University of Nigeria

Research Publications A

utho

r

ONYEKWERE, Eucharia U.

PG/M.Sc/03/34733

Title

Development of Maize-Bambara Groundnut

Complementary Foods Fortified With Foods Rich in Calcium, Iron, Zinc and Provitamin A

Facu

lty

Agriculture

Dep

artm

ent

Food Science and Technology

Dat

e July, 2007

Sign

atur

e

DEVELOPMENT OF MAIZE-BAMBARA GROUNDNUT COMPLEMENTARY FOODS FORTIFIED WITH FOODS RICH IN CALCIUM, IRON, ZINC AND

PROVITAMIN A

ONYEKWERE, EUCHARIA UKAMAKA PG/MSC/03/34733

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY UNIVERSITY OF NIGERIA,

NSUKKA

JULY, 2007.

TITLE PAGE

DEVELOPMENT OF MAIZE-BAMBARA GROUNDNUT COMPLEMENTARY FOODS FORTIFIED WITH FOODS RICH IN CALCIUM, IRON, ZINC AND

PROVITAMIN A

A THESIS PRESENTED TO THE DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTERS OF SCIENCE IN FOOD SCIENCE AND TECHNOLOGY b

ONYEKWERE, EUCHARIA UKAMAKA PG/MSc/03/34733

JULY, 2007.

APPROVAL PAGE

THIS THESIS HAS BEEN APPROVED FOR THE DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY.

7

PROF. P.O. NGODDY SUPERVISOR

INTERNAL EXAMINER

-r

DR. P.O. UVERE CO-SUPERVISOR

EXTERNAL EXAMINER

DR. (MRS.) N.J. ENWERE 1-1EAD OF DEPARTMENT

ACKNOWLEDGEMENT

I thank Almighty God for His guidance, love and mercy without which I could not

have accomplished this research work. My special thanks go to my project supervisors, Prof.

P.O. Ngoddy and Dr. P.O. Uvere who painstakingly supervised this investigation and read

through the work. I also thank them for their patience and encouragement. I wish to thank

Miss. Ifeoma Mbaeyi, Dr. Mrs. N. J. Enwere and Dr. Mrs. J. Ani whose motherly counsel and

encouragement made this work a reality.

My gratitude also goes to Mr. Chima and Mr. Nwankwo of Food Science anc

Technology Department for their encouragement and technical assistance; my colleagues,

Rose and her family, Ify, Ada, Victor, Okey, Chizoba and Lizzy for their assistance and

encouragement at various stages of this work. My sincere thanks go to my beloved husband, #

Mr. Ejike Kay Onwurafor for his patience, understanding, sacrifice, moral and financial

support throughout the period of the study. To our daughter Bera, I owe immensely for her

patience, endurance and sacrifice.

I am gratefbl to my sisters and their families (Mrs. A. Okeke, Mrs. A.N. Nwajagu,

Mrs. N. C. Obisike, Peace and Gini) for their prayers and encouragement, and especially to

Mrs. H.A. Chinda and her family for their encouragement, financial assistance and prayers.

My special thanks go to my mother, Mrs. C. Onyekwere and my mother in-law, Mrs. R.

Onwurafor who patiently and prayerfully waited for the end of this journey, I am also grateful

to all brethren of the S.U. family and ACM family Oji River for their prayers particularly

Daddy Juli Nwagwu and his family, Stanley Ogbonna and family, D.D, Sis. Chiamaka, Sis.

Bola, Dr (Mrs) Aronu and family, Nancy, Mercy and Tonia, who had actually contributed

immensely to the success of this work. I thank everyone else who helped me in one-way or the

other in seeing that this work was completed.

To God be the glory.

ONYEKWERE E. U

TABLE OF CONTENTS

Title page - Approval page - Certification page Dedication page Acknowledgment Table of contents List of figures - List of tables - Abstract -

INTRODUCTION - - - LITERATURE REVIEW - - - Complementary Foods - - Raw Materials for Complementary Foods

Anti-Nutritional Factors in Plant Foods-

Micro-Nutrients Critical for Infants

Calcium - Iron - - Zinc - - Vitamin A -

Micro-Nutrient Interactions - Nutrient Bioavailability - Food Fortification - - MATERIALS AND METHODS

Materials - - - Methods - - - Processing of Foods Used as Fortificants Production of Complementary Food Blends - Fortification of Complementary Food Blends Chemical analysis of fortified complementary foods -

3.2.4.1 Anti-nutrient factors - - - - - 3.2.4.2 Proximate composition - - - - 3.2.4.3 Micronutrients - - - - -

4. RESULTS AND DISCUSSION - - -

1

ii iii iv v vi viii ix X

1

4.1 Effects of processing on selected micronutrient content of food fortificants.-33

4.2 Production of maize-bambara groundnut complementary food blends by malting and fermentation - - - - - 37

LIST OF FIGURES

Figure 1: Chemical structures of vitamin A - - - - 12

Figure 2: Flow chart for production of fortified complementary foods 26

Figure 3: Average root length and malting loss of bambara

groundnut seeds - - - - - - 3 8

Figure 4: Diastatic activity of bambara groundnut malt - - 40

ABSTRACT

Cattle bone, roselle calyces, red palm oil emulsified with Brachystegia eurycoma or sodium

sesquicarbonate were processed to release calcium, irordzinc and vitamin A respectively. Flours from

malted and wet dehulled bambara groundnut and degermed maize were used to develop

complementary infant formulae in a ratio of 70:30 (wlw, maize:bambara groundnut). Red palm oil

emulsified with Brachystegia eurycoma or sodium sesquicarbonate were incorporated before or after

fermentation in a ratio of 1 :9.6 wlw, (emulsified oi1:composite flour). The processed fortificants were

mixed in a ratio of 1 :1.60:2.25 (wlwlw emulsified red palm oi1:roselle ca1yces:bone meal) and mixed

with the complementary food blends in a ratio of 1 :2 (wlw) prefermentation, post-fermentation or as

dry-mix. The samples were fermented by backslopping and dried at 5 0 ' ~ for 12 hours. The products

were analyzed for their micronutrients (calcium, iron, zinc and vitamin A) and proximate

composition. Nutrend a commercial infant food produced by Nestle (Nig) PLC served as control. The

moisture content of the fortified blends varied from 3.80-5.04%. The fortified complementary food

blends had protein contents of 18.30-18.85%, ash contents of 2.75-3.40%, fat contents of4.00-12.60%

and the carbohydrates of 59.30-60.62%. Fortification of maize-bambara groundnut complementary

foods with red palm oil stabilized with Brachystegia eurycoma and sodium sesquicarbonate gave

vitamin A values of 436.27-868.72 pgRE1100g and 356.78-538.82pgRE/lOOg, respectively. Addition

of the fortificants to the post-fermentation products increased their calcium, iron, zinc and vitamin A

contents significantly compared to the products of pre-fermentation and dry mixing. The calcium,

iron, zinc and vitamin A contents of post-fermentation were 353-569.60 mg/100g, 16.14-

38.16 mg/100g, 13.47-18.79 mg/100g and 592.97-858.37pgRE/lOOg, respectively; the values for pre-

fermentation samples were 326-422.5 mg/100g, 18.67-33.4 mg/100g, 13.20-1 5.28 mg/100 and 485.53-

749.35 pgRE1100g for calcium, iron, zinc and vitamin A, respectively while the dry mix samples had

calcium, iron, zinc and vitamin A contents of 352.06-559.20 mg/100g, 12.61- 21.1 1 mg/100g, 9.47-

17.60 mg/100g and 33.02-843 pgREIlOOg respectively. The calcium, iron and zinc contents of the post

fermentation fortified blends were significantly (p<0.05) higher than that of Nutrend by 13.30-

179.60mg/lOOg, 2.61-28.97mgllOOg and 2.47-1 1.7mg/100g, respectively, but the vitamin A contents

of the fortified blends were lower than that of the Nutrend (1494pgRE1100g).

1. INTRODUCTION

Complementary foods are foods given to infants in addition to breast milk when

breast milk nutrients become inadequate for their energy and growth needs.

Complementary feeding starts from 4-6 months of a child's life, during which infants

should receive complementary foods 2-3 times a day between 6-8 months and 2-4 times

daily between 9-1 1 months (WHO, 2004). Poor complementary feeding is the immediate

direct cause of malnutrition which manifests as protein energy malnutrition (PEM) and

micronutrient deficiencies leading to growth faltering and high rate of infections during

infancy and early childhood (Dewey, 2001). In Nigeria micronutrient deficiency is

reported to be endemic (World Bank, 1996).

Ideal complementary foods should have an easy-to-swallow semi-liquid copsistency

of 1000-3000 cP (Nout and Ngoddy, 1997), rich in micronutrients that are bioavailable

and affordable. Cereal-legume mixes have been used in producing complementary food

(Uvere et al., 2002, Maduko, 2002; Ikujenlola, 2005); but the micronutrient densities fall

below the Recommended Dietary Allowance (RDAs) for infants (Lutter and Dewey,

2003). These deficiencies could be remedied by fortification of the foods (Klemm, 2001;

SCN, 2003). Addition of milk solids to combinations of cereals, legumes and other foods

for infants have been culturally utilized; in some cases fortification with vitamins and

mineral premix have been used, but the high cost of these products make them out of

reach for the average low income earner.

This necessitated the use of local foods rich in micronutrients for fortification of

complementary foods. The decision to fortify maize-bambara groundnut composites was

based on a careful assessment of criteria for food fortification and how this study fits into

the country's larger critical micronutrient (Ca, Fe, Zn and Vitamin A) deficiency

prevention strategy for infants. It is against this background that this study was designed

to produce fortified maize-bambara groundnut complementary foods. In doing this,

specific attention was directed at:

I. Producing low-cost, nutrient-dense complementary foods from maize and

bambara groundnut that meet the macronutrient needs of infants. . . 11. Fortification of the foods with food sources rich in Ca, Fe, Zn and Vitamin A.

iii. Determination of the effect of fortification on the Ca, Fe, Zn and vitamin A

contents of the maize-bambara groundnut complementary food blends.

2. LITEFUTURE REVIEW

2.1 Complementary foods

Complementary foods are transitional foods consumed by infants between the

time when the diet is composed exclusively of mother's milk and when it is mostly made

up of family foods. They are mostly produced from plant foods which include cereals

such as wheat, maize and rice; roots and tubers such as cassava, yam etc; legumes such as

soybeans, cowpeas, bambara groundnuts etc. Cereals can be used individually or in

combination with legumes to produce high-energy protein formulations (FAOIWHO,

1994). Examples of cereal-legume formulations are cowpea and maize, soya-ogi, maize-

bambara groundnut (Akpapunam and Sefa-Dedeh, 1995; Uvere et al., 2002). Certain #

problems are, however, associated with plant based complementary foods.

1. Local complementary foods in developing countries have thick consistencies with

viscosities ranging from 3000-20000cP, which exceed the easy-to-swallow, semi-liquid,

ideal consistency of 1000-3000cP (Nout and Ngoddy, 1997). Their starchy nature makes

them bind so much water thus yielding a bulky gruel with decreased nutrient content. The

bulky nature of the complementary foods cause choking in infants and discourages them

from eating enough (Akpapunam and Sefa-Dedeh, 1995).

2. High cost of complementary foods especially when animal product(s) are included.

This makes industrially processed complementary foods out of reach to low-income

earners and poor families.

3. Cereal-legume combinations contain anti-nutrient factors that interfere with the

utilization of macro- and micronutrients; processing techniques such as fermentation,

sprouting, soaking and heat treatments had been used to solve the problem (Anderson and

Wolf, 1995; Gibson et al., 1998). Even with the use of processing techniques to enhance

nutrient bioavailability, plant-based complementary foods by themselves are insufficient

to meet the infant needs during the period of complementary feeding (Daelmans and

Saadeh, 2003), hence the need for fortification of the product.

2.2 Raw materials for complementary foods

Cereals Cereals are the fruits of cultivated grasses belonging to the monocotyledonous family

Gramineae. They are high in carbohydrates, low in fat and have a fair content of protein.

They supply about 70% or more of energy requirements of the poorer people in the

developing world. The protein content of some cereals varies from 7.9% in rice to 11.6%

in wheat (Table 1).

Table 1: Chemical composition of major cereal grains

Wheat Maize Sorghum Millet Rice Crude fibre (g) 2.0 2.8 2.0 2.3 1 .O Calories (Kcal) 348 Carbohydrates (g) 7 1.0 Proteins (g) 11.6 Fat (g) 2.0 fkh (g) 1.6 Calcium (mg) 30 Iron (mg) 3.5

Riboflavin (mg) 0.10 Thiamine (mg) 0.4 1 Niacin (mg) 5.1 Source: F A 0 (1995).

The minerals in the edible kernels of cereals include potassium, magnesium and

calcium, mainly in the form of phosphates and sulphates. Some of the phosphorus may be

present as phytic acid, which may restrict the availability of calcium in the diet. The

limitation in the use of cereals as food is that the amino acids lysine and sometimes

tryptophan are deficient in their proteins. A common example of cereals is maize.

Maize (Zea mays L) is a staple food for majority of the world people especially in

sub-Saharan Africa and Latin America. Maize production is mostly for human

consumption and feeding of animals. Its domestic food utilization follows two main

paths: One is when the dry maize grains are steeped for two or three days, milled, sieved

and used as porridge and stiff pastes. The alternative use is dry milling of the grains,

which may pass through a pre-dehulling process to produce the dehulled and degermed

maize flour. Yellow maize contains p-carotene (a precursor of vitamin A) and its fatty

acids are made up of 56% linoleic, 30% oleic and 0.7% linolenic acids and are rich in

methionine and cysteine (FAO, 1992).

Legumes

Legumes are the edible seeds of leguminous plants and can be classified into

those relatively low or high in edible lipids; the latter are often described as oil seeds.

They are important and economical sources of dietary protein, which are cheaper than

animal products such as meat, fish, poultry and egg. In developing countries, they serve

as a major source of food protein and are often regarded as "poor man's protein" (Singh,

1991). The crude protein content of most legume seeds (Table 2) varies between 16.0% in

barnbara groundnut to 35.1% in soybeans. Their proteins are limiting in essential sulphur-

containing amino acids (methionine and cysteine) but are rich in lysine and tryptophan

(FAO, 1982a). Therefore, a combination of cereal and legume proteins comes close to

providing ideal dietary proteins for human beings. The oil content of most legumes are

low and range fkom 1 to 6% except soybean and groundnut which have above 18% and

45% respectively.

Table 2: Selected nutrients in legumes

Water Enerev Protein Fat CHO Crude Ash Ca Fe Thiamine

Broad beak 13.8 Cowpeas 11.5 Groundnuts 7.3 Jack beans 11.2 Kidney beans 12.1 Lima beans 10.5 Pigeon peas 11.5 Soybeans 10.2 Chick peas 11.0

Source: F A 0 (1982a)

All legume grains contain substantial amounts of minerals and vitamins with cowpea,

(Vigna unguiculata), soybean (Glycine max L) and bambara groundnut (Voandzeia

subterranean Thouars) being good sources of calcium and iron (Elegbede, 1998).

Bambara groundnut is consumed in Nigeria especially in Enugu state and the Northern

states where they also serve as an important article of trade. A compositional study of the

beans showed that they contain crude protein, crude fat, ash and total carbohydrate of 16-

21%, 4.5-6.5%, 2.9% and 50-60%, respectively (Enwere and Hung, 1996); their calcium

and iron contents are higher than those of meat, fish or egg (Elegbede, 1998).

2.3 Anti-nutritional factors in plant foods

A major factor limiting the wider use of many tropical plants as food is the

occurrence in them of a diverse range of natural compounds capable of precipitating

deleterious effects in -man and animals. They include: protease inhibitors, phytate,

tannins, oxalates, flatulence factors and dietary fibre. These interfere with digestive

processes and reduce food intake andlor efficient utilization of the nutrients. The levels of

deleterious substances in tropical legumes vary with the species of plant, cultivars and

post harvest treatments such as drying, soaking, autoclaving and malting of the seed

(Osagie, 1998).

Protease inhibitors: Protease inhibitors are found in all legumes in varying degrees and

inhibit the functions of proteolytic enzymes especially trypsin and chymotrypsin in man,

hence reduction in protein digestibility and nutritive value (Osho, 1989). Cooking, hot

water soaking and other forms of heat treatment inactivate trypsin inhibitor activity.

Phytates: Phytic acid, a hexaphosphate derivative of inositol is an important storage form

of phosphorus in plants. It forms complexes with multivalent, mineral elements such as

calcium, iron, zinc etc and hinder their absorption and bioavailability (Anderson and

Wolf, 1995). This can result in mineral deficiency in populations that depend on whole 8 .

grain and legume-based products as staple food (Harland and Moris, 1995). Malting of

grain cereals and legumes result in the reduction of phytate contents (Nzefibe and

Onyeniran, 200 1).

Tannins: Tannins are polyphenolic substances responsible for the colour of the seed coat

of certain cereals and legumes especially in the testa of pigmented legumes. White beans

have very low tannins while darker coloured beans (red or black seeded) have more

tannin content (Beebe et al., 2000). It forms complexes with protein, carbohydrates and

iron. Tannin-protein complexes are responsible for growth depression, low protein

digestibility and decreased amino acid availability while its complexes with iron hinder

iron absorption and precipitate iron in food preparations (Beebe el al., 2000). Soaking,

malting, and fermentation result in its reduction (Obizoba and Egbuna, 1992).

Oxalates: Oxalic acid is a dibasic acid that is widely distributed among vegetables and

legumes (Quinteros el al., 2003). Oxalates are mostly present in plants as salts of calcium,

potassium, sodium, magnesium and iron. They form non-absorbable insoluble complexes

with calcium, iron and magnesium and cause their deficiency in animals. Its

high consumption in food can lead to calcium deficiency (hypocalcaemia). Soaking in

water and changing the soaking water and fermentation reduce levels of oxalates

(Aworth, 1993; Quinteros et al., 2003).

Flatulence factors: Flatulence factors are oligosaccharides (raffinose, stachyose and

verbascose) that are not digested but fermented in the gut with the production of gas

(hydrogen, methane or carbon dioxide). Their presence in the diet can lead to diarrhoea,

nausea and cramp (Nout, 1990). Proper heat treatment, fermentation, germination and

dehulling are used to eliminate flatulence factors.

Reducing anti-nutrient factors

Antinutritional factors are reduced in foods by processing using methods such as

soakinglsteeping, malting, fermentation and heat treatment.

soakinglsteeping: It involves allowing the seeds to imbibe sufficient water from plain

water or mineral solution over a period of time. The water acts as solvent, reactant and a

medium of transport for nutrients during germination. Tough seed coat legumes (e.g

Voandzeia subtevranea) take 4-12 hours to absorb water enough to aid wet-dehulling

while lighter seed coats (e.g Vigna unguiculata) take about 1-2 minute(s) and above to

imbibe water (Enwere, 1998). Soaking contributes to the significant reduction in the

levels of toxins and antinutritional factors such as phytates, oligosaccharides, tannins, etc

(Kadam and Salunkhe, 1985).

MaltinglGermination: Germination is a natural process in which dormant bu)t viable

seeds are induced to grow into seedlings with the production of hydrolytic enzymes such

as amylases needed for mobilization of endosperm matter in the grains. Proteins are

broken down to peptides and amino acids by protease activity; carbohydrates to simpler

sugars by amylase, phytic acid to inositol and phosphoric acid and tannin-protein

complexes are broken down during germination of seeds (Nout and Ngoddy, 1997).

Germination reduces anti-nutritional factors such as oligosaccharides, lectins, tannins and

phytate (Nzelibe and Onyeniran, 2001).

Fermentation: Fermentation is a process of anaerobic or partially anaerobic oxidation of

carbohydrates especially sugars by the action of microorganisms andlor endogenous or

exogenous enzymes to produce desirable biochemical changes. Complex substances are

hydrolytically broken down into simpler absorbable forms by enzymes such as amylases,

proteases and lipases. The pH is mostly reduced from 6.0 to 3.5; titratable acidity

increases due to the action of lactic acid bacteria and this precludes proliferation of

contaminating acid tolerant species of bacteria and fungi thus imparting robust stability,

safety to the product and pre-empts disease infections such as diarrhoea, salmonellosis,

and high spoilage velocity of porridge in unsanitary environments (Nout and Ngoddy,

1997). Other advantages of fermentation include enhancement of nutritional value,

texture, shelf-life, tastes of food products and micronutrient availability because of

significant reduction in phytates, tannins by as much as 90%. Reported advantages of

fermentation include increase in iron availability during lactic acid fermentation of white

sorghum, the level of which doubled when dehulled sorghum flour was fermented and a

six fold increase occurred when a combination of germination and fermentation were

employed (Obizoba and Atti, 1991). Calcium availability increased due to fermentation

through hydrolysis of phytate and oxalate during daddawa production from locust bean

seeds (Aworth, 1993).

Heat treatment: This eliminates pathogens (WHO, 1998) when heated above ~o'c, denatures and inactivates protease inhibitors; reduces levels of oxalate significantly and

could therefore favour the availability of divalent cat-ions from legumes (Quinteros et al.,

2003). It improves shelf-life, organoleptic attributes and nutritional characteristics of

foods.

2.4 Micro-nutrients critical for infants

Vitamins and minerals cannot be synthesized in sufficient quantity by the human

body and must be provided by the diet. They are necessary for the regulatory syrtems in

the body, for efficient energy metabolism and for such functions as cognition and

immunity. The following key nutrients are critical for infants: calcium, iron, zinc and

vitamin A (Brown, 199 1).

2.4.1 Calcium

Calcium is a divalent cation with a radius of 0.95~'. It is the major cation of bone

mineral and more than 99% of the calcium in the body is used as a structural component

of bone and teeth. Its rate of deposition in the skeleton is highest in the newborn infant,

decreasing to a very low level by the time people stop growing.

The amount of calcium absorbed in the body depends on the habitual calcium

intake. Calcium must be in a soluble form (ionized ca2+), at least in the upper small

intestine or bound to a soluble organic molecule before it can cross the wall of the

intestine. When calcium intake is adequate, differences in bioavailability as in increased

solubilization play no or only minor role in the amount of calcium that is absorbed or

deposited in the skeleton. Good solubility in water is, therefore, an advantage but is not

absolutely necessary. Calcium absorption within the upper small intestine depends on the

vitamin D hormone, calcitriol (1, 25 (OH)z D3). The most striking effect of calcitriol is its

control of the expression of the gene encoding calcium binding protein (CaBP), causing

the synthesis of the protein thereby regulating the migration of calcium across intestinal

cells (Gueguen et al., 2000). Lactose in high doses increases the passive absorption of

calcium in the absence of vitamin D and, consequently, decreases intestinal calcium

binding protein (CaBP) concentration and active transport of calcium

Humans absorb about 25% of the calcium in the food eaten; however, when the

body needs extra calcium such as during infancy and pregnancy, absorption might reach

as high as 60%. Other factors that enhance the absorption of calcium include: parathyroid

hormone, dietary glucose, lactose and normal intestinal mobility (flow). Phytate found in

the bran of most cereals and seeds and oxalate can form insoluble complexes with

calcium thereby reducing its absorbability. About 99% of the body calcium is stored in

the skeleton depending on its needs. The physiology related to growth, pregnancy and

lactation are main factors that affect the efficiency of calcium storage in the bone. Excess

absorbed calcium that cannot be stored in the bone is excreted in urine, faeces and sweat.

The calcium balance in adult human is zero; all absorbed calcium is excreted by these

routes after being released from bone (Gueguen et al., 2000). b

The major roles of calcium in the body include:

1. Forming and maintaining bones: Bone is very active metabolically; when a diet is

deficient in calcium, calcium is released fiom the bone so it can enter the blood.

2. Transmission of nerve impulses to target cells such as muscle, other nerve cells or a

gland are performed across the junction between the nerve and its target cells. The arrival

of the impulse of the target site stimulates an influx of calcium ions into the nerves from

the extracellular medium. The rise in intracellular calcium ion then triggers the release of

neurotransmitters from the synaptic vesicles (Kenney, 1989).

3. Calcium ions help regulate metabolism in the cell by participating in the calmodulin

system. Calcium that enters a cell, binds to the protein calmodulin to form protein-

calcium complex, which regulates the activities of various enzymes such as Ca-ATPase.

Calcium deficiency causes rickets in children characterized by knock-knees and

bowlegs; tetany (low levels of free ionized calcium in the blood) characterized by poor or

stunted growth. Osteoporosis (the failure to maintain adequate bone mass in the body),

arterial hypertension and colon cancer are also associated with low levels of calcium

intake (Gueguen et al., 2000). Calcium toxicity occurs normally with individuals using

excessive amounts of supplemental calcium (Sandstead, 1995).

Good food sources of calcium are shown in Table 3.

Table 3: Calcium content of selected foods

Food item Quantity Calcium (mg/lOOg) Parmesian cheese 2og 6 70 Roman cheese 2og 600 Low-fat yogurt, 240ml 300 Milk 240ml 280 Butter milk 240ml 280 Cooking spinach 170g 220 Cooked burn up greens 170g 200 Canned salmon (with bone) 3012g 180

Source:. :. -, Food and Drug Administration (1994),

2.4.2 Iron

Iron is an important trace mineral found in every cell

combined with protein. It has unfilled "d" orbitals. Its oxidation

of the body usually

state in most natural

forms is from +2 to +6. In biological systems, iron exists primarily as the ferrous (Fe2+)

and ferric (Fe3+) forms. Ferrous iron has six 'd' electrons while ferric iron has five. In

aqueous solution under reducing (low pH) conditions, the ferrous form predominates.

Ferrous iron is quite soluble in water at physiological pH levels. In the presence of

molecular oxygen, however, aqueous Fe2+ may be oxidized to ~ e ~ +

2 FC:~, + 0, -+ 2 FC& + 0; The hydrated Fe3+ will then undergo progressive hydrolysis, to yield increasingly

insoluble ferric hydroxide species.

F ~ ( H , 0): + H ,O + Fe(H,O), ( 0 ~ 3 ~ ' + H30' +-+ -+ Fe(OH),

Because this hydrolysis reaction occurs readily except at very low pH, the concentration

of free ferric ion in aqueous systems is vanishingly small. The predominance of low

solubility forms of iron [Fe(OH)3] explains why it is so poorly available (FAO, 1997).

The primary form of iron used in supplementation programmes is the ferrous

sulphate tablet, due to its low cost and high iron bioavailability. However, the colour of

the tablet may influence perception and acceptability in certain cultures. Other

bioavailable forms of iron such as liquid iron preparation, parenteral iron preparations,

which are less absorbed but costlier than ferrous sulphate also exist.

Absorption: Iron enters the body as heme and non-heme iron. Heme iron is derived from

haemoglobin and myoglobin in meat and is transferred to intestinal cells as the intact

porphyrin complex. The iron released by heme oxygenase mixes with other iron taken up ',

by the cell before regulated transfer to the bloodstream occurs. Heme iron is virtually well

absorbed (20-25%). The rest of the dietary iron is in inorganic form. Non-

heme iron is derived from vegetable foods, inorganic contaminant iron and inorganic

fortificants added to the diet. Solubilization of iron is the key factor that influences its

absorption irrespective of the source. Non-heme iron pool containing soluble iron is

formed in the lumen of the gastro-intestinal tract and iron is extracted from this pool via

absorptive mechanisms. The amount of iron absorbed depends also on mucosal behaviour

in the intestinal wall, the presence of ligands such as chelating agents (citric acid, ascorbic

acid) in the meal, which either promotes or depresses iron absorption from the pool.

Factors that affect absorption of iron are shown in Table 4. #

Table 4: Factors that influence iron absorption

a. Physical state (bioavailability) Heme > ~ e ' + > Fe3+ b. Inhibitors Phytates, tannin, antacid, starch etc c. Facilitators Ascorbic acid, amino acids, iron deficiency,

citric acid d. Competitors Lead, cobalt, manganese zinc

Iron enters the stomach from the oesophagus and is oxidized to the Fe3+ state when taken

orally. Mucins bind iron in the acid environment of the stomach, thereby maintaining it in

solution for later uptake in the alkaline duodenum. Mucin-iron complexes subsequently

cross the mucosal cell membrane in association with integrins. A cytoplasmic iron-

binding protein, dubbed "mobilferrin", accepts the elements once inside the cell and

shuttles it to the basolateral surface of the cell where it is delivered to plasma. The

absorption of iron occurs mainly in the duodenum by an active yet unexplained process,

which transports iron from the gut lumen into the mucosal cells. Iron passes directly

through the mucosal cell into the blood stream where it is transported by transferrin

together with the iron released from old blood cells when the body requires iron. Iron is

stored in the mucosal cell as ferritin and excreted in faeces when mucosal cells are

exfoliated if not required by the body. Absorbed iron in excess of the need is stored as

ferritin in the liver, spleen or bone marrow.

Iron is an intrinsic part of haemoglobin and is required for the transport of oxygen

which is critical for cell respiration and storage in the muscle. Iron is also a component of

tissue enzymes and enzymes necessary for immune system hnctioning.

Iron deficiency causes anaemia, which may be due to severe loss of blood,

malnutrition, infections and excessive drugs and chemicals. Deficiency of iron in the diet

may cause nutritional anaemia, lowered resistance to disease, pale complexion, preterm

delivery, production of abnormal developmental performance and poor growth (Booth

and Auckett, 1997).

Food sources of iron include clams, oysters, liver, poultry, fish and eggs. The non-

heme iron is found in cereals, legumes/pulses, dark green leafy vegetables, fruits and

roselle calyces, etc (Langenhoven et al., 2001). Iron supplementation in excess may cause

gastrointestinal side effects such as nausea, vomiting, constipation, diarrhoda, dark

coloured stools, and stomach distress. Iron fortification lacks the side effects that have

often affected iron supplementation (Darnton-Hill, 2005).

2.4.3 Zinc

Zinc occurs in nature as ZnS, ZnSi04 and ZnO. It is the most abundant trace metal

inside most cells and an essential trace element in human nutrition; its deficiency is a

world nutrition problem. Zinc absorption at an intestinal level takes places by active

transport and passive non-saturable diffusion. The first pathway of absorption is saturable

to high zinc concentrations in the lumen while the second pathway depends on the ,

concentration gradient of the cation and is a non-saturable process. Protein is considered

one of the main sources of dietary zinc and could also improve zinc absorption

(Lonnerdal, 2000). The zinc content of foods is low. Food sources of zinc are red meat,

poultry, beans, nuts, green vegetables, cereals and legumes (Institute of Medicine, 2001).

Zinc plays an essential role in (1) gene expression (2) nucleic acid metabolism

(3) normal growth and development during childhood (4) regulation of cellular growth.

Zinc has the greatest effect on growth in children and is one of the key nutrients proven to

reduce mortality from diarrhoea and measles by 30-50% for children under five years.

Zinc deficiency result to growth retardation, mental retardation, poor appetite, increase in

infectious disease and morbidity in children, which may be closely related to incidence of

diarrhoea (Blum, 1997).

2.4.4 Vitamin A

Vitamin A refers to a group of nutritionally active unsaturated hydrocarbons

including retinol and related compounds and certain carotenoids. Its activity in animal

tissues is predominantly in the form of retinol or its esters, retinal and to a lesser extent

retinoic acid (Figure 1). Beta-carotene is a precursor of retinol and when foods

containing it are consumed, it is converted into retinol in the body and serves the same

functions as vitamin A from animal sources.

Retinoid is a ring structure with a fatty acid tail and four isoprenoid units in

which the tail terminates in one of the groups (retinol, an alcohol); (retinal, an aldehyde)

and (retinoic acid) and vary from cis to trans configurations.

a. retinol

b. retinal Re

CHOOH

c. retinoic acid

Figure 1: Chemical structures of vitamin A; Source: FA0 (1997)

The release of vitamin A from foods requires bile, digestive enzymes from the

pancreas and the intestinal tract, and integration into micelles. Vitamin A as retinyl ester

is absorbed into the lymph via chylomicrons (Wardlaw and Kessel, 2002). Absorption of

provitamin A (carotenoids) occurs via the lymph and depends on bile salts and also fat

content of the diet. Some carotenoids can be converted to retinoid forms within the

intestinal wall and both forms of vitamin A are transported via chylomicrons and released

to the liver. Carotenoids are stored in both liver and adipose tissue and are released from

the adipose tissue when needed back to the liver where they are converted to retinoid.

Vitamin A is not normally excreted by the body (Wardlaw and Kessel, 2002). Many

factors that influence the absorption and utilization of provitamin A are amount, type and

physical form of caroteniods in the diet, intake-fat, vitamin E, fibre, protein and zinc

status (Rodriguez-Amaya, 1997). The use of oil in preparation of food is important for

carotene absorption.

Vitamin A is a critical micronutrient essential for night vision and for the

maintenance of skin and mucosal integrity. Vitamin A deficiency in children causes

retarded growth and xerophthalmia. Severe deficiency of vitamin A predisposes children

to anaemia and increases the risk of fatality, decreased appetite and poor growth in

children (SCN, 2003). These disorders are worse, during the weaning period because

most traditional weaning foods are low in fats and vitamin especially vitamin A. Selected b good food sources of vitamin A are shown in Table 5.

Table 5: Good food sources of vitamin A

Food Vitamin A (pgRE/ 1 00g Tomatoes Carrots raw 430 Fresh red palm oil 5500 Sweet potato (orange) 422

Apricots, dried 362 Papaya 6 8 Cooked egg 21 1

Liver (beef) 20357 Egg yolk 709 Source: Erhardt (2004).

Red palm oil is one of the richest natural plant sources of carotenoids and has more than

15 times retinol equivalent than carrot and 300 times more than tomatoes (Erhardt, 2004).

2.5 Micro-nutrient Interactions

Calcium can interact with other nutrients such as iron and zinc, but evidence to

date fails to demonstrate that excess intake of calcium contributes to deficiencies of these

minerals in human when their intake is adequate. Calcium also inhibits the absorption of

iron in a dose-dependent and dose-saturable fashion. However, available human data fail

to show cases of iron deficiency or even decreased iron stores as a result of high calcium

intake. The inhibitory effect of calcium on iron absorption is independent of the amount

of phytate in a meal and is not pronounced when these nutrients are consumed together in

the absence of food (Cook et al, 1991 b). However, when calcium was given as citrate and

phosphate in the absence of foods, iron absorption was reduced by 49% and 62%

respectively.

Calcium excess, ie. (> Ilg) in diet could impair zinc availability and reciprocally,

zinc supplementation reduces calcium absorption in ZdCa relationship higher than 0.7.

However the degree of calcium-zinc interactions seems to be conditioned by the presence

of phytic acid in the diet. Excessive calcium in the diet could imply lower solubility for

Ca-phytate-Zn complexes and subsequently reduces zinc availability (Carnara and

Amaro, 2003).

An association exists between vitamin A, iron and zinc metabolism. Addition of

vitamin A to iron supplements influences iron metabolism positively and reciprocally,

iron affects vitamin A metabolism. Iron supplementation alone or with zinc i'ncludes a

redistribution of vitamin A resulting in low plasma retinol concentrations, but high

vitamin A liver stores (Wieringa, 2003). The reason for the fall in plasma retinol may be

due to "toxic" effects of iron. Hence, the addition of vitamin A to iron supplements is not

only needed because the two nutrients are more effective in reducing the prevalence of

anaemia than iron alone, but vitamin A is also needed to counteract possible negative

effects of the supplemented iron on vitamin A status.

Zinc improves the utilization of p-carotene and its conversion to retinol and

possibly in the metabolism of p-carotene from tissue deposits (Dijkhuizen, 2003). Zinc-

iron interactions can affect availability of both elements. The metals (ca2+, Fe2+) are

capable of influencing zinc absorption at various concentrations. Nevertheless, this

antagonistic effect depends on the cation concentration and it is coincident with the

affinity of these cations by this metallic channel of transport (zn2+>> Fe2+>>ca2>.

Fortification of food with iron does not significantly affect zinc absorption except when

the iron and zinc molar ratio is 25:1, a ratio that is highly unlikely to occur in fortified

foods (Whittakar, 1998).

2.6 Nutrient Bioavailability

Bioavailability may be defined as the proportion of a nutrient in ingested food that

is available for utilization in metabolic processes. The bioavailability of mineral nutrient

is determined primarily by the efficiency of absorption from the intestinal lumen into the

blood. Virtually all iron in plant foods and approximately 40 - 60% in animal tissue is

non-heme iron and its bioavailability varies depending on composition of the diet. The

bioavailability of heme iron is relatively unaffected by composition of the diet and is

greater than that of non-heme iron.

The absorption of calcium fkom foods is determined by the concentration of

calcium in the food, the presence of inhibitors or enhancers of calcium absorption. The

main dietary inhibitors of calcium absorption are oxalate, tannins and phytate, which can

form insoluble complexes with calcium, thereby reducing its absorbability (Gueguen et

al., 2000). Total iron intake, composition of the diet, and iron status of the individual

consuming the diet play a role in determining the amount of iron absorbed. Non-heme

iron from all sources of meal enters a common pool during digestion and absorption from

this pool is determined by the totality of ligands present in the small intestine at the site of

absorption. #

Vitamin A absorption and utilization are impaired by the amount, type and

physical form of carotenoids in the diet, intake fat, vitamin E, protein and zinc content

(Rodriguez-Amaya, 1997). Some factors that may influence mineral bioavailability from

foods are summarized as follows:

1. Chemical form of the mineral in food:

Highly insoluble forms are poorly absorbed.

Soluble chelated forms may be poorly absorbed if chelate has high stability.

Heme iron is absorbed more efficiently than none heme iron.

2. Food Ligands:

Ligands that form soluble chelates with metals may enhance absorption from

some foods (e.g. EDTA enhances iron absorption).

High molecular weight ligands that are poorly digestible may reduce

absorption (eg. dietary fibre, proteins)

Ligands that form insoluble chelates with minerals may reduce absorption

(e.g. oxalate inhibits calcium absorption, phytic acid inhibits zinc, iron and

calcium absorption).

3. Redox activities of food components:

Reductants (eg. ascorbic acid) enhance absorption of iron but have little effect

on other minerals.

Oxidants inhibit the absorption of iron.

4. Mineral-mineral interactions

High concentrations of one mineral in the diet may inhibit the absorption

of another, e.g. calcium inhibits iron absorption, and iron inhibits zinc

absorption.

5. Physiological state of consumer

1. Homeostatic regulation of minerals in the body may operate at the site of

absorption resulting in enhanced absorption, in deficiency and reduced

absorption in adequacy. This is the case with iron, calcium and zinc. . . 11. Malabsorption disorders may reduce absorption of minerals.

iii. Iron and calcium absorption are reduced in achlorhydria (reduced gastric acid

secretion).

iv. Age: absorption efficiencies may decline in the elderly. #

2.7 Food Fortification

Nutrients are inherent in foods. However, during processing, some of these may

be lost depending on the severity of treatment. As a result nutrients, minerals and

vitamins are usually added to maintain and improve the nutritional quality of food

supplies and correct or prevent nutritional problems in populations or specific population

groups (Caballero, 2003). Food fortification, enrichment and restoration of nutrients are

means of delivering sufficient amounts of these nutrients to consumers to prevent or avert

nutritional diseases or raise the level of the nutrients to that found in the natural

product(s) as consumed (FAOIWHO, 1994). It is the most accessible intervention method

to increase nutrient intake of the population without changing dietary and cultural habits

provided that appropriate food vehicles, particularly food staples are identified

(Klemm, 2001). Food fortification is one of the methods of combating malnutrition across

the varying socio cultural groups (World Bank, 1994).

The most outstanding advantages of food fortification include.

(1) Affordability: The additional cost of fortifying a food product is commonly less

than 2% of the retail price of the unfortified product.

(2) Effectiveness: The effect is immediate. The ultimate impact of fortification on

the micronutrient status of the population can be detected as early as in 3 to 6

months.

(3) The benefits per unit of investment is greater than with many nutrition

interventions.

Behavioural changes are not required: This means that there will be no

changes in feeding patterns.

Fortification is socially acceptable and politically attractive

High population coverage can be achieved especially when staple foods consumed

regularly by the majority of the population are used as a fortification vehicle.

The risk of toxicity is negligible when compared with the risks associated with

supplementation. This is an important feature of food-based interventions,

including dietary diversification.

Food fortification is sustainable: Sustainability of fortification is basically

contingent upon political commitment, legislation, enforcement and monitoring. It

is enhanced when fortification becomes a good quality criterion (Mora, 1995).

Methods of food fortification: Various methods of food fortification include food-to-food

fortification, single nutrient to food fortification and double/multiple nutrient-to-food

fortification. Food-to-food fortification is used when a food substance is added to another

food as a source of nutrients. The food substance is known as the fortificant while the

food to which it is added is known as the vehicle. Food-to-food fortification is a food-

based strategy and has been a very appropriate tool for solving malnutrition problems.

Single nutrient to food fortification involves the addition of a single micronutrient such as

a vitamin or mineral to food or a food mixture. Iron, calcium and vitamin A are added as

micronutrients in complementary foods for infants to overcome the associated nutrient

deficiencies. This had been done using pure chemicals, which may have increased the

cost of the formulation and might not be good for infants (Brown, 1991). Double/multiple

nutrients fortification, are used when two or more macro/micronutrients are added to a

food mixture and these also involved the use of pure chemicals. Nutrient-to-food

fortification as seen with vitamins and minerals are usually applied in industrially

produced complementary foods.

The selection of an appropriate vehicle is a critical step in successful fortification

(FAO, 1995). Important criteria for vehicle selection are that the population at risk for the

deficiency regularly consumes food carriers and that the amount consumed does not vary

widely in the larger population (Klernrn, 2001). Selecting an appropriate fortificant

involves identification of one that does not appreciably alter the dietary carrier's

appearance, color, texture or organoleptic properties and one whose potency remains high

under usual conditions of processing, transport and storage (Bauernfeind, 1980).

Fortified foods are expected to meet certain optimal daily nutrient requirements

for each micronutrient fortified for and for a particular age group. The nutrient

requirements for infants aged 6-12 months are based on Adequate Intake (AI) except for

iron and zinc, which are based on Recommended Dietary Allowance (RDAs) (Lutter and

Dewey 2003). Table 5 is the summary of the recommended nutrient composition of

selected nutrients for fortified complementary foods.

Table 6: Proposed Recommended Nutrient Composition of Selected Nutrients for Fortified Complementary foods

Nutrients Per daily ration Per 1 OOg 6-1 1mo 12-23m0 6-23mo 6-1 1mo 12- 23mo 6-23m0 (40d (60g) (50g)

Vitamin A (%RE) 200 300 250 500 500 500t Calcium ( W ) 100-200 100-200 100-200 250-500 170-330 200-400 Zinc (mg) 4-5 4-5 4-5 10-12.5 6-7 8.3 Iron (mg) 11 7 7-1 1 27.5 11.7 14

Source: Lutter and Dewey (2003).

The RDA is the average daily dietary intake level that is sufficient to meet the nutrient

requirements of nearly all (97-98%) healthy individuals in a particular life stage. Given

the advantages of food-to-food fortification, the following foods have been found to be

rich in calcium, iron, zinc and vitamin A known to be important for linear and weight

growth in infants (Brown, 1991; Daelmans and Saadeh, 2003):

(a) Cattle Bone: Bone is a special form of connective tissue with a collagen framework

impregnated with ca2+ and PO:- salts, particularly hydroxyapatite [C~IO(PO&(OH)~]. The

defatted and dried bones are composed of organic and inorganic salts, in a ratio of 1:2, i.e.

the inorganic is about 65 to 70% and almost all of which is a compound called

hydroxyapatite, which has 10 calcium, 6 phosphorus, 26 oxygen, and 2 hydrogen atoms. It

is found in the matrix of bone and teeth and confers rigidity to these structures. Finely

ground bone is a good source of calcium and phosphorus. Bone meal, which is composed

of finely crushed and processed bone, usually from cattle are used as supplement for

calcium and phosphorus in pediatric foods.

(b) Hibiscus sabdariffa (roselle calyces): Roselle calyces (Hibiscus sabdarga) are of the

Family Malvaceae. Of the 300 species of Hibiscus found around the world, only Hibiscus

sabdarifa provides the swollen red calyces known as roselle, which is attracting the

attention of food and beverage manufacturers and pharmaceutical concerns because of its

high nutrient content especially that of iron and zinc. The dried calyces are brewed into

teas, used in processing of juices, jellies, jams, ice cream, flavours and fiequently cooked

and eaten with pulverized peanut. Dried red coloured Roselle calyces have iron content of

37.8mgl100g (Babalola et al. 2001) and zinc content of 3 1.86mgl100g (Ojokoh et al.,

2000). The chemical constituents of fiesh roselle calyces are shown on Table 7.

Table 7: Analysis of fresh calyces made in Guatemala

Nutrient Valueh 00g (wet weight basis)

Moisture Protein Fat Fiber Ash Calcium Phosphorous Iron Carotene Thiamine Riboflavin Niacin - Ascorbic acid 6.7mg

Source: Morton (1987).

(c) Red Palm Oil: Palm oil is reddish-orange oil extracted fiom the pulp of the fruit of

the Afiican palm. It is extensively cultivated in tropical countries. The seeds of the fruit

and the seed kernels also contain oil. The dominant cooking (red palm) oil is considered

the world's richest plant source of provitamin A (Choo, 1994), and contains about

5SOOpgRE/100g of vitamin A mainly p-carotene (Erhardt, 2004), is rich in the antioxidant

tocopherols and tocotrienols (Ong and Goh, 2002). The distinctive colour of the oil is due

to fat-soluble carotenoids, which are responsible for the high vitamin A content that

promotes good night vision, health of mucous membranes and skin, and the growth of

bone. Palm oil is added to babies' porridge to improve their energy density, palatability

and increase the vitamin A content (Home Garden, 2003).

3. MATERIALS AND METHODS

3.1 Materials

Healthy, mature seeds of bambara groundnut (Voandzeia subterranea Thouars),

yellow maize (Zea mays L), cattle bones, roselle calyces (Hibiscus sabdariffa), red palm

oil, and Nutrend (a commercially available cereal-legume mix produced by Nestle

Nigeria, PLC) were purchased fiom Nsukka market, Nigeria.

3.2 Methods

3.2.1 Processing of foods used as fortificants

Determination of optimum ashing temperature of cattle bone

Cattle bones used as source of calcium were cracked open using Bench vise Ma

(Model HI- Duty Vice, Paramo, England), washed with hot water at 90°C to remove the

marrow and oil. It was sun-dried (31*0.31°C) and further dried in a convection

Gallenkamp oven (Model IH-150) at 50°C for 12 hours. Cattle bone (lg) each was ashed

at 500°C, 600°C and 700°C in a furnace where the optimum ashing temperature was

established and subsequently used in the study.

Processing of cattle bones into meal

The dried cattle bones used as source of calcium were transformed into bone meal as

follows:

(9

( ii)

(iii)

(iv)

A one hundred gram (1 00g) portion was autoclaved at 12 1 OC for 2 hours, dried

at 50°C in a convection Gallenkamp oven (Model 1 H-150) and milled into

powder and designated as sample (a).

A one hundred gram (1 00g) portion of cattle bone was milled and fermented

by backslopping (Nout et al., 1989) for 72 hours in which the ratio of flour to

water was 40: 60. During the fermentation, the products fiom the previous day

were recycled as 10% starter culture for the next 24hrs fermentation.

Recycling was done two more times and dried in a convection Gallenkamp

oven (Model IH- 150) at 50°C (F).

One gram (lg) was ashed at 6OO0C and milled (A)

A one hundred gram (1 00g) portion was ashed at 600°C (Abolude and

Abdullahi, 2005), fermented by backslopping (Nout et al., 1989) for 3days,

dried at 50°C in a convection Gallenkamp oven (Model IH-150) and milled

( A n

A one hundred gram (100g) portion was fermented by backslopping (Nout et

al., 1989) for 72 hours, dried at 50°C in a convection oven, ashed at 600°C

before milling (FA).

A one hundred gram (100g) portion was autoclaved at 121°C for 2 hours and

lg ashed at 600°C (A).

A one hundred gram (1 00g) portion was autoclaved at 12 1°C for 2 hours and

fermented by backslopping for 72 hours, dried at 50°C and lg ashed at 600°C

(aFA)

A one hundred gram (100g) portion was fermented by backslopping for 72

hours, autoclaved at 121°C for 2 hours, dried in a convection Gallenkamp

oven (Model IH-150) at 50°C and 1 g ashed at 600°C (FA). #

A one hundred gram (100g) portion was autoclaved for 2 hours, ashed at

600°C and fermented by backslopping (Nout et al., 1989) for 72 hours, (aAF).

Raw bone (1 g) was milled and used as control (C).

Processing of Roselle calyces into $our

Roselle calyces (Hibiscus sabdarrpa) used as source of iron and zinc were

hand -sorted to remove dirt and extraneous materials:

(a) Four hundred grams (400g) was dried to a moisture content of 10% and

weight was 200g at 5 0 ' ~ in a convection Gallenkamp oven (Model IH- 150);

(i) A portion (100g) of the dried calyces was ashed at 450°C (RCd).

(ii) A one hundred gram (100g) portion was fermented by backslopping

(Nout et al., 1989) for 72 hours, dried at 50°C and ashed at 450°C

Wdb)

(b) A one hundred gram (100g) portion of flesh calyx was milled in a Bentall

attrition mill (Model 200L090) (C).

(c) The fiesh calyx (100g) was fermented by backslopping (Nout et al., 1989) for

72 hours, dried at 50°C and the cake milled using a laboratory mortar (RCb).

Processing of redpalm oil used as source of vitamin A

1. Using Brachystegia eurycoma as emulsifier

Brachystegia eurycoma ('achi') seeds were roasted in a w ing pan over a kerosene

stove 14S°C using a thermometer for 30 minutes, soaked in excess water for 3 hours,

dehulled by abrasion and milled into powder. The powder was used in emulsification of

red palm oil.

A 24-hour stable emulsion of red palm oil, water and Brachystegia eurycoma

(1 : 1 : 2 V/V/W) was formed, dried in a convection Gallenkamp oven (Model

IH-150) at 50°C and the cake milled into flour.

A 24-hour stable emulsion of red palm oil, water and Brachystegia eurycoma

(1 : 1 :2 V/V/W) was formed and fermented by backslopping (Nout et al., 1989)

for 72 hours.

A portion of the emulsified fermented sample (b above) was dried in a

convection Gallenkamp oven (Model IH-150) at 50°C and the cake milled in a

laboratory mortar.

2. Using sodium sesquicarbonate as emulsifier/stabilizer

Sodium sesquicarbonate (trona) was ground into powder and sieved through a

traditional nylon cloth. #

A 24-hour stable emulsion of red palm oil, water and trona (1:1:2 v/v/w) was

formed by mixing, dried in a convection Gallenkamp oven (Model IH-150) at

50°C and the cake milled into flour in a laboratory mortar

A 24-hour stable emulsion of red palm oil, water and trona (1: 1:2 v/v/w) was

formed and fermented by backslopping (Nout et al., 1989) for 72 hours.

A portion of the fermented sample (b above) was dried in a convection

Gallenkamp oven (Model IH-150) at 50°C and the cake milled in a laboratory

mortar.

3.2.2 Production of complementary food blends

Processing of raw materials:

The seeds of bambara groundnuts and maize were cleaned by winnowing and

hand sorting to remove non-viable (broken, insect infested) seeds, stones, empty pods and

other foreign materials.

Maize grains:

Seven kilograms (7kg) of cleaned maize grains were tempered in 15 litres of water

for 15 minutes, drained, dehulled and degermed using a Bentall attrition mill (Model 200

L090). It was dried at 50°C to a moisture content of 10.5%, in a convection Gallenkamp

oven (Model 1H-150), winnowed, the grits milled into flour and sieved using traditional

nylon cloth to get degermed maize flour (M).

Bambara groundnut seeds

Barnbara groundnut seeds (5.3kg) were steeped in 10 litres of tap water for 8

hours at ambient temperature (28*0.56"C), after which they were wet dehulled and sun-

dried at 3 lrt0.26OC. Finish drying was carried out in a Gallenkamp oven (Model 1H- 150)

at 50°C. The samples were milled into flour with a Bentall attrition mill (Model 200

L090), and sieved using a traditional nylon sieve to get bambara groundnut flour (B).

Malting of bambara groundnut seeds

A batch of 5.4kg of bambara groundnut seeds was divided into 27 lots of 200g in

j# malting bags, steeped in 10 litres of tap water at 28st0.56°C using a modification of the

3 (gtwo-step wet-steep method of Etok-Akpan and Palmer (1990). The steeping schedules

c,? were based on the time for maximum water absorption characteristics of bambara

8 groundnut seeds (Maduko, 2002). The seeds were steeped at room temperature 28*0.56OC $& for 14hours, air rested for 4 hours and re-steeped in fresh water for 14 hours. The out-of-

$ 1

steep grains were spread to germinate in a dark room for 72 hours, the ~amples~being

moistened on alternate days by dipping the bags containing the germinating grains in

water for 30seconds. The grains were turned once every 24 hours.

The root length was determined on a daily basis using a metre rule. The green

malts were dried in a convection Gallenkamp oven (Model IH-150) at 50°C for 12-hours,

after which the malts were cleaned of sprouts by abrasion in-between the palms followed

by winnowing. Malting loss was calculated and the cleaned malts milled into flour using

a Bentall attrition mill (Model 200 L090) and sieved using a traditional nylon cloth to get

bambara groundnut malt (B,) flour. The flours were packed in polyethylene bags, sealed

and stored at 4OC until needed.

The diastatic activity of the malts was determined by the method of Hulse et al.

(1980). The slurry of each of the flour (5g) was made with distilled water (1:4.5 wlv). The

temperature of the slurry was gradually raised from 3S°C to 70°C over 1 hour by heating

in a regulated Gallenkamp water bath. The slurry was maintained at 70°C for 1 hour. The

digest was filtered using Whatman filter paper No 1. The concentration of sugar in the

filtrate was determined using a hand-held refractometer (Berlington and Stanley Ltd,

London). The percent total sugar was expressed as diastatic activity in degrees Lintner

(OL).

Production ouermen fed composite flour blends

Composite flours were formulated in a ratio of 70:30 (Canneron and Hofivander,

1983) to give the maize-bambara groundnut (MB) and maize-bambara groundnut malt

(MB,) blends. The blends were fermented for 72 hours by backslopping (Nout et al.,

1989), dried in a convection Gallenkamp oven (Model IH- 1 SO) at 50°C for 12 hours to a moisture content of 5.25% (MB)f and 5.00% (MB,)f

3.2.3 FortijZatwn of complementary food blends

Incorporation of red palm oil into complementary food blends

Palm oil was incorporated into the complementary food blends after emulsification with Brachystegia eurycoma or sodium sesquicarbonate and added pre- or post

fermentation. 1. An emulsion of red palm oil was prepared with Brachystegia eurycoma in the ratio

of 1 : 1 : 1.9 (red palm oi1:water: Brachystegia eurycoma). (a) The emulsified red palm oil

(lorn1 of oil: l0ml of water:20g of Brachystegia eurycoma) was added into the #

complementary food blends in a ratio of 1 :9.6 and fermented by the backslopping method of Nout et al., (1 989) for 72 hours before drying at 50°C in a Gallenkamp oven (Model

IH- 150) for 12 hours. (b) Emulsified Brachystegia eurycoma (20g) and the composite

flours (100g) were fermented separately by backslopping for 72 hours and then mixed in

the ratio of 1:9.6 before drying in a Gallenkamp oven (Model IH-150) at 50°C for 12

hours. The cakes were milled into flour and stored at 4°C until needed for analysis.

2. Sodium sesquicarbonate was used to prepare a stable emulsion of red palm oil and

an equal amount of water and fermented as described for the Brachystegia eurycoma

samples

3. Unemulsified red palm oil was added into the composite flours and fermented by

backslopping (Nout et al., 1989) for 72 hours, dried at 50°C in a convection Gallenkamp oven (Model IH- 150) for 12 hours. The composite flours were fermented by backslopping

for 72 hours and then mixed with unemulsified red palm oil before drying in a

Gallenkamp oven (Model IH-150) at 50°C for 12 hours. The cakes were milled into flour

and stored at 4°C until needed for analysis. The proportion of red palm oil in the formulation blend is shown in Table 8 (Appendix I)

Incorporation of fortificants (ashed bone meal, ashed roselle calyces, red palm oil emulsified with Brachystegia eurycoma or trona) into the complementary food blends.

The processed food fortificants were mixed with the composite flours pre- or post-

fermentation and as a dry mix. The food fortificants were ashed bone used as source of

calcium, ashed; dried roselle calyces used as source of iron and zinc and red palm oil

emulsified with Brachystegia eurycoma or trona used as source of vitamin A. The

quantity of fortificants used was based on the proposed Recommended Dietary

Allowance (RDA) of calcium, iron, zinc and vitamin A for fortified complementary foods

(Lutter and Dewey, 2003). The fortificants were mixed in the ratio of 1 .O: 1.6: 2.25: (red

palm oil: roselle calyces: bone meal) on weight basis and mixed with the complementary

food blends in the ratio of 1 :2.

(i.) For pre-fermentation sample, bone meal, ground roselle calyces and red palm oil

emulsified with Brachystegia eurycoma or trona and the composite flours were mixed in

a bowl and fermented for 72 hours by backslopping (Nout et al., 1989) and dried.

(ii.) The fortificants (bone meal, ground roselle calyces and red palm oil emulsified with

Brachystegia or trona) and composite flours were fermented separately by backslopping

(Nout et al., 1989) for 72 hours, mixed together and then dried.

(iii.) The bone, roselle calyces and red palm oil emulsified with Brachystegia eurycoma #

or trona and the composite flours were fermented separately by backslopping (Nout et al.,

1989), dried at 50°C separately and mixed together.

Drying was carried out at 50°C in a convection Gallenkamp oven (Model IH-150).

The cakes were then milled into flour using a laboratory mortar, packed in polyethylene

bags, sealed and stored at 4OC until needed for analysis. The formula blends and the

proportion of fortificants are shown in Table 9 (Appendix 11). The flow chart for

fortification of complementary foods is shown in Figure 2.

3.2.4 Chemical analysis of fortified complementary food blends

3.2.4.1 Anti-n utrient factors

The following anti-nutritional factors were determined in the complementary

foods:

(i) Oxalate: Oxalate content was determined using the method of Fassett (1973).

Two grams (2g) of the sample were dissolved in 200 ml of 30% HC1. The

extract was titrated with 0.1M KMnO4 (potassium tetraoxomanganate (IV)

and the solution read at 490 nrn in a UV BIOCHROM 4049 spectrophotometer.

The concentration of oxalate was read off a standard curve prepared with

standard solution (Appendix IV) and the value expressed as rng oxalate11 00g.

(ii) Tannin: This was determined using the method of Price et a1 (1980). Ten

millilitres (10 ml) of 2M HC1 was added to 0.5g samples in a conical flask and

shaken for 5 minutes. The content were quantitatively transferred into a test tube

to which 3ml of O.lM FeC13 in 0.1N HCI and 3 ml of O.OO8M potassium

Fort i ficants

(70:30) Maize: Bambara groundnut flourlmrlt I +

Fermentation

1 Drying at SOOC

Milling

Prefermentation fortified complementary foods

Fermented fortificants

Milling

4 Post fermentation fortified

complementary foods

Milling

Dry mix fortified complementmy

foods

Figure 2: Flow chart for the production of fortified complementary foods

ferrocyanide (K3Fe(CN)6) were added. It was allowed to stand for 3 minutes and

read in a UV spectrophotometer, BIOCHROM 4049 within ten minutes at 500

nm. The concentration of tannin was read off a standard curve prepared using

standard tannic acid solution and the value expressed as the tannin1100g using the

formula:

Conc. (mglg) from standard curve x dilution factor mg Tannin =

weight of sample

where mg/100g sample = mg of tannin x 100

(iii) Phytate: Phytate content was determined using the method of Latta and Eskin

(1980). Half a gram (0.5g) of each sample was extracted with 2.4% HCl. An

aliquot (2 ml) of a mixture of 0.1M NaOH and 0.7M NaCl was added arfkl passed

through a resin (200-400 mesh) to remove inorganic phosphorus and other interferring

compounds. Modified Wade reagent (0.03% FeC13.6H20 and 0.3% sulfosalicyclic acid)

in distilled water were added and the solution was mixed on a votex mixer for 5 secs. The

mixture was centrifuged for 10 minutes and the supernatant was read at 500nm in a W

spectrophotometer, BIOCHROM 4049. The concentration of phytate was read off a

standard curve prepared with standard sodium phytate and the value expressed in

mg/l OOg using the formula:

Conc. (mg/g) from standard curve x dilution factor mg phytate =

weight of sample

where mg/100g phytate = mg phytate x 100

3.2.4.2 Proximate Composition

Moisture content

Moisture contents of the samples were determined by the method described in

AOAC (1990). Two gram (2g) of sample was weighed into an aluminium dish with

cover. The covered dishes and the covers were placed in an oven previously regulated to

135h2"C and the samples dried for 2 hours. The cover was placed on dishes and

transferred to a desiccator to cool and then re-weighed. The percentage moisture content

of the samples was calculated using the formula:

Weight difference 100 X-

original weight of the sample 1

Where weight difference = original sample weight - final sample weight.

Crude protein content

The crude protein content of the samples was determined using the Kjeldahl

procedure described in AOAC (1990). One gram (1 g) of the sample was weighed into a

250 ml Kjeldahl digestion flask to which 16.7g K2S04, 0.01g anhydrous CuS04, 0.6g

T102 and 20 ml of H2SO4 were added. An additional 1.0 ml of H2S04 was added. The

digest was heated until the solution was colourless, cooled and 250 ml of water was

carefully added and then cooled to room temperature. The distillate was transferred

quantitatively into a 100 ml volumetric flask and made up to the mark. Five millilitre

(5ml) of the diluted digest was poured into a 50 ml conical flask and 10 ml of mixed boric

acid and 3 drops of methyl red indicator were added. The conical flask with it6 content

was placed under the collection spigot of the distillation apparatus. Five millilitre (5ml) of

60% NaOH was added slowly into the solution.

The distillation was carried out until the distillate volume was about 50 ml and the

solution became green. The green solution or the distillate was titrated with 0.01N HCl

until the solution turned purplish. The titre value was read from the burette. The nitrogen

content of the sample was calculated. The value obtained was multiplied by 6.25 to obtain

the protein content.

% N = [@acid) (V~lacid) - (V~lbk) (NNaoH) - (VolNaoH)] (1400.67)/mg) sample.

Where V0lNaoH = Volume of standard based needed to tirtrate sample; VolXid =

volume of standard acid used for that sample; Volbk = volume of standard based needed to

tirtrate 1 ml standard acid minus Volume of standard based needed to titrate reagent blank

carried through method and distilled into 1 ml of standard acid, NNaoH = normality of

standard acid; Nbase = normality of standard based.

% crude protein = 6.25 x % N.

Fat content

The crude fat content of the samples was determined using the method described

in AOAC (1990). Two gram (2g) of the sample was weighed into a thimble. Flat

bottomed fat extraction cups were weighed and placed on the platform of the tecator fat

extraction unit. The thimbles were attached to the Soxtec extractor and the samples

extracted with petroleum ether (60-80°C) for 1 hour. The solvent free fat in the cups was

dried in an air oven for 30 minutes at 80°C. The cup with its content was cooled in a

dessicator and reweighed

Weight of extract + cup - weight of cup 100 % Fat = X-

original weight of sample 1

Ash content

The ash content of the samples was determined using the method described in

AOAC (1990). Two grams (2g) of each sample was weighed into a porcelain crucible and

ignited in a temperature-controlled furnace at 600°C and held for 2 hours. The crucible

with its content was removed, cooled in a desiccator and weighed.

The percentage weight of the ash was calculated as shown below.

Weight of the sample + crucible - weight of crucible difference 100 %Ash = X - original weight of sample 1 '

Crude fibre content

The crude fibre content of the samples was determined using the method

described in AOAC (1990). Two grams (2g) of the sample (WI) was put in a 250 ml

beaker, boiled for 30 minutes with 100 ml of 0.12M &So4 and filtered through a funnel.

The filtrate was washed with boiling water until the washing was no longer acidic. The

solution was boiled for another 30 minutes with 100 ml of 0.012M NaOH solution,

filtered with hot water and methylated spirit three times. The residue was transferred into

a crucible and dried in the oven for 1 hour. The crucible with its content was cooled in a

desiccator and then weighed (W2). This was taken to a furnace for ashing at 600°C for lh.

The ashed sample was allowed to cool in the furnace, then removed and put into the

desiccator and later weighed (W3). The percentage crude fibre was calculated thus:

% Crude fibre = 2 - x 100 w ,

W1 = original weight of sample taken before treatment

WZ = weight of crucible with dried residue of digested sample before ashing

W3 = weight of ashed sample

Carbohydrate content

The percentage carbohydrate in each sample was obtained by difference, that is,

by subtracting the amount of moisture, protein, fat, ash and crude fibre from 100%

(AOAC, 1990).

3.3.4.3 Micronutrients

Calcium

The calcium content was determined using the method described in Pearson

(1976). One gram (lg) of the complementary food blend was digested with 2 ml of conc.

HC1 and evaporated to dryness. Ten millilitre (10 ml) of normal HCl was used to boil the

sample and then 5ml of distilled water added, filtered through Whatman No. 1 filter paper

and the resultant solution made up to 20 ml. The solution was shared into two 10 ml

portions in centrifuge tubes. To each tube, 0.5 ml of 5% ammonium oxalate and a drop of

methyl red indicator were added, followed by 1 ml of 5% ammonia and lml of acetic

acid. The resultant solution was allowed to stand for 4 hours, centrifuged twice at

360.r.p.m for 10 minutes and the supernatant discarded after each run. Two millilitres

(2nd) of dilute &SO4 was added to the sediment and the sample heated to 4 5 0 ~ and

titrated with 0.02N potassium permangnate. The calcium content (g) of the sample was

calculated using the relationship: 1 ml of 0.02N potassium permangnate = 0.00040g Ca.

The amount of calcium found in 10 ml aliquot was multiplied by 2.0 to obtain the weight

of calcium.

Iron

The iron content of the food blends was determined by the method of AOCS

(1 993). One gram (lg) of each sample was ashed at 4 5 0 ' ~ and digested in 10 rnl of O.1N

HC1, heated for 30 minutes, cooled and filtered into a 25 ml standard flask and made up

to the mark with distilled water. To 5 rnl of the above solution duplicated in test tubes, 2

ml of acetate buffer (pH 7.4), 1 ml of hydroquinone and 1 ml of a-a' dipyridyl solution

were added. The resultant solution was measured into a 1-cm micro-cuvette and the

absorbance was read at 520nm using a UV BIOCHROM 4049 spectrophotometer. The

concentration was read off a standard curve prepared with standard iron solution

(Appendix IV). The iron content of the sample was calculated using the formula:

concentration mg Fell 00g samples = x dilution factor.

weight of sample

The dilution factor = 2.5

Zinc

The zinc content of the samples was determined by the method described by

Hibbard (1937). Five grams (5g) of the sample was weighed into a flat-bottomed flask

and 10 rnl of a 1:l (vlv) mixture of concentrated nitric acid and perchloric acid were

added to the samples and digested in a fbme chamber. The solution was then made up to

100 ml with distilled water. To 5 ml of the digest in a test tube were added 5 ml of acetate

buffer (pH 7.4), 1 ml of 1N sodium thiosulphate solution and then 10 ml of 1N dithizone

solution The mixture was allowed to stand for 4 minutes for colour development and the

absorbance read at 535 nm in a UV spectrophotometer (BIOCHROM 4049). The

concentration of zinc was read off a standard curve prepared with standard zinc

(Appendix IV) and the value expressed as mg zinc11 00g sample using the formula: b conc. (pg/d) fiom standard curve

mg zinc = x dilution factor weight of sample

where mg zinc11 00g sample = mg zinc x 100.

The dilution factor = 10.

Vitamin A

The vitamin A content was determined by the method of Arroyave et a1 (1982).

Ten rnillilitres ( 10ml) of 95% ethanol and an equal volume of hexane were added into a

test-tube containing lg of the sample, followed by the addition of 10ml of normal saline

to dilute it. The tube was stoppered and the contents mixed vigorously on a vortex mixer

for 2 minutes to ensure complete extraction of carotene and vitamin A before

centrifbgation for 10 minutes at 3000xg to obtain a clean phase separation. Thereafter,

lOOpl of hexane extract was transferred to a microcuvette and the absorbance due to

carotene at 450nm was read against hexane blank. The sample was then transferred fiom

the microcuvette to a test tube and the cuvette rinsed with 5 0 ~ 1 hexane and the solution

added to the sample in the test tube. The extract was evaporated to dryness under a gentle

stream of nitrogen in a 6 0 ' ~ water bath while avoiding splashing on the test tube wall.

The residue was immediately redissolved in lop1 of chloroform-acetic anhydride (1:1,

VIV) reagent and 100p.l of fieshly prepared TFA-chloroform chrornagen reagent was

added. The solution was rapidly transferred to the microcuvette using a microtransfer

pipette. The blank consisted of chloroform-acetic anhydride mixture and TFA-chloroform

chrornagen (1 : 1, v/v) reagent. A W spectrophotometer (BIOCHROM 4049) was used to

read the absorbance of the sample at 620nm after 1 Sseconds (t15) and again at 30seconds

(f30) after addition of the chromagen. The concentration of vitamin A was read off a

standard curve prepared by diluting vitamin A standard with hexane (Appendix IV) and

the calculation was made as below:

Vitamin A (as pg REIdl) = A, - 2 A,, x FC450 x FA,, x 75 FC620

Where A620 = Absorbance reading taken at 620nm

= Absorbance reading taken at 450nm

FC450 = Calibration factor for carotene at 450nm = pg carotenelml &50 b

FC620 = B-carotene A620 correction factor = pg carotene/ml A620

FA620 = Factor for vitamin A at 620nm = pg vitamin Ntube A620

In measuring Vitamin A, the absorbancy correction at 620nm for carotenoids is:

2x xFC450 , in which the faetor of 2 derives &om the difference in the

FC620

dilution of carotenoids and vitamin A in their respective assays.

Data Analysis

Data obtained were statistically analyzed using the Completely Randomized

Design (CRD) and Randomized Complete Block Design (RCBD) as applicable

(Steeland Torrie, 1980). Duncan's New Multiple Range Test was used to detect

differences among the treatment means.

4. RESULTS AND DISCUSSION

4.1 Ashing Temperature For Cattle Bone

Table 10 shows the calcium content of cattle bone ashed at 500 to 700°C.

Table 10: Calcium content of bones ashed at three different temperature

Ashing temperature (OC) "Calcium content (mgI100g) 500 680 k 0.000 600 680 k0.300 700 664af 0.141 * Results are the means of two replications; Values carrying different alphabet in the same column are significantly different (p<0.05).

There were significant differences (p4.05) among the samples that received different

treatments. Ashing at 700°C resulted in a significantly (pK0.05) lower calcium content

(664mg/100g) compared to 500°C and 600°C. The lower calcium content of 700'C could

be attributed to loss of the mineral through decomposition and formation of whitish

calcium oxide, which may not have been completely estimated under the conditions of the

test. Ashing at 600°C resulted in a significant increase in calcium content (680mg/100g),

however the value was not significantly (p<0.05) different from that of 500°C. This

suggests that 500-600°C could be the optimal temperature for the decomposition of

hydroxyapatite beyond which some losses could occur. Visual comparison of the colour

of the ashed samples showed that samples ashed at 500°C were darker than 600°C and

both of them appeared darker than samples ashed at 700°C, which was the whitest of all.

Ashing of cattle bone at 600°C could be ideal in terms of calcium content and colour of

the ashed sample as reported by Abolude and Abdullahi (2005).

Effect of Processing on Selected Micronutrient Content of Food Fortificants

The effect of processing on the selected micronutrients content of food fortificants

are presented in Tables 1 1,12 and 13.

Calcium content of bone

The results for the calcium content of processed cattle bone are shown in Table

11. The values ranged from 336a0.283mg/lOOg in the raw bone to 764ct0.212 mg/100g in

the autoclaved, ashed and fermented (aAF) samples.

Among the samples that received single treatments, ashing (A) released the

highest amount of calcium (344mg/100g) over and above the control; fermented and

autoclaved samples released 184mg/100g and 4mg/100g respectively. This may be due to

thermal decomposition of calcium hydroxyapatite, which has a decomposition

Tables 1 1 : Calcium content of processed cattle bone samples

Treatment *Calcium content (mg1100g) *Calcium content released (mg/lOOg) C 336 a zt0.283 0 a 340 *0.353 4 F 52Oc k0.141 184 A 680 *0.141 344 FA 572 *0.071 236 aA 608 *0.000 272 AF 760 zt0.071 424 FaA 632 *0.000 296 aFA 632 *0.141 296 aAF 764 ' *0.2 12 428

*Results are the means o f duplicate determinations; Values carrying different alphabet in the same column are significantly different (p<0.05); C (control) = raw bone; a = autoclaved bone; A = ashed bone; F = bone fermented by backslopping; FA = bone fermented by backslopping and then ashed; aA = bone autoclaved and ashed; AF = bone ashed and fermented by backslopping; FaA = bone fermented by backslopping, autoclaved and ashed; aFA = bone autoclaved, fermented by backslopping and ashed; aqF = bone autoclaved, ashed and fermented by backslopping.

temperature of 800°C at which the organic content is removed (Chai and Ben-Nissan,

1994). Similar high values of calcium (998.3mgflOOg) were reported in the analysis of

fish bone ashed at 600°C (Abolude and Abdullahi, 2005).

When the treatments were paired with ashing, the ashing and fermentation (AF)

caused the release of the highest calcium content (424mg/100g). Compared to ashing (A)

alone, the relatively low increase of 80mg/100g shows that fermentation may not be an

effective method of decomposing calcium hydroxyapatite. When compared to the FA

sample, the results indicate that thermal decomposition of calcium hydroxyapatite before

fermentation encouraged more effective release of the calcium content by fermenting

microorganisms and enzymes. The higher value (424mg/100g) of the AF sample

compared to the FA (236mg/100g) may be due to increased surface area provided by

milling of the ashed sample before fermentation. This may have contributed to ease of

accessibility of fermenting microorganisms to the calcium hydroxyapatite.

The introduction of autoclaving as a third treatment gave the highest calcium

release of 428mg/100g, which marginally differed from the 424mg/100g of the AF

sample. The result indicates that order of treatment affected calcium released from

hydroxyapatite. The lower values for both aFA and FaA (296mg/100g) may be due to

leaching during fermentation and reduced surface area of the raw bone. The marginal

differences between aAF and AF samples (4mg/100g), though statistically significant

(pC0.05) but may not be economical in terms of energy used; this, however, suggests AF

as the choice method of processing cattle bone into meal.

Iron and zinc contents of Hibiscus sabdariffa

The iron content (Table 12) ranged ftom 40.24 rng1100g for the ftesh leaves

(control) which had a moisture content of 68.75% to 13.96rngIlOOg sample for the dried

roselle calyces fermented by backslopping (RCdb moisture content =lo%).

Table 12: Iron and Zinc contents of processed roselle calyces

TREATMENT EFFECT ON Treatment *Iron content Differences from *Zinc content Differences from

(mg/lOOg) control (mg/100g) (mg/100g) control (mgl100g) C 40.24 a * 0.004 0 20.00a* 0.100 0

RCdb 13.96 '* 0.056 9.936 38.40 0.030 18.40

*Results are the means of two replications; Values carrying different alphabets in the same q~lumn are significantly different (p<0.05); C = fresh roselle calyces (control); RCa= Roselle calyces dried at 50°C; RCb = Roselle calyces fermented by backslopping; RCdb = Roselle calyles dried at 50°C and fermented by backslopping.

Roselle calyces dried at 50°C had significant w0.05) increase in iron content (8.846

mg/100g) over the control (C) and may be due to moisture removal. The higher values

reflect a 200% increase in concentration. When the ftesh roselle calyces were fermented

by backslopping (RCb), the increase in iron content was 5.806mgllOOg reflecting an

increase of over 125%. This lower value compared with that for drying suggests that

microorganisms that require iron for their metabolism may be associated with the

fermentation process. This is more apparent ftom the iron content of RCdb (13.96

mg/lOOg), which suggests that some of the fermenting microorganisms associated with

the fiesh leaves may have been eliminated during the initial drying process. This is why

the combined treatments gave a value less than could be expected ftom the addition of the

effects of the individual treatments. The significant increase in iron content of RCdb

(l.lOmg/lOOg) over and above RCd may be attributed to phytate reduction during

fermentation (Nout and Motarjemi, 1997). Babalola et al. (2001) and Nnam and Onyeke

(2003) reported a higher value of 37.8mg/100g and 833.0mg/100g during the

fermentation of roselle calyces dried at 60°C and 55OC respectively. The differences in

results may be attributed to variation in soil fertility, fermentation and analytical methods.

It is possible that pre-fermentation fortification of maize-bambara groundnut

complementary food with roselle calyces could yield higher values of iron.

The zinc content (Table 12) of the processed roselle calyces differed

significantly (p<O.O5)among the treatments and the values ranged from 20.0Omg/100g

for fresh leaves (control) with moisture content (68.75%) to 3840.0mgAOOg for roselle

calyces dried and fermented by backslopping (RCdb). The dried roselle calyces (RCd),

fiesh roselle calyces fermented by backslopping (Rcb) and roselle calyces dried and

fermented by backslopping (RCdb) had zinc values significantly @<0.05) higher than the

control. The increase ranged fiom 53.6%-92.W; RCd and RCb had 68.0% and 53.6%

increases in zinc content respectively over the control. Drying increased zinc

concentration by the removal of water. Fermentation of the fresh calyces resulted in a

reduced zinc content suggesting that microorganisms requiring zinc for their metabolism

may be associated with the fermentation process. This is because fermentation is

supposed to lead to loss of tissue structure, water binding capacity and reduced phytate

(Nout and Motarjemi, 1997). This is more apparent fiom the zinc content of the dried and #

fermented sample (RCdb) suggesting that most of the initial microorganism associated

with fiesh leaves may have been eliminated during the drying process. RCdb had

significantly @<0.05) higher zinc content release (18.40mg/lOOg) reflecting a 92.0%

increase over the control. The significant increase in the zinc content of RCdb (48

O.Omg/lOOg) over and above the dried roselle calyces (RCd) could also be due to

fermentation-induced phytate reduction. Ojokoh et al. (2002) reported zinc content of

3 1.86mgAOOg during fermentation of roselle calyces dried at 50°C while Nnam and

Onyeke (2003) reported a lower value of 1.17mg/100g.

Vitamin A content of processed red palm oil

The results (Table 13) show the vitamin A content of red palm oil emulsified with

Brachystegia eurycoma or trona.

Table 13: Vitamin A content (pgREl100g) of palm oil emulsified with Brachystegia eurycoma and sodium sesquicarbonate.

Treatments Vitamin A content (pgREIl OOg) *Brachystegia *Vitamin A *sodium *Vitamin A Eurycoma content released sesquicarbonate content released

C 262 1 'H.007 0 2621 'M.007 0

Efd 2417bk0.381 -204 2175~*0.007 -446 *Results are the mean of two replications; Values carrying different alphabets in the same column are significantly different (p<O.O5); C (control) =unemulsified red palm oil; Ed = red palm oil emulsified and dried at 50°C; Ef = red palm oil emulsified and fermented by backslopping; Efd = red palm oil emulsified, fermented by backslopping and dried at 50°C.

Emulsification of red palm oil followed by drying led to reduced vitamin A content, the

amount of vitamin A lost was more in palm oil emulsified with Brachystegia eurycoma

than sodium sesquicarbonate (822pgRE/100g vs 484pgREllOOg) suggesting that the use

of Brachystegia eurycoma and sodium sesquicarbonate as emulsifierslstabilizers reduces

vitamin A probably by sequestering or destroying it. This implies that the use of these

emulsifierslstabilizers in traditional foods may be suspect except that subsequent cooking

during soup making with Brachystegia eurycoma may lead to release of vitamin A.

Drying at 50°C contributed to reduce vitamin A content as had been reported by Erhardt

(2004) and Uzoma et al. (2005).

On fermentation of the emulsion (E,), vitamin A contents increased; the amount

released from the trona sample (722 pgRE1100g) was 6 times higher than the #

Brachystegia eurycoma sample (127 pgREIlOOg), suggesting that the reduction on

emulsification of palm oil was by complex formation; a more complex binding system is

suggested in Brachystegia eurycoma.

When the fermented stabilized emulsion was dried, values less than the control but

higher than the dried sample were obtained, showing that the heating contributed to

destruction of vitamin A. The carotenoids are highIy unsaturated and are therefore prone

to isomerization and oxidation. Isomerization of the trans-carotenoids to the less active

cis-isomer during drying is promoted by contact with acids (Rodriguez-Amaya, 2002),

which could be the case when the fermented emulsions were dried. The heat of drying at

50°C may possibly contribute to the loss of the carotenoids through accelerated oxidation

to products, which usually break down to low molecular mass compounds (Rodriguez-

Amaya, 2002). The results suggest that (i) the stabilizers bind vitamin A and/or P- carotene, (ii) drying at 50°C results in a loss of vitamin A and that sodium

sesquicarbonate induced a greater loss of vitamin A on drying after fermentation This is

consistent with the results of Uzoma et al. (2005) that vitamin A activity for "enriched

palm oil gar?' (EPOG) decreased from 2000 pgRE/100g to 46.1 pgRE1100g during

toasting alone.

4.2 Production of maize-bambara groundnut complementary foods by malting and fermentation

Malting of bambara groundnut seeds

The root length and malting loss of bambara groundnut malts are shown in Figure

3. The results show that the root length increased with germination time. The highest

0 1 2 3 4 5 6 7 Malting time (days)

Fig. 3: Average root length and malting loss of bambara groundnut seeds

increase in root length between successive days (2.01cm) was observed on the third day

of malting while the first day had the lowest. The increase (87.69%) on day 3 was

significantly (p<0.05) higher than other malting days. The length of root indicates growth

and probably the hydrolysis of higldcomplex molecular structures within the kernel. The

highest change in root length on third day suggests improved modification of endosperm

resulting from the improved enzyme secretion and activity (Palmer and Bathgate, 1976).

This could explain the increasing malting loss, which also peaked on day 3 (Figure 3),

with a value of 11.86% and could be attributed to the root and shoot outgrowth and

respiration of the embryo (Pollock, 1962). Maduko (2002) made a similar observation and

attributed it to leaching of nutrient and degree of modification of the grains. Abiodun

(2002) observed malting loss between 15.5% and 33.9% in sorghum malt on day 3 of

malting. The higher malting loss in cereals could reflect the higher starch contdnt of the

grains.

The diastatic activity (Figure 4) increased as the duration of germination increased

with the day 3 malts having the highest value (41.465OL). The high diastatic activity may

be attributed to increased secretion, the extent of breakdown of granule cell wall and

subsequent hydrolysis of starch. Maduko (2002) observed that diastatic activity of

bambara groundnut peaked on malting day 3 and attributed it to the degree of

modification of the grain constituents.

Malting time (days)

Figure 4: Diastatic activity of bambara groundnut. stg = out-of-steep grains malt.

Chemical composition of maize-bambara groundnut complementary foods

The anti-nutrient (tannin, oxalate and phytate) contents of the maize-bambara groundnut

blends are presented in Table 15.

Table 15: Anti-nutrient composition of complementary food

Samples *Tannin (mg/100g) *Oxalate (mg1100g) *Phytate (mg/100g)

*Results are the means of three replications; Values carrying different alphabet in the same cwlurnn are significantly different (pc0.05); m=Untreated maize-bambara groundnut blends; MB= Maize- bambara groundnut wet dehulled; MB,= Maize-bambara groundnut malt; (MB)f = Maize-bambara groundnut fermentaed by backslopping; (MB,Jf = Maize-bambara groundnut malt fermented by backslopping;

Tannin contents decreased as a result of dehulling of bambara groundnuts, degerming of

maize, malting of bambara groundnut and the fermentation of the blends. The reduction

ranged from 25 to 62.5%, a very significant ( ~ ~ 0 . 0 5 ) decrease in value compared to the

control (MB,) and could be attributed to the effectiveness of the food processing

techniques applied. Malting is known to increase polyphenol contents (Hurrell, 2003) but

fermentation reduces them (Nout and Ngoddy, 1997). The reduction in tannin content of

maize-barnbara groundnut malt (MB,) therefore, suggests some loss during soaking with

little synthesized during germination. Moreover, since the white grains were used, it does

suggest that tannins synthesized were used for other metabolic functions.

Ene-Obong (1995) reported that decrease in tannin contents could be achieved

through soaking, dehulling, fermentation and germination. The highest decrease (62.5%)

was observed in fermented maize-bambara groundnut malt [(MB&] and could also be

attributed to the activity of enzymes associated with seeds particularly after malting.

Similar results were obtained by Obizoba and Egbuna (1992).

The results for phytate and oxalate contents followed a similar trend as the

tannins. The phytate composition of the blends was generally low and could be due to the

effect of malting and germination (Nout and Motarjemi, 1997; Akubor and Chukwu,

1999; Nzelibe and Onyeniran, 2001). The decrease observed in oxalate contents of the

blends (50-75%) could be attributed to the effect of steeping and fermentation on the

samples (Aworth, 1993; Quinteros et al., 2003).

The tannin, oxalate and phytate levels observed were lower than the safe levels of

2.0g/1 OOg, 2.2g/100g and 5.0g1100g respectively as reported by Munro and Bassir (1 969)

and could ensure no adverse physiological effects when the products are consumed; the

low levels of these antinutrients would also ensure availability of divalent cation

(calcium, iron and zinc) in the product (Quinteros et al., 2003; Beebe et al, 2000).

The nutrient composition of the processed maize-bambara groundnut food blends

is presented in Table 16.

Table 16: Nutrient Composition of unfortified maize-bambara groundnut food blends

Nutrient *MB, *MB *MB, *(MB)r *@mh

Crude protein (%) 15.75"+0.05 17.95'+0.02 17.25 b0.02 18.35 d+0.15 18.7fi0.25

Moisture (%) 6.00C+0.04 5Zd+0.0 1 5.00c+0.06 4.7sb+0.0 1 4.508+0.01

Crude fat (%) 7.OOe+0. 10 5.00~+0.02 4.1 5b+0. 15 4.25'+0.04 4.008+0.02

Crude fibre (%) 2.37 '50.50 1.38'+0.04 1.38'+0.02 2.00 b+0.02 2.05 b+0.02

Ash (%) 1.90 b+0.50 1 .508+0. 13 1-95 bC+0.04 1.95 bC+0.03 2.05 'g.03

Carbohydrate (%) 69.63d+o.06 68.92'+0.02 70.27'+0.06 68.60~20.5 68.6sb+1 .25

Calcium (mg/l OOg) 80.00b+0. 15 68.008+0.0 1 1 06.00C+0.09 124.00~+0.00 126.67'9.02

Iron (mg1100g) 2.67 'fl.10 4.25 b+0.07 6.87dC+0.2 1 6.65'3.0 1 6.88d+0.02

Zinc (mg/100g) 1.93 b+0.06 1.833.0 1 3.8OC+O.35 2.63'+0.02 3.65d+0.02

Vitamin A (pgRe/lOOg) 43.05'+0.03 25.758+0.50 30.00 b+0.02 33.0 1'9.02 4 1.1 7d+0.~4

*Results are means o f three replications; Values carrying different alphabet in the same row are significantly different (p<O.OS); MB, = (control) untreated Maize-bambara groundnut flour; MB =Maize bambara groundnut wet-dehulled; MB,=Maize-bambara groundnut malt; (MB),= Maize-bambara groundnut fermented by backslopping; (MB,)f =Maize-bambara groundnut malt fermented by backslopping;

From the results, malting, dehulling, degerming and fermentation increased the

protein content of the blends compared to MB, (control). The percent increases were

13.97, 16.5 1, 14.60 and 17.78 for MB, MB,, (MB)f ,d (MB,)f respectively. MB, had

the least increase in protein content and this may indicate that bambara groundnut malt

used may have lower protein content than the unrnalted. This may result from the short (3

days) malting time. Germination is known to be a cheap and effective method of

increasing protein content and its digestibility. Reduction in protein content of bambara

groundnut malted for 3 days had been reported by previous workers (Obizoba and Atti,

1991; Obizoba and Egbuna, 1992). The high protein content of all the fermented maize-

bambara groundnut blends could be due to the release of proteins bound as complexes

and contributions from microbial biomass. The highest increase in protein content

observed in fermented malt blends (MB,)f could be attributed to its low moisture

content, loss of anti-nutrients, release of more protein fiom organic complexes, synthesis

during germination and denaturation of native protein (Liener, 1976).

The moisture content ranged fiom 4.5-6%. The decrease in moisture content of

(MB)f and (MB,)f (9 and 10%) compared with MB and MB, respectively is attributable

to fermentation during which hydrolysis of the starch molecules of the grains caused the

release of bound water which was lost during drying. The malting process further

hydrolysed the complex starch and protein molecules of the blends resulting in a dryer

malt which when fermented, resulted in a further decrease in moisture content of

fermented maize-bambara groundnut malt [(MB&].

Crude fat contents ranged fiom 4.0-7.0%. The decrease in fat content observed in

fermented maize-bambara groundnut (MB)f and fermented maize-bambara groundnut

malt [(MB,)d could be attributed to the activities of lipolytic enzymes during mahting and

fermentation.

The crude fibre content ranged between 1.38 to 2.37% and reflected a 13.50-

42.19% reduction below the untreated blends (MB,). (MB)f had the highest decrease in

fibre content (2.00%) among the fermented samples which did not differ significantly

(p<0.05) fiom [(MB,)f] (2.00%). The low fibre content of the blends may have been due

to the activities of enzyme inherent in the grains and the use of dehulled maize and

bambara groundnut seeds. Nnarn (2001) reported a decrease in the fibre content of

sprouted dehulled bambara groundnut.

The ash content of the food samples ranged fiom 1.50 to 2.05 % and represented

an increase of 2.63-21% over the control. This may be attributed to the migration of

minerals fiom the bran into the endosperm of the grain during malting and hydrolysis of

phytates (which chellate some of the mineral ions) during fermentation (Nout and

Motarjemi, 1997). The high ash content could also be due to loss of dry matter caused by

the activities of enzymes and microorganism during malting and fermentation.

The carbohydrate contents ranged between 68.60-70.27%. The decrease in

fermented maize-bambara groundnut (MB)f and fermented maize-bambara groundnut

malt (MB,)f samples could be attributed to the use of carbohydrate as a source of energy

by microorganism. Fermented maize-bambara groundnut malt [(MB,)d had the highest

reduction in carbohydrates and could be attributed to the dual effect of malting and

fermentation which caused increased degradation of gelatinized starch granules to much

more soluble and easy-to-digest sugars.

The mineral contents of the processed blends differed significantly (px0.05)

among the samples. The malted blends [(MB,, and (MB,Jf] had higher calcium and iron

contents than MB, (control). The increases ranged from 32-69% for calcium and 157%-

164% for iron with [(MB,)f] having the highest values. Zinc values followed the same

trend as iron with malted blends having a higher zinc content than the unmalted and

control. The highest increase in mineral content of malted and fermented blends [(MBm)f]

could be attributed to the combined effect of fermentation and malting which caused

increased phytase activities (Nzelibe and Onyeniran, 2001; Akubor and Chukwu, 1999;

Nout and Motarjemi, 1997) and reduced moisture contents of the products.

The vitamin A content of the samples varied from 25.75 to 43.05pgREIlOOg. The

vitamin A contents of fermented blends were comparable to, but lower than the control.

This could be due to the combined effect of hydrolysis of p-carotene and improved

moisture loss resulting from malting and fermentation. Rodriguez-Amaya (1 997) reported

that increases in carotenoid contents during drying are consequences of analytical

process, such as loss of caroteniods in fresh samples due to enzymatic activity, greater

extractability of carotenoids from processed samples and unaccounted loss of water.

4.3 Fortification of maize-bambara complementary foods

Effect of emulsification and stage of incorporation of red palm oil on the vitamin A content of maize-bambara groundnut complementary foods.

The results (Table 17) show the moisture content of maize-bambara groundnut

blends to which red palm oil emulsified with Brachystegia eurycoma and sodium

sesquicarbonate were added.

Table 17: Moisture content of maize-bambara groundnut blends fortified with emulsified red palm oil.

Sample Moisture contents (%) *Trona samples *Brachystegia eurycoma samples

Pre (P) Post (,,) Pre (,) Post (,,) (MB) 5.25d*0.05 5.25k0.05 5.2SC*0.05 5.25'*0.05

( ~ ~ m j e p 4.3740.04 4.0Oa*O.03 4.00a*0. 15 3.75a*0.08 *Results are the mean of three replications; Values canying different alphabet in the same column are significantly different (p<0.05); MB (control) =~&e-bambara boundnut without oil; MB, =Maize-bambara groundnut malt without oil; MB, = Maize-bambara groundnut fortified with unemulsified red palm oil; (MB,,,), = Maize-bambara groundnut malt fortified with unemulsified red palm oil; (MB),= Maize-bambara groundnut fortified with emulsified red palm oil; (MB&,=Maize- bambara groundnut malt fortified with emulsified red palm oil.

The moisture content of the fortified blends ranged from 4.37-5.25% and 4.0-5.25% for

pre-and post-fermentation, respectively in trona samples and 4 to 6% and 3.75-6.25% for

pre- and post-fermentation samples respectively in Brachystegia eurycoma samples. The

least values were observed with emulsified palm oil samples suggesting that emulsifiers

could hydrolyze macromolecules. This could be due to loss of water holding capacity as a

result of hydrolysis of starch and protein by malt enzymes. The Brachystegia eurycoma

samples have lower values suggesting that Brachystegia eurycoma had more hydrolytic

capabilities. Post-fermentation incorporation yielded lower moisture content than pre-

fermentation suggesting that some component in palm oil or the samples inhibited the

hydrolytic capability of the emulsifiers. The low level of moisture in all the blends

suggests higher keeping quality of the blends if stored. Similar observations were

reported on dehydrated complementary foods (Akpapunam and Sefa-Dedeh, 1992).

Table 18 presents the vitamin A content of maize-bambara complementary foods

to which red palm oil (RPO) emulsified with sodium sesquicarbonate or Brachystegia

eurycoma was added.

Table 18: Vitamin A content of maize-bambara groundnut complementary food blends fortified with emulsified red palm oil

Sample Vitamin A content (pgRE1100g) *Trona samples *Brachystegia eurycoma samples

Pre Post P r e Post (MB) 33.01a*0.02 33.01a*0.02 33.01a*0.02 33.01a*0.02 (MBm) 4 1.1 7b*0.04 4 1.1 7b*0.04 4 1.1 7b*0.04 4 1.1 7b*0.04 (MBh 344.3Oc*0.O0 37O.OOc*0.O3 344.3Oc*0.O0 370.0Oc*O.03 (MBm)p 356.78d*0.76 472.94d*0.56 456.78d*0.76 472.94d*0.56 (MWep 490.09k0.76 5 1 1 .4de*0.03 53 6.27k2.05 634.42k2.83 (MBm)ep 502Xf*1 .76 538.82f*0.76 740.12Q1.05 868.72Q0.00 *Results are the means of three replications; Values carrying different alphabet in the same column are significantly different (p<0.05); MB (control) =Maize-bambara groundnut without oil; (MB,) =Maize-bambara groundnut malt without oil; (MB),= Maize-bambara groundnut fortified with unemulsified red palm oil; (MB,), = Maize-bambara groundnut malt fortified with unemulsified red palm oil; (MB),= Maize-bambara groundnut fortified with emulsified red palm oil; (MB&=Maize- Bambara groundnut malt fortified with emulsified red palm oil.

The vitamin A content varied from 33.01-502.92 pgREIl00g and 33.01-538.82 pgRE1100g

for pre- and post-fermentation fortification, respectively in samples containing palm oil

emulsified with trona and 33.01 -740.12 pgRE1100g and 33.01 -868.72 pgRE1100g for

pre- and post-fermentation fortification respectively in samples containing palm oil

emulsified with Brachystegia eurycoma. These were significantly (p<0.05) higher than

the values for samples without oil, indicating that addition of palm oil improved the

vitamin A content of the blends. The vitamin A content of the maize-bambara groundnut

malted samples were significantly (p<0.05) higher than their unmalted counterparts with

values that ranged from 8.16-12.83 pgREl100g and 8.1 6-27.38 pgRE/lOOg for pre- and

post-fermentation fortification, respectively in samples containing palm oil emulsified

with trona and 8.16 - 203.85 pgRE1100g and 8.16-234.3 pgRE1100g for pre- and post-

fermentation fortification respectively in samples containing palm oil emulsified with

Brachystegia eurycoma. This indicates that malting of the grains may have produced

enzymes that contributed to the hydrolysis of p-carotene or other vitamin A precursors in

the grain and palm oil.

The vitamin A contents of fortified blends were 3 1 1.29-827.55 pgRE1100g higher

than unfortified blends and could be due to fortification with palm oil. Addition of 6

unemulsified red palm oil to the maize-bambara groundnut flourlmalt (MBIMB,) blends

gave lower vitamin A values than when either Brachystegia eurycoma or trona emulsified

red palm oil was added. The increase over the former ranged from 78.80-79.31%,

indicating that the emulsifiers served to stabilize the retinol formed or served as an ideal

matrix for fat-soluble vitamin A (Day and Mora, 2003).

Addition of Brachystegia eurycoma and trona emulsified palm oil was better

when added post-fermentation suggesting that prefermentation addition might have

resulted in vitamin A utilization and/or loss during fermentation and drying at 50°C. The

reduction in value for pre-fermentation fortified blends (21.35-128.6pgREllOOg)

compared to post-fermentation could also be attributed to reduced acidity of the

fermented samples; vitamin A is very unstable at pH less than 5.0 (Damton-Hill, 2005),

similar values of which was obtained during fermentation of maize-bambara groundnut

flours (Uvere et al., 2002). Brachystegia eurycoma emulsified red palm oil added post

fermentation to (MB,) had the highest vitamin A content. The lower values for trona-

fortified samples indicate that Brachystegia eurycoma is a less effective sequestrant of

vitamin A than sodium sesquicarbonate and could, therefore, serve as emulsifier of

choice.

Effects of time of fortification on the calcium, iron, zinc and vitamin A content of maize-bambara groundnut blends

The calcium, iron, zinc and vitamin A contents of fortified maize-bambara

groundnut blends are shown in Table 19.

Table 19: Calcium, iron, zinc (mg/100g) and vitamin A (pgRU100g) contents of fortified maize-bambara groundnut food blends

Micronutrient Concentrations *Pre fermentation (p) *Post fermentation (,) *Dry Mix (d)

Samples Calcium Iron Zinc Vitamin A Calcium Iron Zinc Vitamin A Calcium Iron Zinc Vitamin A

I

*Results are the means of three replications; Values carrying different alphabet in the same column are significantly different (p<0.05) (MB)r(control) = Maize-bambara groundnut fermented by backslopping; (Ml3& = Maize-bambara groundnut malt fermented by backslopping; (m)b = Maize-barnbara groundnut fortified with bone, roselle calyces and red palm oil emulsified with Brachystegia eurycoma; (MB& = Maize-bambara groundnut malt fortified with bone, roselle calyces and red palm oil emulsified with Brachystegia eurycoma; (MB), = Maize-bambara groundnut fortified with bone, roselle calyces, and red palm oil emulsified with trona; (MB& = Maizebarnbani groundnut malt fortified with bone, roselle calyces, and red palm oil emulsified with trona.

Calcium Content

The calcium contents of fortified blends increased by 202.00-295.83 mg/100g,

228.65-442.93 mg/100g and 225.39-432.53 mg/100g for pre-fermentation post-

fermentation and dry mix fortification respectively. The increases were significant

w0.05); they were some 150-300% over the unfortified and could be attributed to the

high calcium contents of cattle bone (760 mg1100g) used in fortification. Abolude and

Abdullahi (2005) reported similar high calcium contents (998.3 mg/100g) in fish bone.

The calcium content of the fortified malt blends (MBm)b and MB& were higher than their

corresponding maize-bambara blends ((MB)b and (MB)t) and could be attributed to the

enhanced phytase activities during fermentation and germination and the reduction in

tannin and oxalate concentrations. This would suggest that the calcium content would be #

readily available in the body.

Addition of processed bone meal post-fermentation but before drying gave the

highest calcium content. The increase was 11.90-34.82% and 1.86-3.30% higher than pre-

fermentation and dry mix fortified blends respectively. Compared with pre-fermentation

the increase would be attributed to non-utilization of calcium by fermentation

microorganisms. For the dry-mix it could be due to non-incorporation into microbial a-

amylase during fermentation and leaching during drying. Fortified blends containing

trona-emulsified palm oil had 20.09-48.90% reduction in calcium content compared to

fortified blends containing Brachystegia eurycoma-emulsified palm oil and this could be

ascribed to a lag in the activity of the micro-flora enzymes and the low rate of hydrolysis

of calcium-phytate complexes. All the samples containing red palm oil emulsified with

Brachystegia eurycoma except (MB)b fortified post-fermentation had significantly

(p<0.05) higher calcium content than Nutrend with values ranging fiom 13.30-

179.60 mg/100g.

Iron Contents

The increase in iron content after fortification with processed roselle calyces

varied from 180.75-385.46%, 134-454.7% and 89.60-206.83% over the control ((MB)f

and (MBm)f) and was highly significant (p<0.05). Post-fermentation fortification gave the

highest iron values in samples containing red palm oil emulsified with Brachystegia

eurycoma while in those containing trona-emulsified red palm oil, pre-fermentation

fortification gave the highest iron values. The post fermentation increase in iron content

in Brachystegia eurycoma-emulsified blends varied between 6 1.53- 133.40% and 16.68-

84.60% over the pre-fermentation and dry mix samples respectively.

The lower values for pre-fermentation could be attributed to loss of iron through leaching

and nutrient utilization by the fermenting micro-flora while the higher iron content of pre-

fermentation-trona emulsified blends could be ascribed to hydrolysis of iron from their

organic complexes to their much more absorbable and soluble form caused by the

presence of fermenting organism. In general, the formulated fortified foods contained

more iron than Nutrend which served as control with increases ranging from 2.61-28.97

mg/100g. These high iron values suggest that the maize-barnbara groundnut products

could meet the iron requirements of growing infants.

# Zinc Contents

The zinc content of the fortified complementary food blends followed the same

trend as iron. The zinc contents of the fortified blends were significantly (p<0.05) higher

than the control samples (unfortified blends). The increases varied from 10.57-

1 1.63 mg/100g, 10.84-1 5.14 mg/100g and 6.84-13.95 mg/100g for pre-fermentation,

post-fermentation and dry mix fortification, respectively thus indicating that fortification

with roselle calyces improved the zinc content of complementary foods.

Post-fermentation fortification gave the highest zinc content compared to pre-

fermentation and dry mix incorporation in Brachystegia eurycoma-emulsified blends. The

increases were 40.53% and 6.76% over pre-fermentation and dry mix fortified blends

respectively. The decrease in pre-fermentation fortified blends compared to the post-

fermentation could be attributed to nutrient utilization by the fermenting microorganisms

and loss of zinc through leaching while the reduction in the dry mix fortified blends could

be due to a lag in the activity of the micro-flora enzymes.

The increase in the zinc value of the samples containing red palm oil emulsified

with trona and fortified pre-fermentation over post-fermentation and dry mix blends could

be attributed to moisture loss, which concentrated the zinc. However, the values for trona

emulsified pre-fermentation fortified malt bends were lower than Brachystegia eurycoma

emulsified post-fermentation fortified malt blends with a 6.95% reduction.

All the fortified blends had zinc contents significantly (p<0.05) higher than the

Nutrend. The increase over Nutrend varied by 2.47-1 1.7mgl100g with maize-bambara

groundnut malt containing palm oil emulsified with Brachystegia eurycoma and fortified

post-fermentation having the highest increase (1 1.7mg/lOOg). The increase could be

attributed to high zinc content of roselle calyces (38.40mgllOOg) used in fortification. It

could be inferred that addition of roselle calyces post fermentation could improve the zinc

content of infant formula.

Vitamin A Content

The vitamin A content of fortified maize-bambara groundnut complementary food

blends increased by 452.52-708.2pgRE/lOOg, 559.96-8 17.2pgREIl OOg and 500.0 1 - 80 1.83pgREIlOOg for pre-fermentation, post-fermentation and dry mix incorporation

respectively. These values were significantly (p<0.05) higher than the control (unfortified

blends), indicating that addition of red palm oil to the maize-bambara groundnut blends

significantly improved the vitamin A content. Post fermentation fortified blends had the

highest increase in vitamin A content in blends containing red palm oil emulsified with * Brachystegia eurycoma. The increases varied &om 1 09- 124 pgREI100g and 1 5.37-

69.93 pgRE/lOOg above pre-fermentation and dry mix samples, respectively.

However, the dry mix fortified blends had the highest vitamin A increase in the

sample containing red palm oil emulsified with trona. The variation in vitamin A content

caused by time of incorporation could be attributed to compositional differences between

trona and Brachystegia eurycoma and their effects on palm oil carotene. The malt blends

containing red palm oil emulsified with Brachystegia eurycoma and added post

fermentation had highest vitamin A content (858.37 pgRE1100g) and could be attributed

to loss of moisture content or to greater extractability of carotenoids from the processed

samples. It could also be due to the high zinc content of the blends which could enhance

the conversion of p-carotene to vitamin A (Dijkhuizen, 2003). However, the vitamin A

values were lower than that of Nutrend. The higher value of vitamin A content of Nutrend

than fortified blends may be attributed to fortification of Nutrend with high doses of

vitamin A.

Comparing the vitamin A values of the fortified blends for pre- and post-

fermentation with values in Table 18, the vitamin A values in Table 18 were higher in

samples containing palm oil emulsified with trona and also samples containing palm oil

emulsified with Brachystegia eurycoma and fortified pre-fermentation. The increase in

values ranged from 4.56-82.9 pgRE/l OOg and 8 1.53-1 52.74 pgRE/l OOg for pre- and post-

fermentation fortification, respectively in blends containing trona emulsified palm oil and

9.25-47.83pgREllOOg for pre-fermentation fortification in samples containing

Brachystegia eurycoma emulsified palm oil. The observed higher values in Table 19,

could be attributed to the effect of zinc content on the vitamin A content as suggested by

Dij khuizen (2003). The post-fermentation fortification for sample containing palm oil

emulsified with Brachystegia eurycoma gave higher vitamin A than values in Table 18.

Effect of fortification on proximate composition of maize-bambara groundnut

complementary food

The results (Table 20) show the proximate composition of fortified maize-bambara

groundnut complementary food blends. The moisture contents of blends were low (3.80-

5.04%) compared to the unfortified blends and could be due to loss of water holding

capacity as a result of the hydrolysis of starch and protein.

Similar results were reported on moisture content of some dehydrated fermented foods

(Akpapunam and Sefa-Dedeh, 1995). The very low moisture contents suggest that 'when

properly packaged and stored even under ambient conditions, these samples would have

long shelf life.

The ash content of the fortified blends varied fiom 2.75%-3.40%, which were

43.58 to 70% higher than the unfortified blends. This might be attributed to the effect of

fortification and loss of organic matter during processing. Obizoba and Atti (1991)

observed increases in ash content in fermented unsprouted and sprouted sorghum seeds.

The higher ash content in samples containing sodium sesquicarbonate could be due to its

inorganic content.

The Eat content of the fortified blends ranged fiom 11.83-12.60% and were

significantly (p<0.05) higher than the unfortified blends. The high values could be due to

fortification with red palm oil and synthesis of lipids during malting. The high fat content

could improve vitamin A absorption fiom the food.

The crude protein contents of the fortified samples varied fiom 18.30-19.05%. The

increases were 1-3.25% above the unfortified blends and could be attributed to protein

denaturation during drying. Mild heat treatment has been reported to release bound

nutrients fiom the food matrix and binding proteins thereby increasing their

bioavailability (Gibson et al., 2000; Liener, 1976). The increase were more in samples

containing fortificants with red palm oil emulsified with Brachystegia eurycoma samples

than sodium sesquicarbonate and could be attributed to contribution fiom protein content

of Brachystegia eurycoma.

Table 20: Proximate composition of fortified blends e

Dietary blendshtage of *% Moisture *% Ash *%Crude protein *%Crude fat *%Crude fibre *%Carbohydrate

(MBm)tpo 4.50dM.01 3.4OeM.O5 1 8.6SeM. 1 1 l2.lofM.04 2.1OC*0.02 S9.3Oaa.02

(MBm)td 4.3OcM.05 3 .30dM.02 18.SodM.02 12.15%0.02 2.OSaM.02 59.7Oc*0.O5 *Results are the means of three replications; Values carrying different alphabet in the same row are significantly different (p<0.05) Key: (MB)~Maize-Brambara groundnut fermented by backslopping; (ME%& = Maize-bambara groundnut malt fermented by backslopping; (ME3)bp=Maize-bambara groundnut fortified pre-fermentation with fortificant containing Brachystegia eutycoma emulsified palm oil (ME3)bp=Maize-bambara groundnut fortified post- fermentation with fortificant wntaining Brachystegia eutycom emulsified palm oil (MB)M= Maize-bambara groundnut fermented and fortified with fortificant containing Brachystegia eutycoma emulsified palm oil as dry mix; (ME3m)b= Maize-bambara groundnut malt fortified pre-fermentation with fortificant containing Brachystegia eutycom emulsified palm oil (MB,&p= Maize- bambara groundnut malt fortified post-fermentation with fortificant containing Brachystegia eutycom emulsified palm oil (ME3Ad =Maize-bambara groundnut malt , fermented and fortified with fortificant containing Brachystegia eutycoma emulsified palm oil as dry mix; (MB)@=Maize-bambara groundnut fortified pre-fermentation with fortificants containing trona emulsified palm oil (MB&=Maize-bambara groundnut fortified post-fermentation with fortificants containing trona emulsified palm oil (M&= Maize-bambara groundnut fermented and fortified with fortificants containing trona emulsified palm oil as dry mix; (MBmb= Maize-barnbara groundnut malt fortified pre-fermentation with fortificants containing tmna emulsified palm oil (ME3&= Maize-bambara groundnut malt fortified post-fermentation with fortificants containing trona emulsified palm oil (MB,bd = Maize- bambara groundnut malt fermented and fortified with fortificants containing trona emulsified palm oil as dry mix.

The fibre content of the fortified blends ranged from 1.98-2.15% and does not reflect

any significant changes compared to unfortified blends. The low fibre content may be due to

the fact that dehulled raw materials were used in the formulation. Low fibre influences

nutrient availability positively while high fibre lowers plasma cholesterol levels (Nwokolo,

1996).

The carbohydrate content of fortified blends varied from 59.35-60.62%. The values

were 0.85-0.35% lower than unfortified blends. The decrease could be attributed to increased

degradation of gelatinized starch granules to much more soluble and easy-to-digest sugars

& caused by malting and fermentation treatments as reported by Obizoba and Egbuna (1992). g 5 The higher carbohydrate contents of samples containing red palm oil emulsified with

Brachystegia eurycoma could be due to its high carbohydrate contents. #

5. CONCLUSION AND RECOMMENDATIONS

Conclusion The findings of this investigation which showed that adequately processed cattle

bone, roselle calyces and red palm oil were used as fortificants for calcium, iron and zinc, and vitamin A respectively to develop micronutrient-rich maize-bambara groundnut complementary foods, could be summarized as follows:

1. The traditional low cost and easily available processing treatments (dehulling,

degerrning, malting and fermentation) caused significant increases in the macro-nutrients (protein, carbohydrate, fat, ash and crude fibre) and micro-

nutrients (calcium, iron, zinc and vitamin A) contents and reduction in

antinutrients (tannin, oxalate and phytate) of maize-bambara groundnut

complementary food blends and are recommended for the household preparation of these complementary foods. @

2. Red palm emulsified with Brachystegia eurycoma and incorporated post-

fermentation into maize-bambara groundnut malt blends gave the highest vitamin A contents compared to pre-fermentation incorporation. Vitamin A

activity was higher in Brachystegia eurycoma emulsified and fortified malt blends than sodium sesquicarbonate emulsified and fortified malt blends.

3. Fortification of maize-bambara groundnut complementary foods with bone meal, roselle calyces and red palm oil emulsified with Brachystegia eurycoma improved significantly the calcium, zinc and iron, and vitamin A contents of

the developed complementary foods. These could be incorporated post-

fermentation for higher nutrient retention.

4. The fortification of the product increased the macronutrient contents more

than the unfortified blends.

Recommendations 1. The use of bone meal, roselle calyces and red palm oil emulsified with

Bracychstegia eurycoma in the fortification of maize-bambara groundnut

complementary food should be encouraged because the food ingredients are

natural and have no health risk to consumers. 2. Further research should be carried out to instantize and determine the physical

and functional properties, rheological characteristics, shelf-stability and

sensory quality of the developed products.

3. Animal studies to investigate the toxicity level, if any, and micronutrient bioavailability of the fortified complementary foods should be carried out.

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APPENDIX I

Table 8: Proportion of red palm oil in the composite blends

Samples Red palm oil: composite flour Percentage composition MB 0 1 OO%MB

1 : 9.6 90.6%MBm : 9.4%~ Key: MB (control) = Maize-bambara groundnut without palm oil; MB, (control) = Maize-bambara groundnut malt without palm oil; (MB), = Maize-bambara groundnut fortified with unemulsified red palm oil; (MB,), = Maize- bambara-groundnut malt lortified with unemulsified red palm oil; (MB)w = ~aize-bamba.groundnut fortifikd with red palm oil emulsified with troua or Brachystegia eutycoma; (MBm), = Maize-bambara groundnut malt fortified with red palm oil emulsified with trona or Brachystegia eurycoma; p = red palm oil

APPENDIX I1

Table 9: Proportion of different fortificants in the complementary food

Samples Fortificants: composite flour Percentage composition (MB)f 0 100%MB : O%b : O%R : O%,

Nutrend 0 100% Nutrend 0 0 0 Key: (MB)f (control) = Unfortified Maize-bambara groundnut; MB,= Unfortified maize-bambara

groundnut malt; (MBh, = Maize-bambara groundnut fortified with fortificants containing red palm oil emulsified with Brachystegia euqvcoma; (MB&=Maize-bambara groundnut malt fortified with fortificants containing red palm oil emulsified with Brachystegia euqvcoma; (MB), = Maize-bambara groundnut fortified with fortificants containing red palm oil emulsified with trona; (MB&=Maize-bambara groundnut malt fortified with fortificants containing red palm oil emulsified with trona. Nutrend (control); p = red palm oil; R = roselle calyces; b = bone.

APPENDIX I11

Table 14: Germination time, root length, malting loss and diastatic activity of bambara groundnut malt

Germination Root length Malting loss 1 (%) Diastatic activity time (days) (cm) (OL)

0 0.00 0.000 17.235 stg 0.00 0.000 23.278 1 0.52 4.752 26.056 2 1.95 6.955 29.497 3 3.96 1 1.985 41.465 4 5.42 12.108 38.108 5 5.99 12.342 37.364 6 6.54 13.094 34.388 Key: stg = out-of-steep grains; malting loss 1= malting loss of undehulled barnbara groundnut.

#

APPENDIX IV

DATA FOR STANDARD CURVE

Concentration of iron (pglml) a Absorbance 2.5 0.3 1 5 .O 0.66 7.5 0.93 10.0 1.28

Concentration of zinc (pglml) Absorbance 1 .OO 0.06

C Concentration of vitamin A (pghl) Absorbance 3 0.04 6 0.36 9 0.60 12 0.95 15 1.21

Concentration of tannin (mdl O O d Absorbance

Concentration of phytate (mgf 100g) Absorbance 0.0 0.012 1 .O 0.025 2.0 0.040 3.0 0.082 4.0 0.095