirectly. The data strongly
suggested that germination improved cysteine bioavailability, but it cannot be concluded
with 100% confidence that germination was the only factor increasing growth, since
small differences in food intake may be the most important factor in improved growth.
Therefore a separate study is needed where food intake is closely controlled to evaluate
the difference in the protein (cysteine) bioavailability in germinated and non-germinated
millet plus cowpea. Further protein in germinated food could be hydrolyzed with
immobilized proteases to determine if more of the free amino acid cysteine is produced.
Further, a separate study is needed to investigate why conventional open kettle cooking

significantly reduces protein digestibility of millet, but not cowpea.

56

APPENDICES

57

VII APPENDICES

Appendix I: The hemagglutinin levels of different diets as read at absorbance
740nm.

 

 

DIET ABSORBANCE‘
Millet non- germinated and cooked 0.066 t 0.066
Millet non- germinated and extruded 0.078 i 0.066
Millet germinated and cooked 0.074 :1: 0.026
Millet germinated and extruded 0.083 :1: 0.004
Cowpea non-germinated and cooked 0.081 t 0.004
Cowpea non-germinated and extruded 0.090 :I: 0.002
Cowpea germinated and cooked 0.097 t 0.013
Cowpea germinated and extruded 0.093 t 0.051
Raw Millet 0.142 i 0.004
Raw Cowpea 0.234 i 0.031

 

rMeans :1: Standard deviation

MNGC- millet non germinated and cooked, CNGC- cowpeas non germinated and
cooked, MCNGC- millet + cowpeas non germinated and cooked, MNGE- millet non
germinated and extruded, CNGE- cowpea non germinated and extruded, MCNGE- millet
+ cowpeas non germinated and extruded, MGC- millet germinated and cooked, CGC-
cowpeas germinated and cooked, MCGC — millet + cowpeas germinated and cooked,
MGE- millet germinated and extruded, CGE — cowpeas germinated and extruded, MCGE

—millet + cowpeas germinated and extruded, RM- raw millet.

58

 

 

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59

Appendix 111: Three way factorial AN OVA on protein digestibility

 

 

 

Factorial AN OVA Statistics Report on protein digestibility

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Factorial ANOVA
Source DF SumOqu MeanOqu F-Value P-Value
C1 1 724.889 724.889 6.703 0.011
C2 1 1742.430 1742.430 16.1 13 0.000
C3 2 57909.337 28954.669 267.749 0.000
Main Eff. 4 60376.656 15094.164 139.578 0.000
C1*C2 1 19.423 19.423 0.180 0.673
C1*C3 2 128.965 64.482 0.596 0.553
C2*C3 2 96.815 48.408 0.448 0.640
C1*C2*C3 2 12.040 6.020 0.056 0.946
Error 96 10381558 108.141
Total 107 71015.457 663.696

 

60

 

 

 

 

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61

Appendix V: Three way factorial AN OVA on food intake

Factorial AN OVA Statistics Report for food intake

 

 

 

 

 

 

 

 

 

 

 

 

 

Source DF SumOqu MeanOqu F-Value P-Value
C1 1 61.111 61.111 1.917 0.169
C2 1 178.332 178.332 5.595 0.020
C3 2 6834.729 3417.364 107.213 0.000
Main Eff. 4 7074.172 1768.543 55.485 0.000
C1*C2 1 13.356 13.356 0.419 0.519
C1*C3 2 318.388 59.194 4.994 0.009
C2*C3 2 48.707 24.353 0.764 0.469
C1*C2*C3 2 54.632 27.316 0.857 0.428
Error 96 3059.942 31.874
Total 107 10569.196 98.778

 

62

 

 

 

 

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63

Appendix VII: Three way factorial AN OVA on growth

 

Factorial ANOVA Statistics Report on growth

 

 

 

 

 

 

 

 

 

Factorial ANOVA
Source DF SumOtSq MeanOqu F-Value P-Value
C1 1 724.889 724.889 6.703 0.011
C2 1 1742.430 1742.430 16.113 0.000
C3 2 57909.337 28954.669 67.749 0.000
Main Eff. 4 60376.656 15094.164 139.57 8 0.000
C1*C2 1 19.423 19.423 0.180 0.673
C1*C3 2 128.965 64.482 0.596 0.553
C2*C3 2 96.815 48.408 0.448 0.640
C1*C2*C3 2 12.040 6.020 0.056 0.946
Error 96 10381.558 108.141
Tota1 107 71015.457 663.696

 

64

 

Appendix VIII: Growth pattern for rats fed millep, cowpea and millet + cowpea

diets

 

 

 

 

 

 

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65

REFERENCES

66

VIII REFERENCES

Alonso R, Aquirre A, and Marzo F. 2000. Effects of extrusion and traditional processing
methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney
beans. Food Chemistry 68: 159-165.

Ariahu C C, Ukpabi U and Mbajunwa K O. 1999. Production of African breadfruit
(Treculia Africana) and soybean (Glycine max) seed based food formulations, 2: Effects
of germination and fermentation on microbiological and physical properties. Plant Foods
for Human Nutrition 54: 207 — 216.

Association of Official Analytical Chemists International, 1995. Official methods of
Analysis, 6th Edn. AOAC, Washington, DC.

Bau H M, Villaume C and Nicolas J P. 1997. Effect of germination on chemical
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