nutritional, sensory and keeping properties of solar …
TRANSCRIPT
NUTRITIONAL, SENSORY AND KEEPING PROPERTIES OF
FERMENTED SOLAR-DRIED COWPEA LEAF VEGETABLES.
Charity Caroline Njambi Muchoki
A Thesis Submitted in Partial Fulfillment for the Degree of Master of Science in
Food Science and Technology.
Department of Food Science, Technology and Nutrition,
University of Nairobi.
2007
ii
DEDICATION
To my husband; Nicodemus N. Musembi, daughter; Melanie Mumbi
and to my parents; Rosemary Muthoni and Wilson Muchoki.
iii
DECLARATION
I, Charity Caroline Njambi Muchoki, declare that this thesis is my original work and has not
been presented for a degree in any other University.
……………………………….. ………………………
Charity C. N. Muchoki Date
This thesis has been submitted for examination with our approval as University Supervisors.
………………………………… ………………………
Mr. P. O. Lamuka Date
Dept. of Food Technology and Nutrition
……………………………….. ………………………
Prof. J. K. Imungi Date
Dept. of Food Technology and Nutrition
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ACKNOWLEDGEMENTS Many thanks to God, for His mercies and goodness thus far. His presence was real throughout
the entire venture.
My gratitude and deep appreciation go to my Supervisors, Prof. J. K. Imungi and Mr. P. O.
Lamuka for their invaluable guidance, comments, suggestions, criticisms and friendship
throughout the research and the writing of the thesis manuscript.
Deserving no less gratitude are the technicians of the Department Food Technology and
Nutrition for their technical help during the laboratory analyses. My sincere thanks go to the
technical staff of the Soil Science Department, who without their invaluable assistance, the
analyses of minerals would have been a problem. Special thanks to Catherine Njeri and Christine
Ogola for the efforts they took to ensure the laboratories remained clean.
To the MSc. Food Technology students, I acknowledge, with deep appreciation, the help you
rendered to me in one way or another during the period of my studies.
I am sincerely grateful to HELB (Higher Education Loans Board) for financing my studies and
IPGRI (International Plant Genetic Resources Institute) for financing my research work.
Special mention must be made to my parents without whose toil, devotion, sacrifice and
encouragement, I would not be what I am. I also thank my brothers B. Njigua, S. Maina and J.
Muriu and my sisters C. Wanjiku, M. Nyambura, S. Njeri, M. Wairimu, T. Njoki and J.
Waitherero, for the unknowing help and immeasurable moral support they gave me throughout
this venture.
Last but not least, I am indebted to my wonderful husband, Nicodemus N. Musembi, who stood
by me in every way and whenever I needed him. A friend indeed.
To all these wonderful people, who went out of their way for my success, I salute you. May the
God Almighty whom I serve, bless you mightily!
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TABLE OF CONTENTS
DEDICATION.............................................................................................................................. II
DECLARATION........................................................................................................................ III
ACKNOWLEDGEMENTS........................................................................................................IV
TABLE OF CONTENTS............................................................................................................. V
LIST OF TABLES .................................................................................................................... VII
LIST OF FIGURES ....................................................................................................................IX
ABSTRACT .................................................................................................................................. X
CHAPTER 1........................................................................................................................... 1
INTRODUCTION......................................................................................................................... 1
1.1. PROBLEM STATEMENT................................................................................................... 3
1.2. OVERALL OBJECTIVE ..................................................................................................... 3
1.3. SPECIFIC OBJECTIVES..................................................................................................... 4
1.4. HYPOTHESIS...................................................................................................................... 4
CHAPTER 2........................................................................................................................... 5
LITERATURE REVIEW............................................................................................................. 5
2.1. THE IMPORTANCE OF INDIGENOUS GREEN LEAFY VEGETABLES...................... 5
2.2. THE COWPEA..................................................................................................................... 9
2.3. NUTRIENTS...................................................................................................................... 11
CHAPTER 3......................................................................................................................... 32
MATERIALS AND METHODS ............................................................................................... 32
3.1. PROCUREMENT AND PREPARATION OF RAW MATERIALS…………………32
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3.2. OPTIMAL LEVELS OF SALT AND SUGAR FOR FERMENTATION .................... 32
3.4. CHEMICAL, MICROBIAL, SENSORY AND PHYSICAL ANALYSES. ................. 36
3.5. EXPERIMENTAL DESIGN AND STATISTICAL DATA ANALYSIS..................... 50
CHAPTER 4......................................................................................................................... 51
RESULTS AND DISCUSSION.................................................................................................. 51
4.1. PROXIMATE COMPOSITION OF RAW COWPEA LEAVES................................. 51
4.2. OPTIMAL LEVELS OF SALT AND SUGAR............................................................. 51
4.3. NUTRIENT LEVELS IN COWPEA LEAVES.................................................................. 55
4.4 LEVELS OF ANTI-NUTRIENTS IN COWPEA LEAVES ......................................... 58
4.5. MICROBIOLOGICAL RESULTS .................................................................................... 59
4 .6. STORAGE RESULTS....................................................................................................... 68
4.7. REHYDRATION PROPERTY.......................................................................................... 86
4.8. SENSORY EVALUATION ............................................................................................... 88
CHAPTER 5......................................................................................................................... 92
CONCLUSION AND RECOMMENDATION ........................................................................ 92
5.1. CONCLUSION .................................................................................................................. 92
5. 2. RECOMMENDATIONS ................................................................................................ 93
REFERENCES............................................................................................................................ 95
APPENDICES ........................................................................................................................... 108
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LIST OF TABLES
Table 1: Some of the commonly used Kenyan green leafy vegetables…………….……………6
Table 2: Leaves yield and nutritional values of some Kenyan indigenous and local
vegetables………………………………………………………………………………8
Table 3: Some food values of cowpea leaves compared with sweetpotato and cassava
leaves…………………………………………………………………………………..11
Table 4: Nutrient content of cowpea leaves…………………………………………………….12
Table 5: Proximate composition of raw cowpea leaves ………………………………………..51
Table 6: Moisture, dry matter and sugar levels in cowpea leaves from three sources………….53
Table 7: Mean scores for sensory attributes of fermented cowpea leaves treated with
different levels of sugars……………………………………………………………….55
Table 8: Levels of vitamins, minerals and chlorophyll of raw, fermented-, acidified-, and
control-dried cowpea leaves …………………………………………………………..56
Table 9: Recommended daily intakes by World Health Organization (WHO) for some
nutrients……………………………………………………………………………….57
Table 10: Anti-nutrients levels in raw, fermented-, acidified-, and control-dried cowpea
leaves………………………………………………………………………………….59
Table 11: Mean number of total microorganisms at different days during spontaneous
fermentation of cowpea leaves…………………………………………………………62
Table 12: Effect of fermentation and acidification on minerals and anti-nutrients during
storage……………………………………………………………………………..…..84
Table 13: Effect of storage temperature on minerals and anti-nutrients………………………..85
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Table 14: Effect of packaging material on minerals and anti-nutrients during storage…………85
Table 15: Rehydration of fermented-, acidified-, and control-dried cowpea leaves soon after
drying………………………………………………….……………………………..86
Table 16: Effect of duration on rehydration for fermented-dried stored samples using hot
water………………………………………………………………………………….87
Table 17: Effect of length of storage on rehydration for fermented-dried stored samples using
hot water ……………………………………………………………………………88
Table 18: Effect of storage temperature on rehydration for fermented-dried stored samples using
hot water……………………………………………………………………………..88
Table 16: Effect of packaging material on rehydration for fermented-dried stored samples using
hot water……………………………………………………………………………88
Table 20: Mean scores for sensory attributes for freshly processed cowpea leaves………..…...89
Table 21: Mean scores for sensory attributes after three months of storage………………….....90
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LIST OF FIGURES Figure 1: Product manufacture flow diagram………………………………………………35
Figure 2: Development of acidity during fermentation of the vegetables with varying levels
of added salt………………………………………………………………………52
Figure 3: Development of acidity during fermentation of the vegetables with 3% added salt
and varying levels of sucrose and glucose ……….……………………………….54
Figure 4 (a): Acid development during spontaneous fermentation of cowpea leaves……….60
Figure 4 (b): pH changes during spontaneous fermentation of cowpea leaves………………60
Figure 5 (a): Standard plate count during fermentation……………………………………...64
Figure 5 (b): Slime formers development……………………………………………………64
Figure 5 (c): Gram-negative bacteria development………………………………………… .66
Figure 5 (d): Lactic-acid-forming bacteria development……………………………………..66
Figure 5 (e): Yeasts and molds development…………………………………………………67
Figure 6 (a): Retention of beta-carotene in fermented-dried cowpea leaves during
storage……………………………………………………………………………71
Figure 6(b): Retention of beta-carotene in acidified-dried cowpea leaves during storage.…..72
Figure 6(c): Retention of beta-carotene in control-dried cowpea leaves during storage……..73
Figure 7(a): Retention of ascorbic acid in fermented-dried cowpea leaves during storag…....76
Figure 7(b): Retention of ascorbic acid in acidified-dried cowpea leaves during storage……77
Figure 7(c): Retention of ascorbic acid in control-dried cowpea leaves during storage……...78
Figure 8 (a): Retention of chlorophyll in fermented-dried cowpea leaves during storage……81
Figure 8(b): Retention of chlorophyll in acidified-dried cowpea leaves during storage….…..82
Figure 8(c): Retention of chlorophyll in control-dried cowpea leaves during storage………..83
x
ABSTRACT
This study was conducted to determine the effect of fermentation, solar drying and packaging on
the nutritional, sensory and keeping properties of cowpea leaf vegetables. The cowpea leaves
were purchased from the local markets, sorted to remove the blemished, leaves and foreign
materials, washed in running tap water then drained. The vegetables were divided into three
batches of 16kg. One batch was blanched in hot water, salted and solar-dried. The second portion
was blanched, salted, acidified to a pH of 3.8. and then solar dried. The third portion was salted
and addedd sugar. It was then fermented for 21 days, heat-treated in its own liquor, and solar
dried. The three batches of vegetables were spread at different times on drying trays at the rate of
4kg/m2 and dried in a solar drier to approximate moisture content of 10%. The dried vegetables
were packaged in either polyethylene bags or kraft paper bags and stored for three months at
18oC, 22o-26oC or 32oC.
Fermentation-solar-drying of vegetables retained substantial levels of nutrients; beta-carotene
(88%), ascorbic acid (15%), calcium (70%), iron (73%) and chlorophyll (68%). There was also
substantial reductions in the antinutrients; nitrates (72%), oxalates (11%) and total phenolics
(28%). Storage of the treated vegetables led to loss in beta-carotene, ascorbic acid and
chlorophyll. The retention of beta-carotene, ascorbic acid and chlorophyll at the end of storage
ranged from 23 to 52%; 4 to 7% and 12 to 23%, respectively, depending on storage conditions.
Samples stored at 32oC had the highest losses, while those stored at 18oC had the lowest.
Samples stored in kraft paper bags had the highest losses in beta-carotene and ascorbic acid,
while those packaged in polythene bags had the highest losses in chlorophyll. Rehydration in hot
water was highly significant (P<0.001) compared to that in cold water and there was significant
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difference (P<0.05) in durations of rehyration. The rehydration of fermented-solar-dried sample
was significantly higher (P<0.05) than for acidified- and control-solar-dried samples. Storage for
three months did not have significant effect on the sensory attributes of the dried vegetables.
Fermentation and solar drying can be used by the local communities for preservation of these
vegetables, as it is of low cost and effective. Increased acceptability of the fermented-solar-dried
vegetables should be promoted among the rural communities, where the deficiency of vitamin A
and iron is likely to be rampant during the period of drought.
1
CHAPTER 1
INTRODUCTION
Malnutrition due to nutritionally inadequate diets is one of the major concerns in Kenya and
many other developing countries (Imungi, 1984). The prevalence rates of micronutrient
malnutrition remain high, with devastating consequences for health and productivity
(Underwood, 2000). Among the micronutrient deficiencies, the most important are those of
vitamin A, iodine, iron and zinc. Vitamin A deficiency (VAD) generally causes retardation of
growth and development and increases the risk of death due to childhood diseases. It has also
profound effects on sight (UNICEF/ GoK, 1998). In a micronutrient survey done in 14 districts
of Kenya, moderate VAD was found in 3 while severe VAD was found in 11 of the districts. The
study showed that the children aged 6 - 24 months are at the greatest risk of this deficiency
(Ngare et al., 2000). The incidence of anaemia varies greatly throughout Kenya, but the Coastal
areas are the most severely affected, where according to criteria established by the World Health
Organization (WHO), 90% of the population are affected. The other region with high levels of
anaemia is around Lake Victoria, where 25 - 35% of the population is believed to be anaemic
probably because of malaria and intestinal worms (UNICEF/ GoK, 1992). In infants and
children, nutritional anaemia can impair learning and lower resistance to disease, while in adults;
it causes weakness and decreases the capacity to work.
In Africa, people have always depended on traditional leafy vegetables to meet their nutritional
needs. The vegetables are compatible with starchy staples main meal and represent cheap but
quality nutrition for large segments of the populations in both urban and rural areas. The
vegetables are rich in vitamins especially A, B, and C, and minerals such as iron, zinc, calcium
2
and phosphorus (Mnzava, 1997). The vegetables, being accessible to the low-income
communities, offer opportunity for improving nutritional status of many poor families whose
health and nutrition are at risk (Chweya and Eyzaguirre, 1999). Their contribution to fibre,
carbohydrates and proteins in diets can also not be ignored. The main problem with traditional
vegetables is the lack of availability due to seasonality. However, in areas where seasonality is a
critical factor in limiting availability, promotion of home gardening and appropriate local
preservation technology can improve availability (Nest and Sommer, 1993).
The cowpea (Vigna unguiculata or Vigna sinensis) is one of the most important legumes in
Kenya. It is cultivated all over Kenya mainly for seeds but the leaves are popular as local
vegetable. In some parts of the country, however, some types are grown primarily for leaves. For
vegetables, the leaves are harvested starting at 3 - 4 weeks from planting (Maundu et al., 1999).
The young leaves are often cooked alone or in a mixture with other leafy vegetables to prepare a
side dish for ugali (paste made of maize meal and water). In some communities, it is cooked with
maize, pulse and potatoes and/or bananas and mashed to make a tasty mixture.
Little information on processing of cowpea leaves has been documented. Therefore, the
vegetables are only seasonally available (Imungi, 1984). Processing the leafy vegetables like
cowpea for preservation would ensure year-round supply of the vegetables. Foods have from
antiquity been fermented to improve palatability, taste, aroma and texture, extend the keeping
quality, increase nutritional value and improve level of safety. Fermentation of indigenous foods
is considered to be an effective, inexpensive and nutritionally beneficial household technology,
especially in the developing world where most vegetables have been preserved by fermentation.
3
Comprehensive reviews are available on the fermentation of sauerkraut, olives and cucumbers
(Carr et al., 1975). Other vegetables and fruits that have been fermented include, beets, turnips,
radishes, chard, Brussels sprouts, mustard leaves, lettuce, fresh peas and vegetable blends
(Pederson, 1971) and kales (Mutegi, 2002). Sun drying is the oldest method of drying food and
its cost is low. Drying has been a means of preserving food from earliest times (Mehas and
Rodgers, 1989). The primary objective in drying any food is to reduce its weight, hence bulk,
leading to cost effective transport, handling and distribution. The other objective is to improve its
keeping quality by reducing the moisture level, which reduces microbial attack (Kordylas, 1990).
The sun’s ultraviolet rays have also been reported to inhibit the growth of microorganisms
(Mehas and Rodgers, 1989).
1.1. PROBLEM STATEMENT
The problems that prompted this research include:
1. The seasonality of the cowpea leaf vegetable, which is only available during the rainy
season.
2. Lack of food security, which involves lack of vegetables during the dry season and
lack of post harvest processing.
3. Malnutrition, especially that of micronutrient such as anaemia and vitamin A
deficiency.
1.2. OVERALL OBJECTIVE
To study the effect of fermentation, solar drying and packaging materials on the nutritional,
sensory and keeping properties of cowpea leaf vegetables.
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1.3. SPECIFIC OBJECTIVES
1. Determination of the proximate composition, vitamins A and C, minerals (iron and calcium)
and anti-nutrients (nitrates, oxalates and phenolic compounds) of raw cowpea leaves.
2. Determination of levels of vitamins A and C, minerals (iron and calcium) and anti-nutrients
(nitrates, oxalates and phenolic compounds) in fermented and solar dried and stored cowpea
leaves.
3. Determination of microorganisms involved in the fermentation of cowpea leaves.
4. Production of fermented and solar dried cowpea leaf vegetables.
5. Assessment of the rehydration properties of fermented and solar dried cowpea leaves.
6. Determination of the sensory characteristics and acceptability of fermented, solar dried and
stored cowpea leaves.
7. Determination of the keeping quality of fermented, solar dried and packaged cowpea leaves.
1.4. HYPOTHESIS
The null hypothesis (Ho): The nutritional, sensory and keeping quality of fermented-solar-dried
cowpea leaves does not differ significantly from that of acidified- and control-solar-dried cowpea
leaves.
Alternative hypothesis (H1): The nutritional, sensory and keeping quality of fermented-solar-
dried cowpea leaves differs significantly from that of acidified- and control-solar-dried cowpea
leaves.
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CHAPTER 2
LITERATURE REVIEW
2.1. THE IMPORTANCE OF INDIGENOUS GREEN LEAFY VEGETABLES
Green leafy vegetables are an important source of food. They have been grown for their foliage
from ancient times and are considered excellent sources of the essential nutrients. In recent years,
nutritionists have strongly emphasized their use in the human diet due to their health-giving
qualities (Salunkhe and Desai, 1984). The green leafy vegetables play an important role in the
human diet as the vital sources of protein, fiber, minerals and vitamins.
In Kenya, indigenous green leafy vegetables form a substantial proportion of the diets of most
low and middle income Kenyans (Onyango et al., 2000). These vegetables have been recognized
as inexpensive and easily accessible sources of food and essential micronutrients (Mwajumwa et
al., 1991). A variety of these vegetables are gathered in the wild and some like Amaranthus
graecizans (michicha), Corchorus trilocularis (murere), Asystasia schimperi (atipa), Digera
muricota and Coccinia grandis are found as weeds in cultivated fields. Others like Gynandropsis
gynandra (chinsaga), Solanum nigrum (rinagu), Vigna unguiculata (kunde) and Amaranthus
species are now considered as commercial crops and are cultivated for sale (Onyango et al.,
2000).
Traditional vegetables have, however, been given little attention by the policy makers,
researchers, extension workers and farmers. Some reasons for the negligence include myths and
beliefs that the vegetables are nutritionally inferior, difficult to prepare, cannot be domesticated
and that some types are poisonous. Hence popularization of these vegetables has been
6
Table 1: Some of the commonly used Kenyan green leafy vegetables (scientific, common and local name)
Scientific name Common name Local name Gynandropsis gynandraa Spider flower Chinsaga5, Akeyo1, Tsisaka,
Mgangani 8 Crotalaria brevidensa Sunhemp Mito1, Miro2, Crotalaria ochroleucaa Giant sunhemp Mito1, Miro2 Corchorus olitoriusa Jute vegetable Murere2, Apoth1, Mlenda 3 Corchorus trilocularisc Jute vegetable Murere2, Apoth1, Mlenda 3 Amaranthus spp(oleraceaeaa, lividusa hybridusa, graecizansc
African spinach Michicha3, Livokoi2, Dodo5
Solanum nigrumb Black nightshade Mnavu3, Lisutsa2, Rinagu5, Managu4, Ndunda8
Asystasia schimperib Atipa1 Vigna unguiculataa Cowpea Kunde3, Egesare5, Boo1, Ngunyi6 Cucurbita sppa Pumpkin leaves Susa1, Lisebebe2, Risosa5,
Marenge4, Nenge6 Basella albab Indian spinach Nderema2, Enderema Brassica intergrofolliab Ethiopian kale Khajira3 Phaseolus vulgarisa Bean leaves Mboso6, Rikuneni5 Solanum tuberosuma Irish potato Waru4, Maluu6 Ipomea batatasa Sweetpotato Makwasi6 Colocasia esculentaa Arrow root (Taro) Nduma3, Maguru 7 Manihot esculentaa Cassava Muhogo1, Manga6,8 Commelina africanac Wondering jew Odielo1, Linyoronyoro2, Itula6 Oxygonum sinuatumc - Awayo1 Lagenaria vulgarisc - Agwata1 Portulaca quadrifidac - Obwanda1 Bidens pilosac Black jack Anyiego1 Thunbergia alatac - Nyawend agwata1 Sonchus schiveinfurthic - Achak1, Mnyinya8 Tribulus terrestrisc - Okuro1 Symphytum peregrinuma Russian comfrey Mabaki4,7 Stinging nettle Thabai4 a - fully domesticated, b - partially domesticated, c - wild and/or weeds 1 Luo, 2 Luhyia, 3 Kiswahili, 4 Kikuyu, 5 Kisii, 6 Kamba, 7Meru 8 Taita Source : Onyango et al., 2000
7
met with obstacles (Opole et al., 1991). The importation of exotic agro-technology for producing
exotic vegetables, colonial legacy of plantation farms and food habits are some of the obstacles.
Other limitations include introduction of commercial food production systems and the
Government policy of placing greater emphasis on exotic vegetable because they generate
income and foreign exchange; and the notion that consumption of exotic vegetables enhances
social status in society. Indigenous green leafy vegetables, however, have several advantages
over their exotic counterparts: i.e. adapted to local climatic conditions; more resistant to pests
and disease; can be intercropped easily since they are found growing in the wild as weed; some
have medicinal properties; have high yield potential and have high nutritional value (Onyango et
al., 2000). The number of indigenous green leafy vegetables present in Kenya is enormous
(Table 1). At least 200 varieties have been documented but only 20 have been recognized
(Mandu and Kabuye, 1993). Many of these are either gathered from the wild or cultivated, and
consumed by most rural people (Mwajumwa et al., 1991). A wide variety of these vegetables are
available in all parts of Kenya, with more consumption being concentrated in the Coast, Nyanza
and Western Provinces.
2.1.1. Nutritional Value and Utilization
Studies on the nutritional importance of various indigenous green leafy vegetables in Kenya and
elsewhere have been done (Table 2). Some of the indigenous vegetables have been known to
have medicinal value. A survey done by Opole et al. (1991), showed that Gynandropsis
gynandra leaves extracts can cure scurvy, stomach ache, stomach upset, ear ache, and for the
treatment of conjunctivitis and severe infection of thread-worms. Solanum nigrum leaves may be
8
Table 2: Leaves yield and nutritional values of some Kenyan indigenous and local vegetables,
per 100g fresh edible portion.
Scientific name
Yield ton ha-1
Crude protein (g)
ß-Carotene (mg)
Vitamin C (mg)
Calcium (mg)
Iron (mg)
Dry matter (g)
Vigna unguiculata 5.4 -10 - 6 - 8 70 -100 200- 400 10 -15 15 - 20
Solanum nigrum 30 - 80 3 - 6 8 -10 40 -140 250 5 -17 18 - 22
Gynandropsis gynandra 10 -13 5 -10 6 -19 130 -180 434 11-15 15 - 20
Corchorus olitorus 7 -11 8 4 - 8 170- 210 270 8 20 - 23
Amaranthus 45 4 -5 5 -10 90 -160 800 5 -15 11-15
Crotalaria brevidens 36 4 -5 3 - 9 110 -130 270 4 -
Basella alba 50 5 4 100 250 4 15
Cucurbita - 3 - 5 2 - 6 170 -175 400 9 -11 20 – 25
Brassica oleraceae var.Acephala
20 5 2 - 6 100 250 4 15
Brassica oleraceae var. Capitata
20 1.4 -1.5 0.02-0.06 40 40 - 55 0.5- 0.8 7 - 8.2
Lactuca sativa 10 1.4 0.2-0.6 15 35 1 6
Source: Onyango et al., 2000
boiled and the extract used to relieve abdominal pains, relax uterus and boost health of expectant
mothers, relieve muscular pains in old people. Solanum nigrum is also known to treat burns and
scalds (Kokwaro, 1976; Opole et al., 1991). Crotalaria spp extract can facilitate easy and quick
delivery during childbirth (Opole et al., 1991). Thunbergia alata's leaves and buds when
pounded and mixed with ghee are used for treatment of backache and pains of joints. Bidens
pilosa extract cures diarrhoea in suckling babies (Kokwaro, 1976).
9
2.2. THE COWPEA
The cowpea (or Southern Pea), Vigna sinensis or Vigna unguiculata (L.) is in the family
Leguminosae. The cowpea varieties include the black-eyed pea, cream pea, crowder peas, purple
hull peas, and some other less common types. It probably originated in Central Africa where it is
seen growing wildly. It is an erect, trailing or climbing herb. Has three leaflets of 10 cm long or
more, ovate, rhomboid or lanceolate, entire or lobed at the base. The flowers are of various
colours; pale green to light blue or purple, borne on auxiliary inflorescences composed of a long
stalk, usually held vertically and with several flowers towards the tip. The pods are up to 15 cm
long, straight and usually hanging. Morse (1920) recognized three broad groups of cultivated
cowpea,
i.e. Asparagus bean (Vigna sinensis, type sesquipedalis), Catjang (V.sinensis, type cylindrica)
and Southern pea (V. unguiculata L.). It is an important pulse crop in the tropics and cultivated
throughout Africa and Southern Asia. Cowpea is also grown on a commercial scale in the
southeastern states of the U.S., especially in Texas, Georgia and Alabama. It is one of the
important pulse crops of India. As reported by Maundu et al., (1999), the cowpea is cultivated all
over Kenya as a vegetable and pulse. It is grown mainly from 0 - 1500 metres above sea level,
however, growth is poor at higher altitudes. It requires hot, moderately wet conditions; loam,
sandy and well-drained soils. It grows well in semi-humid to arid agro-climatic zones. Cowpea is
tolerant to drought, low soil fertility and acidity. It is known by various local names depending
on community. Several cultivars differ in seed colour, pod shape and length, habit (creeping or
erect), and leaf shape and size. Fast maturing, erect cultivars are grown for seeds. The creeping,
deeply rooted types are drought resistant and preferred for their leaves among the Kamba,
Tharaka, Mbeere and Meru communities. According to Verdcourt, in the Flora of East Africa,
10
five sub-species. i.e. Dekndtiana (Harms) Verdc., Unguiculata, Cylindrica (L.) Eselt., Mensensis
(Schweinf) Verdc., and Sesquipedalis (L. ) Verdc have been recognized in Kenya.
Although the cowpea is widely grown in Africa for seed, the leaves are also used extensively as a
vegetable. They are incorporated into a variety of dishes, soups and sauces. Young leaves and
shoot tips are harvested and cooked while green or dried for storage. Field harvest strategies
include harvest of the entire vegetative plant before flowering or partial defoliation and later the
seeds are harvested. In some parts of Africa, this removal may promote setting. It has been
estimated that up to two tons/ha. of leaves can be removed this way without adversely affecting
the yield of the seed (Imungi and Potter, 1983).
Utilization of cowpea as a leafy vegetable and a seed crop provides nutritional versatility not
available with purely vegetative crops like lettuce or monocarpic crops like wheat (Bubenheim
and Mitchell, 1987; 1988). The nutrient composition of cowpea foliage is more desirable than
that of many vegetative crops (Table 3), hence cowpea could provide dietary versatility by
utilization of either foliage or seeds.
Controlled Ecological Life Support Systems (CELSS) tried to develop a purely vegetative form
of cowpea as the yield efficiency is suppressed by the combination of leaf and seed harvest.
Yield efficiency was greatest for the vegetative harvest strategy, but the bioavailabilty of
nutrients from foliage must be determined before potential use of this strategy can be adequately
evaluated. Both leaves and seeds of cowpea appear to provide a low-fat high-protein food choice
(Bubenheim et al., 1990). Work done by CELSS found that the leaf carbohydrates content
increased with leaf age, but was greatest in the seed. Protein content of older leaves was similar
11
to that of seeds; however, protein content of young leaves was highest. Fat content was higher in
leaf tissue than in seeds and was not affected by leaf age. Mineral (ash) content of cowpea
foliage was much higher than for seeds regardless of the leaf age (Bubenheim et al., 1990).
Table 3: Some food values of cowpea leaves (per 100g of edible leaves) compared with
sweetpotato and cassava leaves.
Cowpea Sweetpotato Cassava
Dry matter (g) 11.6 13.3 19.0
Calories (Kcal) 34.0 42.0 60.0
Protein (g) 4.2 3.2 6.9
Fiber (g) 1.7 1.6 2.1
Calcium (mg) 110.0 85.0 145.0
Iron (mg) 4.7 4.5 2.8
Carotene (mg) 2.4 2.7 8.3
Ascorbic acid (mg) 35.0 20.0 80.0
Source: Bubenheim et al., (1990)
2.3. NUTRIENTS
The cowpea leaves are a good source of minerals and vitamins (Table 4). Minerals whose levels
have been reported to be high in cowpea leaves include potassium, calcium, magnesium, iron
and manganese (Imungi and Potter, 1983).
2.3.1. Minerals
2.3.1.1. Iron
Iron is a component of human blood haemoglobin and myoglobin (Imungi and Potter, 1983). It is
also a constituent of enzymes involved in energy metabolism. Iron deficiency has been shown to
be the most common cause of anaemia in the world. Anaemia is a major health problem in
12
Table 4: Nutrient content of cowpea leaves (per 100g of edible leaves).
Leaves Fresh Dried
Energy (KJ) 163 950
Water % 86 10.6
Protein (g) 4.5 22.6
Fibre (g) 1.8 -
Ash (g) 2.0 -
Fat (g) 0.3 3.2
Total CHO (g) 4.3 -
Vitamin C (mg) 76 35
β-carotene Eq.(µg) 5185 -
Calcium (mg) 183 1556
Iron (mg) 4.7 -
Phosphorus (mg) 63 348
Source: Maundu et al., 1999).
Africa, particularly for women. Negative iron balance leads to a reduction in the iron content of
all functional components, which results in anaemia. When iron availability to support metabolic
systems in the tissues is reduced, the main physiological consequences are impaired oxygen
delivery and reduced metabolic rate. Anaemia caused by tissue iron deficiency per se results in
mucosal and epithelial abnormalities, deterioration of immunity leading to easy infection,
skeletal muscle dysfunction and behavioral and neurological abnormalities (Sean, 1993).
The main problem with iron metabolism is that, not all of it in the food is physilogically
available. Bioavailability is a term used to describe the proportion of a nutrient that is absorbable
and utilizable by the consumer (Macrae et al., 1993). There are two types of iron in food: - non-
13
haem iron, which is present in both plant foods and animal tissues, and haem iron, from
haemoglobin and myoglobin in animal products. The chemical form of existence in fact is
important in absorption i.e. ferrous salts are more readily absorbed than ferric salts. The
availability from food is influenced by: - the size of the elemental iron particle, type of food
source and other dietary factors. It has been established that iron availability is highest in diets
rich in animal products than plant products (Baker and DeMaeyer, 1979). Addition of meat or
ascorbic acid to a vegetable diet enhances iron absorption (Monsen et al., 1978). On the other
hand, the presence of calcium, phosphate, high roughage content and oxalate may negatively
influence iron absorption (Imungi, 1984). Other dietary constituents, like protein, amino acids,
and carbohydrates, have been suggested as having an effect on iron availability.
The main factors that influence the bioavailability of iron from the diet are the amounts of haem
and non-haem iron, the presence and amounts of dietary factors influencing iron absorption and
the iron status of the individual. It has been shown that increased absorption occur in iron-
depleted subjects or in patients with iron deficiency anaemia. There is evidence indicating that
iron absorption may be greater in women than in men and that iron absorption decreases with
increasing age in children. Good sources of iron include eggs, lean meats, legumes, whole grains
and green leafy vegetables. The amount of total iron available in cowpea leaves is 28.6-
38.8mg/100g solids (Imungi and Potter, 1983).
2.3.1.2. Calcium
Calcium is the most abundant cation in the body. This abundance generally reflects the presence
of hard tissues containing an inorganic matrix, impregnated with salts in which calcium is the
14
major cation. Calcium is usually found in combination with carbonates or phosphates as a
crystalline extracellular deposit. It has a major structural function in skeletons and teeth (Imungi,
1984; Belitz, 1987). i.e. is required in humans for bone and teeth formation, clotting of blood,
enzyme functions, cell membrane integrity, nerve transmission and cellular metabolic controls.
Good sources of calcium include milk, cheese, dark green leaves and dried legumes.
Deficiency of calcium will result in offset in the balance of all processes requiring calcium
(enzyme activation). This can lead to dental caries, stunted growth, rickets, osteoporosis and
convulsions. (Walker, 1972; Imungi and Potter, 1983). The calcium/phosphorus (Ca/P) ratio is
essential for calcium fixation in the human body; the ratio is 0.7 for adults and 1.0 for children
(FAO, 1995). Gomez (1982) reported that the calcium contents of some Kenyan vegetables,
ranged from 55 to 618 mg/100g of edible portion. The amount of calcium in raw cowpea leaves
was reported to be 1520 - 1750mg/100g solids (Imungi and Potter, 1983).
Calcium bioavailability from vegetables is dependant on the specific vegetable itself and vitamin
D is required for efficient calcium absorption (Hegsted, 1973). Oxalic acid is the main
constituent in some foods that limits calcium utilization. However, this is not of practical
importance where the amount of calcium consumed is sufficiently liberal (Imungi, 1984).
2.3.2. Vitamins
2.3.2.1. Vitamin A
In plants, the precursors of vitamin A occurs as very closely related yellow-orange coloured
carotenoid (Belitz, 1987). Beta-carotene is the most effective precursor (Wu and Salunkhe,
15
1974). Caroteinoids are also potent anti-oxidants, scavenging potentially harmful oxy-radicals,
commonly associated with inducement of certain cancers (Gareth et al., 1998). Vitamin A
(retinol) is a constituent of rhodopsin (visual pigment), which is necessary in the maintenance of
epithelial tissues. It also plays an important role in mucopolysaccharide synthesis.
Deficiency of vitamin A is among the most widely spread and serious nutritional disorders that
affect mankind, especially in developing countries (Imungi, 1984; Ricardo, 1993). Deficiency.
can lead to dry-eye disease (xerophthalmia), night-blindness and eventually complete blindness
In the young, it also leads to failure of normal bone and tooth development and diseases of the
epithelial cells and membranes of the nose, throat and eyes. It decreases body’s resistance to
infection (Imungi, 1984; Belitz; 1987; FAO, 1995). Children who are deficient in vitamin A are
more likely to die from infectious diseases than healthy children.
Yellow coloured fruits, carrots, and dark green leaves are good sources of pro-vitamin A (beta-
carotene). Retinol is also present in milk, butter, cheese and fortified margarine. Fats, protein and
zinc are essential for absorption and use of vitamin A, thus a diet low in these nutrients can
contribute to vitamin A deficiency. Total carotene content in raw cowpea leaves has been
reported to be 57mg/100g solids (Imungi and Potter, 1983).
Beta-carotene is fat-soluble and therefore it is not affected by the washing and blanching steps,
but is moderately destroyed (5 to 40%) by retorting during canning (Belitz, 1987). According to
Lee et al. (1982) carotene appeared to be relatively unaffected by heat processing. The beta-
carotene content in blanched and canned samples was found to be slightly higher than in raw
16
samples (Lee et al., 1982). It is well documented that dehydration of leafy vegetables results in
substantial destruction of their carotene contents (Gomez, 1981; Maeda and Salunkhe, 1981).
2.3.2.2. Vitamin C
Vitamin C includes both ascorbic and dehydroascorbic acids, as both forms are biologically
active. In fresh foods, the reduced form (ascorbic) is the major one present, but the amount of the
dehydro-form increases during cooking and processing. Vitamin C is used in maintenance of
inter-cellular matrix of cartilage, bone and teeth. It is also important in collagen synthesis, which
is an important constituent of skin and connective tissue. Vitamin C enhances the absorption of
iron (FAO, 1995). Its deficiency leads to scurvy, degeneration of skin, teeth, blood vessels, and
epithelial hemorrhages.
Fruits and vegetables are excellent sources of vitamin C (FAO, 1995). The ascorbic acid contents
of vegetables vary considerably even within the same species due to variations in season,
climate, agronomical and soil conditions (Imungi, 1984; Ricardo, 1993). Content of ascorbic acid
in raw cowpea leaves has been reported to be 410mg/100 solids (Imungi and Potter, 1983).
Ascorbic acid, is easily destroyed in foods during processing or preparation depending on a large
number of factors such as processing temperature and conditions, and equilibrium relative
humidity, oxygen partial pressure, light, catalysts, package permeability and package
configuration in storage (Labuza, 1972; Ana and Lia, 1997). It is easily lost during processing,
cooking and storage (FAO, 1995). Sometimes, vitamin C losses are due to leaching into the
cooking or processing water, and due to both enzymatic and chemical degradation especially in
17
the presence of traces of heavy metal ions (Belitz, 1987). Conservative processing and low
temperature storage are critical for vitamin C preservation (Barth et al., 1990).
2.4. ANTI–NUTRIENTS IN FOODS
These are also referred to as nutritional stress factors. They are defined as unfavorable factors
that influence nutritional requirements by interfering with the transfer of nutrients from the
environment to the cell of the human or animal organism. They act specifically and exert their
effects by increasing the loss of essential nutrients from the body, interfering with the
metabolism of absorbed essential nutrients, decreasing the digestion of food, or decreasing food
intake (Teutonico and Knorr, 1984).
There is a wide diversity of toxic constituents in plants (Imungi, 1990). These toxicants may
occur either naturally, or as contaminants through environmental pollution (Flegal, 1993). While
most plant species contain low levels of naturally occurring toxicants (Coon, 1973), chronic
ingestion of these compounds may present a potential health hazard. Consequently, reduction of
nutritional stress factors in plant foods increases the availability of nutrients in the plant and thus
improves its quality as a foodstuff, as well as the overall agricultural productivity (Teutonico and
Knorr, 1984). Amongst the most commonly occuring antinutrients in plant foods include nitrates
and nitrites, phenolic compounds, cyanogenic glycosides, glucosinolates, oxalates and saponins.
Many of the toxic constituents of plant products can be destroyed by heat treatment, soaking in
water or fermentation.
18
2.4.1. Nitrates and Nitrites
Nitrate salts occur naturally in many foods. Their occurrence is a consequence of the nitrogen
cycle. Nitrates are natural constituents of many soils and are also found in water and most
growing plants (Mohri, 1993). The accumulation of high amounts of nitrate in plant tissues
grown on heavily fertilized soils is of concern, particularly in infant foods prepared from plant
tissues.
Leafy vegetables are the main contributors of nitrates in diets, accounting for more than 75% of
the total amount ingested (Imungi, 1984, 1990; Michael, 1990; Mohri, 1993). The vegetables that
contain relatively high levels of nitrate and small quantities of nitrites are cabbage, spinach,
celery, lettuce and several root vegetables (e.g. carrot, beets). Increased nitrate and nitrite levels
in these vegetables usually results from the use of nitrogenous fertilizers (Mohri, 1993; Ricardo,
1993). Extreme weather conditions such as prolonged cloudiness or drought can influence the
nitrate level (Graham, 1983). The nitrate contents of plant tissues vary considerably within
varieties (Imungi, 1990) due to differences in season, growth location and age of the plant (Dhan
and Pal, 1991).
The intake of vegetables that have accumulated nitrates may be harmful to humans, especially
when there is gastro-intestinal disturbance (Mengel, 1979). Toxicity to human is due to nitrites
that arise from microbial reduction of nitrates within the gastro-intestinal tract. This reduction is
more likely in infants than in adults, due to low acidity in their digestive tract, which allows the
survival of coliforms and clostridial bacteria (Maynard et al., 1976). Acceptable Daily Intakes
(ADI) according to WHO for nitrate, and nitrite is 220mg and 8mg, respectively, for a person of
19
60kg body weight. Nevertheless, these levels are not applicable to infants under 6 months of age,
who are susceptible to methaemoglobinaemia (Mohri, 1993).
Nitrates per se are not toxic at the levels normally present in food. The toxic chemical form is
nitrite. The toxicity of ingested nitrate is due to its in vivo reduction to nitrite (Mohri, 1993).
Nitrites oxidize ferrous iron in haemoglobin to the ferric form (methemoglobin), which cannot
transport oxygen (Maynard et al., 1976), leading to fatal methaemoglobinaemia.
Methaemoglobinaemia is the most prevalent and potentially the most serious complication,
caused by nitrate and nitrite exposure. Methaemoglobinaemia is characterised by cyanosis (a
bluish-purple color of the skin and lips), stupor and cerebral anoxia. This occurs when 15%
hemoglobin is oxidized. Oxidation of 70% to methemoglobin or more in human blood is fatal
(Lee, 1970). The reported fatal doses for adults as a single dose of 30 to 35g of nitrate or 20mg
of nitrite per kilogram of body weight (Lee, 1970; Imungi 1990).
Nitrates and nitrosamines are linked to various types of cancer (Cuzick and Babiker, 1989). The
major concern is that the nitrogenous compounds can produce N-nitroso compounds,
endogenously and exogenously, by reaction with amines and amides in foods, and that the N-
nitroso compounds may cause human cancer. However, there is no evidence to conclude that
orally ingested nitrate or nitrite is carcinogenic, teratogenic or has other adverse reproductive
effects in species examined, although nitrite can cross the mammalian placenta and induce fetal
methemoglobin production after maternal ingestion (Macrae et al., 1993).
20
Nitrates and nitrites are water-soluble and therefore some degree of leaching is possible during
washing or processing in water. Most of the nitrites present are oxidised to nitrate, and upon
cooking, they leach out of the vegetable (Ricardo, 1993). The degree of leaching depends on
water temperature, surface area of the vegetable and ratio of vegetable to water (Varoquax et al.,
1986).
2.4.2.Oxalates
Oxalates occur naturally in plant materials at relatively high levels, mainly as soluble sodium and
potassium salt or as insoluble calcium oxalate (FAO, 1988; Lathika et al., 1995). High
concentrations of oxalate may be of great nutritional disadvantage to both man and animals.
Oxalic acid is a plant toxicant, which forms an insoluble salt with the essential nutrient calcium
(Hodgkinson, 1977), thus inhibiting its absorption. It also inhibits the absorption of iron
(Bothwell and Charlton, 1982) and, to some extent, zinc (Hughey, 1983). This is manifested as
calcium deficiency even in diets with adequate levels of calcium. This is more significant in
growing children than adults, because of the developing bones and teeth (Imungi, 1990).
Oxalic acid is widely distributed in nature, with appreciable quantities (on dry weight basis)
being found in rhubarb (7.8%), amaranth (7.2%), spinach (5.6%) and swiss chard (5.5%) (der
Marderosian et al., 1980). Variation in oxalate contents of plants can occur depending on season,
species, variety, age, part of the plant and soil condition during growth (Kasidas and Rose, 1982;
Gad et al., 1982). For populations whose diets are rich in these vegetables, oxalate may lead to
loss of bone minerals, particularly if the diet is deficient in calcium or vitamin D (Hodgkinson,
1977). Oxalates possibly block renal function by precipitation of insoluble oxalate (oxalate
21
crystals), which is the major component (two-thirds) of all kidney stones. Death has been
reported from this condition (Graham, 1983; Armesto et al., 1989).
Toxic levels of oxalates in humans have been indicated to be 2 to 5g equivalent of oxalic acid per
day for population consuming low levels of calcium (Ricardo, 1993). Graham (1983) reported
that oxalic acid of 5g or more can be fatal in man, causing corrosive gastroenteritis, shock,
convulsion and renal damage. Oxalate poisoning can be aggravated when the calcium intake is
low. Oxalate toxicity has also been reported in animals fed on vegetable amaranth (Lemos et al.,
1993; Kommers et al., 1996; Torres et al., 1997).
The most commonly held view on oxalic acid metabolism is that:
(1) Oxalic acid is formed in the plant directly from oxaloacetate, ascorbate, or glyoxylate
(Chang and Beevers, 1968) to restore the cation-anion balance disturbed by the assimilation
of nitrate to organic nitrogen (Joy, 1964);
(2) The use of nitrate as the nitrogen source in plant, produces higher oxalate content than when
ammonium is supplied (Joy, 1964). Teutonico and Knorr (1984), however, found that the
level of oxalate accumulation was similar whether ammonium, urea, or nitrate was the sole
source of nitrogen. Therefore, they concluded that anions other than nitrate, such as
phosphate (Dijkshoorn, 1962), must be responsible for the cation excess that prompts the
synthesis of oxalate. It is likely that the factors controlling acid synthesis in relation to cation
excess are related to the accumulation of oxalate and cations to the vacuole of leaf cells
(Hughes et al., 1979).
22
Except for leaching, oxalic acid is minimally changed during processing and preparation of
food. However, it might leach out into the cooking water bound to calcium, which severely
reduces the levels of the element in the cooked materials if the cooking water has to be
discarded. This, however, removes most of the soluble oxalate (Gad et al., 1982; Samuel, 1985;
Imungi, 1990).
2.4.3. Phenolic compounds
The name 'phenol' refers to the monohydroxy derivative of benzene, but it is applied generally to
all derivatives of benzene and its related compounds with a nuclear hydroxy groups. Natural
phenols in foods encompass a diverse group of compounds, including simple phenols
(derivatives of benzoic and cinnamic acids) and polyphenols. Phenolic compounds are
synthesized by plants as secondary metabolites as they are not directly involved in metabolic
pathways for growth and reproduction (Butler, 1988). Their levels in plants vary dramatically,
and are influenced especially by factors such as germination, ripening, storage and type and
extent of processing. Phenols are widespread but the simple phenols are relatively uncommon. In
addition to potential toxicological concern, these compounds have been implicated as influencing
the functional, nutritional and sensory properties of foods with which they are associated
(Macrae et al., 1993). Very high levels of phenolic compounds are undesirable for women
seeking to become pregnant, since these compounds are also known to decrease fertility,
possibly by modulating hormone levels and even by interfering with the critical early stages of
pregnancy (Greenwell, 2000). Polyphenols confer on fruits, vegetables and other plant foods
both desirable and undesirable qualities (Macrae et al., 1993). Phenolic compounds give
vegetables an astringent taste and bind proteins hence can lower protein digestibility and quality
23
(Chweya and Mnzava, 1999). They modulate the action of enzymes by inhibiting certain
digestive enzymes and kinase necessary for cell proliferation (Greenwell, 2000). However, the
risk of serious toxic effects from phenolics compounds commonly present naturally in foods is
small (Singleton, 1981; Butler, 1988).
Cooking reduces significantly the tannin levels and this loss is attributable to the destruction of
polyphenolic compounds by moist heat. It is also due to the formation of some insoluble
complex between the tannin, phenolics compound and protein (Ekpenyong, 1985).
2.5. PROCESSING AND STORAGE OF VEGETABLES
Food processing techniques e.g. fermentation, canning, freezing or drying have been reported to
improve the palatability, nutritional and storage capability, as compared to fresh vegetables
(Belitz, 1987; FAO, 1990).
2.5.1. Fermentation of Vegetables
Fermentation, one of the oldest known methods of preparing and preserving food, has been
reported by several researchers to improve palatability, taste, aroma and texture; extend the
keeping quality; increase nutritional value and improve safety of food products. Fermentation of
indigenous foods is considered to be an effective, inexpensive and nutritionally beneficial
household technology in the developing world. The fermentation process is known to be very
effective in elimination of a number of antinutritional factors in foods. It improves digestibility
and utilization of proteins and fatty acids; improves solubility of minerals and reduces gastro-
enteric upsets. Fermentation also has anti-microbial activities and impacts flavors and
24
functionality attributes into foods. Food fermentation can be used as a tool in alleviating
nutritional defects at household level during food preparation or processing (Sasson, 1988).
Brining or salting or pickling, which permit and favor fermentation, can preserve many
vegetables, through action of lactic acid-forming microorganisms. Brining vegetables in salt, and
the resultant lactic acid fermentation, is an ancient form of vegetable preservation. The two
components of pickling process, acid and salt, are key in the preservation of perishable products.
Acid, which may be added directly or produced through microbial conversion of indigenous
sugars to organic acids, will lower the pH of the product and inhibit spoilage microorganisms.
Salt acts to inhibit the growth of undesirable microorganisms and to delay enzymatic softening.
In addition, it withdraws juice and nutrients from plant tissues which acts as substrates for lactic
acid bacteria. Salt also adds flavour. The changes that occur during the fermentation process are
predominantly the result of enzymatic activity brought about by microorganisms. During
fermentation, microorganisms carry out catabolic processes, altering the organic components of
food to obtain energy for their growth. The lactic acid produced effectively inhibits the growth of
other bacteria that could cause decomposition and spoilage. Such preservation is therefore
dependent upon the combined effect of salt, acid, carbon dioxide, low oxidation-reduction
potential, and other minor factors. The salt added varies from 2 to 15% depending upon the
vegetable, the salting or brining treatment, and environmental factors.
In fermented pickles, microorganisms ferment sugars to lactic acid and produce enzymes, which
modify pickle texture. Therefore low levels or absence of fermentable sugars is a deterrent to
undesirable secondary fermentation initiated by yeast at pH values below 3.8. Residual sugars
25
can also cause gas production and brine turbidity in finished products if yeast and bacteria
growth continues (Fleming and McFeeters, 1981). Lactic acid bacteria are the primary
microorganisms involved in preservation of fermented pickled products. Although these
microorganisms represent only a small proportion of the total microbial flora present on the
surface of the plant materials, they predominate under acidic conditions. Pederson (1971)
reported that the major aerobic species in raw vegetables were of the genera Pseudomonas,
Flavobacterium, Enterobacter, Escherichia and Bacillus. Acidity, salt concentrations,
temperature and sanitary conditions are the primary environmental factors that influence
fermentation. Low temperatures inhibit the growth of lactic acid bacteria and thus slow
fermentation. At 7.5oC, Leuconostoc mesenteroides will grow, but the growth of Lactobacillus
and Pediococcus species is very slow. At temperatures in the range of 18o – 23oC, Lb. brevis, and
Lb. plantarum exhibit active growth, while at 32oC, Lb. plantarum and Pediococcus pentosaceus
predominate. Pasteurization, the final step in pickle processing, inactivates or destroys the
fermenting organisms.
2.5.2. Blanching
Heat-treament, a short heat treatment of fruits and vegetables, prior to processing or preservation
aims at inactivating enzymes in the vegetables. It can be done either by immersion in hot water
or spraying steam. In some cases the blanching water is used repeatedly, with the purpose of
building up the concentration of dissolved solids to the point where leaching losses are small.
Dehydrated vegetables are blanched prior to drying in order to arrest undesirable enzyme action
and that the dried products will refresh more readily. Enzymes are sensitive to moist heat
conditions, especially where temperatures range above the maximum for enzyme activity. Moist
26
heat instantaneously inactivates the enzymes. There are exceptions, but as a rule, exposure for a
minute at 100oC renders enzymes inactive. When exposed to dry heat (dry air) at the same
temperature, enzymes are notably insensitive to the effect of heat (Desrosier and Desrosier,
1987). Blanching shortens drying and rehydration time by relaxing the tissue walls so that
moisture can escape more rapidly. Blanched vegetables take less time to cook because they are
partially cooked (Mehas and Rodgers, 1989). Blanching cleanses the product, causes a great
reduction in numbers of microorganisms as much as 99% in some instances, and decreases its
volume (Frazier, 1967). In some cases, it removes disagreeable odors or flavors, and with some
vegetables, it removes slime-forming substances. Blanching may or may not aid in retention of
the green colour of the vegetables. This depends upon the vegetables, blanching temperature and
preservation method used. Blanching spinach at boiling temperature results in loss of the green
colour. The loss is due to decomposition of the chlorophyll to phaeophytin, which is yellow-
green in colour. Blanching vegetables at 77oC, retains its natural green colour to a remarkable
extent, even when heated to 121oC during the subsequent sterilization. The blanching period
should be sufficient to completely inactivate peroxidase enzyme, except for potatoes, where a
slight residual peroxidase reaction appears to be of little consequence, and with cabbage, in
which catalase but not peroxidase is destroyed during blanching. Un-blanched and under-
blanched green vegetables develop a grayish-green colour as well as disagreeable odor and
flavor during storage (Cruess, 1958). Blanching before drying, gives dried products of tender
cooking character, better flavor and better keeping quality. Blanching by steam, results in lower
leaching losses and greater cleanliness than blanching by hot water. Instant quick heat-treament
(IQB) is a new technique that results in minimal leaching and destruction of nutrients. It uses hot
air with steam injection for a short time.
27
2.5.3. Dehydration of Vegetables
Dehydration or drying has been a means of preserving foods from earliest times. Drying is the
deliberate removal of water from food products. The water removal should be under controlled
conditions causing minimum or no changes in the food properties. A major criterion of quality of
dehydrated foods is that when they are reconstituted in water they be very close to, or virtually
indistinguishable from the original food material used. The primary objectives in removing water
from any food material are to reduce its weight and bulk, leading to economical transportation,
handling and distribution; and to improve its keeping quality by reducing the water activity (aw).
Fruit and vegetables have high moisture content, hence highly perishable. However, when
moisture has been removed, they can be preserved over a long period with minimal microbial
attack. (Kordylas, 1990). Bacteria do not grow below 18% available moisture; yeasts require
20% or more and molds require 13 - 16%. During storage, there is slow decrease in numbers of
organisms, more rapid at first and slower thereafter. The microorganisms that are resistant to
drying will survive best. Therefore, the percentages of such organisms will increase. Spores of
bacteria and molds are resistant to storage under dry conditions and there may be some
opportunity for contamination of the dried food during handling prior to drying and packaging.
Drying is used in production of convenience foods. It is inexpensive in energy forms and dried
products are economical in their storage requirements. The principal disadvantages of dried
products are that they require a longer cooking period than the fresh or canned and do not retain
their flavor.
Sun drying is the oldest method of drying food and its cost is low. The sun’s ultraviolet rays can
also inhibit the growth of microorganisms (Mehas and Rodgers, 1989). The radiant energy of the
28
sun provides the heat to evaporate the water. Drying proceeds well in warm and dry weather,
however, at night and during the rainy seasons sun-drying is not effective. The temperatures of
the food during sun drying are usually 5o – 15oC above ambient temperatures. The time of drying
can be 3 - 4 days or longer depending on the product and prevailing weather conditions. In
dehydration of vegetables, enzyme systems must be inactivated prior to drying. This is
accomplished usually by blanching of vegetables.
2.5.4. Changes of nutritional value of leafy vegetables during processing
Several authors have reviewed the effects of processing on nutritional value of foods. Although
heat may improve the nutritional value of some foods by inactivating harmful substances or by
liberating nutrients from otherwise stable complexes, it usually causes destruction of some
vitamins and may change the digestibility of proteins. Thus, it is well established that heat
damage and leaching are the major factors in nutrient losses from foods during processing (Lee
et al., 1982).
Vitamin A is mainly degraded by chemical oxidative reactions, which is accelerated by light,
heat and metals such as copper. Its loss from foods during preparation is therefore minimal if the
temperatures are kept moderate and the cooking vessel is covered. At high temperatures,
however, even if the cooking vessel is covered, the long polyunsaturated carbon chains undergo
isomerization from the trans- to the cis- form leading to loss of vitamin activity (Tannenbaum,
1976). It has been reported that the effects of cooking, heat-treatment and canning of leafy
vegetables on their carotene contents can be quite variable, and the vitamin contents after the
above processes almost show exclusively an increase (Imungi and Potter, 1983). The apparent
29
increase in the carotene content, when expressed on a solid basis, is attributed to the soluble
solids that result in a concentration effect (Lee, 1945). Solar drying of the vegetables if carried
out without the shade provision results in large losses of both vitamin A and C. Labuza (1973)
indicated that the major loss of fat-soluble vitamins such as vitamin A and E is probably due to
the reaction of peroxides and free radicals, which are oxidation products of lipids with these
vitamins.
Vitamin C is usually oxidized in air under alkaline conditions, but not acidic conditions. Its
degradation is catalyzed by the presence of heavy metals like copper (Tannenbaum, 1976). Loss
of ascorbic acid during processing of vegetables depends on the method of cooking, the volume
of water used and the species of vegetable. Large volume of water lead to loss of the vitamin
through leaching. It has been shown that cooking vegetables in just enough water and retaining
the cooking water optimizes retention of vitamin C (Krehl and Winters, 1972). Kohman (1942)
stated that vitamin C can completely be destroyed in most dehydrated products, and that
dehydrated vegetables retain their vitamin values poorly, however currently this has been
avoided by using modern dehydration processes.
Several minerals in the green leafy vegetables exist either free or bound to the tissue matrix of
the vegetables. The main route of loss of minerals during cooking or processing is through
leaching. If the cooking or processing water however, is consumed almost 100% of the minerals
are ingested. Imungi and Porter (1983) found that cooking cowpea leaves in 5 volumes of water
for 30 minutes resulted in the retention in the drained vegetables of more than 50% of each of the
17 minerals including calcium, iron and phosphorus. The retention of iron and copper were more
30
than 100% each. They also found that the vegetables that had been retorted for 42 minutes and
drained retained 70% each of the 17 minerals. Several minerals including potassium, iron and
zinc showed an apparent increase after retorting of the vegetables.
A common problem with dehydrated vegetables is the colour change (browning), which may be
caused by heat damage during dehydration or poor storage conditions. If the degree of browning
is not great, colour change may be the only notiable effect. When the change proceeds further,
the flavour, rehydration capacity and nutrient content may also be adversely affected. Exposing
cut vegetables to the fumes of burning sulphur or a solution of sodium sulphite or metabisulphite
inhibits darkening or loss of colour. The application of these treatments before drying is a
common practice. Sulphur dioxide is, however, detrimental to vitamin B1 (thiamine) and the
treatment should be avoided when the vitamin is the nutrient of interest (Salunkhe et al., 1974).
Also sulphites have been reported to cause allergic reactions to some consumers. Its use is
therefore not recommended. However, other pre-treatments exist that results in nutrient retention
during dehydration.
2.5.5. Packaging
Packaging has become a specialized study, with many new laminated paper and plastic materials,
appropriate to different food products. Combined with improved traditional methods of
processing, packaging of foods can reduce wastage and lengthen storage life (FAO, 1969). The
aim of packaging foods is to protect them against spoilage, preserve their quality and provide
convenience of handling. In order to protect food against alteration, it is necessary to place a
barrier between it and the environment. This barrier should be adapted to the capacity of
31
adsorption of the food for the factors responsible for the deteriorative reactions. It should also
minimize causes that decrease quality, such as oxidation, permeation of gases, water vapor and
aroma substances and other chemicals, or the transfer of energy (light, heat). The properties of
the packaging material are determined by its permeability to the degradative agents permeating
from outside. Packaging plays an indispensable role in modern society. Benefits of packaging
include: protection, distribution facilitation, promotion of consumer choice, preservation,
provision of consumer convenience, promotion of hygiene and safety, information and
instructions about the products’ contents. It minimizes waste, helps eliminate risk, helps contain
prices, displays and describes the product it contains and is innovative. Foods should be
packaged soon after processing to avoid recontamination or for dried foods to protect against
moisture and infestation with insects. Some foods may, however, be held for long periods
without packaging (Frazier, 1967).
2.5.6. Rehydration of Dried Foods
The quality of dried product is reflected not only in its texture, flavour and colour, but also in its
ability to rehydrate as closely as possible to the original raw material. The rehydration efficiency
is determined by preparation and the method of drying. During rehydration, dehydrated
vegetables should be soaked in water for some time before cooking; otherwise, they are likely to
remain tough and shrinkled. Enough water should be used to permit plumping up to
approximately the original volume of the fresh product, with enough water remaining to almost,
but not quite, cover the vegetable. The same water should then be used for cooking of the
vegetables. Factors that affect rehydration processes of the dehydrated products are time,
temperature, air displacement, pH and ionic strength (Karel, 1963).
32
CHAPTER 3
MATERIALS AND METHODS
3.1. PROCUREMENT AND PREPARATION OF RAW MATERIALS
The fermented-solar dried product was prepared at the Department of Food Technology and
Nutrition, University of Nairobi, Kenya. The fresh cowpea leaves were purchased from the local
market in the morning and transported quickly to the laboratory at the Department of Food
Technology and Nutrition, University of Nairobi. Immediately on reaching the laboratory,
samples were analysed for proximate composition, nutrients and anti-nutrients. For the
fermentation trials, the stalks, withered and dried leaves, weeds, stones and other foreign
materials were sorted out from the rest of the vegetables. The vegetables were then thoroughly
washed and well drained. They were cut manually with a kitchen knife into slices of
approximately 5mm thick.
3.2. OPTIMAL LEVELS OF SALT AND SUGAR FOR FERMENTATION
3.2.1 Determination of the optimal level of salt
The sorted cowpea leaves were divided into equal seven (7) portions and were fermented in lots
of 500g. Each lot was mixed thoroughly with 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0% concentration
respectively of tablesalt, (Salt Manufacturers Kenya Ltd.) followed by tight packing in 4-litre
plastic beakers. They were allowed to stand for 10 minutes, after which a polythene bag full of
water was placed inside each container as a weight to press down on the salted vegetables and
ensure that the vegetables were completely immersed under brine during fermentation.
Fermentation was carried out at ambient temperatures (22o – 26oC). During fermentation,
samples of the fermenting liquor were withdrawn at regular interval i.e. 1, 5, 10, 17 and 24 days
33
for pH and total titratable acidity (TTA) determination. Fermentation lasted for 24 days. The
preliminary experiments were replicated two times.
3.2.2. Determination of the optimal level of added sugar
Preparation of raw materials and fermentation was carried out in similar conditions as in the first
preliminaries (3. 1. 1 above) except that the samples were mixed with only 3% salt (determined
as the best level, based on the results of the first preliminaries) and varying percentages of
glucose (Excel Chemicals Ltd) and table sugar (Mumias Sugar Company Ltd, Kenya) i.e. 2.5%,
3.0% and 3.5%. During fermentation, samples of the fermenting liquor were withdrawn at
regular intervals i.e. 1, 4, 7, 10, 13, and 16 days for pH and Total Titratable Acidity (TTA)
determination. The fermentation was carried out for 16 days and replicated two times. Sensory
analyses were performed on the fermented vegetables to determine the effect of added sugar on
acceptability of the fermented vegetables.
3.3. PRODUCT MANUFACTURE
Procurement and preparation of the raw materials was similar to that carried out in 3.1 and
Figure 1. The vegetables were sliced then divided into three equal portions each of 16 kg. And
then two portions were blanched separately using hot water and one was not. One of the
blanched portions was thoroughly mixed with 3% salt and allowed to stand for two hours. This
was treated as control sample. The second portion was thoroughly mixed with 3% salt and citric
acid (EFF Chemicals Ltd, Kenya) to a final pH of 3.8 and allowed to stand overnight. This was
treated as an acidified sample. The third portion (un-blanched) was thoroughly mixed with 3%
salt and 3% sucrose, which were then tightly packed well in a 60-litre plastic bucket. The salted
34
and sugared vegetable sample was allowed to stand for 10 minutes before a polyethylene bag full
of water was placed inside the bucket as a weight to ensure that the vegetables were immersed
under the brine and fermented for 21 days. Samples of the fermented liquor were drawn
aseptically at regular intervals for microbial, pH and TTA analyses. The temperature inside and
outside the container was also recorded. To avoid contamination of final product, samples for
laboratory analyses were picked from a smaller transparent 8-litre bucket that had the vegetables
fermenting in similar conditions as the big bucket. The fermented vegetable sample was heat-
treated by boiling in its own liquor at 90o – 95oC for 3 minutes. The vegetables were then cooled
and drained immediately after heat-treatment. This was treated as the fermented sample.
Each one of the vegetable samples was loaded onto a solar drier with shade provision (Kordylas,
1990) separately. The vegetables were spread on trays at the rate of 4kg/m2 and the trays inserted
into the drier. They were then dried until the weight was constant, which took on average 5 days.
Samples were taken for vitamins (A and C), minerals (iron and calcium), antinutrients (nitrates,
oxalates and phenolics), chlorophyll and sensory analyses. The sun-dried vegetables were
packaged in either kraft or polyethylene paper. Each package contained 50g of the dried
vegetables. The packaged products were stored at: 32oC, ambient temperatures (22o – 26oC) and
18oC in enclosed dry places for three (3) months. Samples were then taken at 1-month interval
and analyzed for ascorbic acid and beta-carotene while anti-nutrients and minerals were only
analyzed after the third month. Sensory evaluation and chlorophyll analyses were carried out
after the second and third month of storage. Product manufacture was replicated twice.
RAW COWPEA LEAVES
Ferm
Acidified sample
Figure 1. Product manufacture flow diag
5
(Spon
SO(To
(Po
(A
HEA(Boilin
90o –
WASHING
CUTTING mm thickness
SALTING (3% salt)
FERMENTATION taneous, for 21 days at 22o
– 26o C)
ented sample
Control sample
LAR DRYING constant weight)
SALTING (3% salt) T-TREATMENT
g in own liquor at 95oC for 3 min)
PACKAGING lyethylene and kraft)
SORTING AND DISTEMMING
STORAGE t 18o, 22 – 26oC
and 32oC )
ACIDIFICATION (With citric acid
to pH 3.8)
SUGARING (3% sugar)
ram.
BLANCHING (In hot water for
3 min)
BLANCHING
(In hot water for 3 min)
SALTING (3% salt)
35
36
3.4. CHEMICAL, MICROBIAL, SENSORY AND PHYSICAL ANALYSES.
These analyses were carried out on the raw, solar dried and stored products. All the analyses
were performed on triplicate samples.
3.4.1. Determination of Moisture Content
The moisture contents were determined according to AOAC methods (AOAC, 1984). For raw
vegetables, approximately 5g of the sample were weighed in a moisture dish and dried in a
thermostatically controlled hot air-oven at 105oC to constant weight. The weight of the residue
was converted to percent total solids (dry matter) and the moisture content was calculated as the
difference. However, the moisture contents of the dried vegetables were determined by drying
approximately 1g samples using an infrared balance-cum-drier to constant weight and calculated
as percent loss in weight. (The two methods were used because they remove different kinds of
water in sample material. The hot air-oven method removes the loosely bound and superficial
moisture while the infrared method removes the cellular moisture.i.e. both free and bound
water).
3.4.2. Determination of Total Ash
Total ash was determined by AOAC methods (AOAC, 1984). Approximately 2g of the dried
vegetables were weighed accurately in porcelain ashing dishes previously dried in a hot air oven
at 98o – 100oC, cooled and tarred. The dishes were held in a muffle furnace at approximately
600oC for 4 hrs. They were then cooled to room temperature in a dessicator and weighed. The
weight of residue represented the total ash.
37
3.4.3. Determination of Crude Fibre
Crude fibre content was determined following AOAC methods (AOAC, 1984). Approximately
2g of dry ground sample were used to determine the crude fibre as loss on ignition of dried
residue remaining after boiling of the sample with 2.04N sulphuric acid and 1.78N sodium
hydroxide solutions under specific conditions.
3.4.4. Determination of Ethereal Extract
Crude lipid (ethereal extract) was determined by AOAC methods (AOAC, 1984). Five grams of
dry ground sample were accurately weighed in cellulose extraction thimbles (Whatman, 22 x 80
mm) and extracted with petroleum ether (Boiling point 60o – 80oC) in a soxhlet extraction unit
for 16 hours. The ether extract was transferred to a 250 ml round-bottomed flask, which had been
previously dried, cooled and tarred. Excess petroleum ether was evaporated and the residual
extract in the flask was dried to constant weight and converted to percentage of the original dry
weight.
3.4. 5. Determination of Crude Protein
Crude protein was determined as total nitrogen using the semi-micro Kjeldahl method (AOAC,
1984). Dry ground samples of 0.5g were accurately weighed in nitrogen-free filter papers and
placed in 100ml Kjeldahl flasks together with anti-bumping pumise. Sulphuric acid (10ml per
sample) was added followed by two Kjeldahl catalyst tablets. The flasks were heated on a
Kjeldahl heating assembly initially at low setting until all frothing ceased, and later changed to
high setting and the mixture digested until a clear solution remained. After cooling, the clear
38
digest was mixed with distilled water just enough to dissolve it and transferred to distillation
flasks. The Kjeldahl flasks were rinsed using distilled water into the distillation flasks. Distilled
water was added to three quarters of the distillation flask and some drops of phenolphthalein and
zinc powder were added. The flasks were connected to the distillation unit and enough 40%
sodium hydroxide added to turn the mixture alkaline.
Four-hundred millilitre volumetric flasks each containing 25ml of 0.1N hydrochloric acid
solution and some drops of methyl red indicator were placed under the outlet of distillation unit.
The mixture was distilled until a drop of distillate could not react with Nessler’s reagent. The
quantity of ammonia in the distillate was determined by back-titration with 0.1N sodium
hydroxide. A blank determination was carried out for correction of sample titre. The total
nitrogen was calculated as Nitrogen titre = Blank – Sample titre and converted to crude protein
using a factor of 6.25
3.4.6. Determination of Beta-Carotene (Pro-Vitamin A)
Vitamin A was determined as beta-carotene by the method of Astrup et al. (1971) as modified by
Imungi and Wabule (1990). One gram of the sample was ground in a mortar and pestle in
admixture with some acid-washed sand and then extracted completely with acetone. The
homogenate was filtered through glasswool and the residue ground again and rewashed with
acetone until the filtrate was colourless. The volume of the combined extracts was raised to 50ml
by adding acetone. Twenty-five millilitres of this extract were evaporated to near dryness in a
rotary vacuum evaporator in a water bath at 65oC.
39
The separation was carried out in a chromatographic column packed with silica gel to 15cm
depth. The top was filled with 1cm layer of anhydrous sodium sulphate to remove any traces of
water in the sample. The evaporated sample was dissolved in 2mls of petroleum spirit (Boiling
point 40o – 60oC), then quantitatively spotted into the column, and eluted with petroleum spirit.
The first yellow eluate was collected in a 25ml flask and made to the mark with the petroleum
spirit. The optical densities of the beta-carotene fraction was measured at 450nm using a CE 440
UV/Vis Double Beam Scanning Spectrophotometer, that had been calibrated with standard
solutions of pure beta-carotene in petroleum spirit. The results were calculated as beta-carotene
equivalents.
3.4.7. Determination of Ascorbic Acid (Vitamin C)
Ascorbic acid was determined by titration with 2,6-dichlorophenolindophenol dye (AOAC,
1984). Ten gram of the sample were extracted in 30ml of 5% oxalic acid in a mortar and pestle,
and then filtered. Standard indophenol solution was prepared by dissolving 0.05g of 2,6-
dichlorophenolindophenol in distilled water, diluted to 100ml and filtered. Ascorbic acid
standard solution was prepared by dissolving 0.05g of pure ascorbic acid in a small volume of
5% oxalic acid solution and then diluted to 250ml with the same oxalic acid solution. Ten
millilitres of the ascorbic acid standard solution was titrated with the Indophenol solution to a
slight pink end point. Ten millilitres of oxalic acid was titrated as a blank. The amount of
ascorbic acid corresponding to 1ml of indophenol solution was then calculated. Ten millilitres of
the filtered sample extract was pipetted into a 50ml flask and made to the mark with the 5%
oxalic acid solution. It was filtered quickly through glasswool after the first few millilitres of the
40
filtrate were discarded. The standard Indophenol solution was used to titrate 10ml of the filtrate.
The vitamin C content was calculated as mg/100g sample.
3.4.8. Determination of Iron and Calcium
Iron and calcium were analysed using an Atomic Absorption Spectrophotometer (Perkin Elmer,
Model 2380) equipped with an air acetylene flame, hollow cathode lamp and recorder. The
device was operated at standard conditions using wavelengths and slit widths specified for each
element.
One gram of dried ground sample was weighed into 100ml beaker and ashed for 8hrs at 550oC.
The ash was cooled to room temperature and the residue was dissolved in 20ml of 50%
hydrochloric acid by heating. Twenty milliliters of distilled water were added and the boiling
continued until the sample was clear. The contents were filtered through Whatman No. 1 filter
paper into 100ml volumetric flask. One milliliter of nitric acid was added to the extracts to
prevent phosphorous interference. The filtrate was filled to the mark with distilled deionised
water. Appropriate dilutions were carried out for calcium. The amount of elements was
calculated against their standards.
3.4.9. Determination of Total Sugars
The total sugar analyses were performed on triplicate samples of dried whole leaves obtained
from different locations to determine the sugar content before fermentation. Sugars were
determined by the calorimetric method (Dubois, et al., 1956). A dried sample of approximately
41
5g was weighed into 50ml test tube, thoroughly mixed with 25ml of 80% hot ethanol and
centrifuged. The supernatant was filtered using Whatman filter paper No. 41. The extraction was
repeated four times, followed by filtration each time. The filtrate was evaporated on a sand-bath
to remove the alcohol, with a bead inside the beaker to aid in boiling. The water phase was then
diluted to 100ml. An aliquot of 0.1ml of the evaporated and diluted sample was mixed with
4.9ml-distilled water, 5ml of 5% anthrone reagent and 5ml of 96% sulphuric acid. The mixture
was placed in iced water and shaken vigorously on a vortex mixer, and boiled for 15min. The
tube was cooled in cold water to ambient temperatures. A blank was prepared by adding 5ml of
5% anthrone and 5ml of 96% sulphuric acid to 5ml distilled water. The optical density was
measured at 490nm and the results determined from standard curve prepared using pure glucose
solution. The total reducing sugars were calculated as equivalent mg of glucose per 100g.
3.4.10. Determination of Nitrate
Nitrates were determined using the method by Cataldo et al. (1975). A standard curve was
prepared using different concentrations of potassium nitrate and nitrates were calculated as
equivalent milligrams/100g fresh weight. The sample was ground, then re-dried over-night in a
hot air oven at 70oC. A sample of 0.1g was then suspended in 10ml-distilled water in 100ml
beaker and incubated at 45oC for 1hr, to extract the nitrates and then filtered through Whatman
filter paper No. 1. An aliquot of 0.2 ml of the filtrate was pippeted into 50ml beaker and then
0.8ml of 5% (w/v) salicyclic acid in sulphuric acid was added and mixed thoroughly. The
mixture was allowed to stand for 20min at ambient temperatures. 19ml of 2N sodium hydroxide
was added and the mixture allowed to cool for 30min. The absorbance was measured at 410nm
42
against a common blank. The nitrate content was determined from a standard curve and the
nitrates content calculated as mg/100g.
3.4.11. Determination of Total Oxalates
Oxalates were determined by the method described by Marshall et al. (1967). Standard sodium
oxalate solution was prepared by dissolving 3mg of sodium oxalate in 10ml of 0.5M sulphuric
acid. This was followed by titration with 0.1M potassium permanganate at 60oC using a
microburette to a faint violet colour that was stable for at least 15 seconds and a standard curve
was plotted. A dried sample of 0.1g was extracted with 30ml of 1M hydrochloric acid in a
boiling waterbath for 30min. The sample was cooled, then shaken and filtered through No. 1
Whatman filter paper. The filtrate was adjusted to a pH greater than 8 with 8M ammonium
hydroxide followed by re-adjusting it to pH 5.0 – 5.2 with 6N Acetic acid. An aliquot of 10ml
was precipitated with 0.4ml of 5% calcium chloride, shaken thoroughly, allowed to settle at
ambient temperatures for at least 16hrs, and centrifuged at 3000rpm for 15 min. The supernatant
was discarded, rinsed twice with 2ml of 0.35M ammonium hydroxide and then the cake (pellet)
drip-dried. The pellet was dissolved in 10ml of 0.5M sulphuric acid followed by titration with
0.1M potassium permanganate at 60oC using a microburette to a faint violet colour that was
stable for at least 15 seconds. Oxalates content in the sample was determined from the standard
curve prepared earlier as mg/100g.
43
3.4.12. Determination of Total Phenolic Compounds
Total phenolic compounds were determined as tannins by Folin-Denis method (Burns, 1963).
The Folin-Denis reagent was prepared by mixing 100g sodium tungstate, 20g phosphomolybdic
acid and 50ml phosphoric acid with 750ml water. The mixture was then refluxed for 2hrs, cooled
and diluted to 1litre. Saturated sodium carbonate solution was prepared by dissolving 35g
anhydrous sodium carbonate in 100ml water at 70o – 80oC, and allowed to cool overnight. The
supersaturated solution was seeded with crystals of hydrated sodium carbonate and filtered
through glasswool after crystallization. Tannic acid solution was prepared by dissolving 100g
tannic acid in 1litre distilled water. Fresh solution was prepared for each determination. A
standard curve was prepared by pipetting 1 – 10mls aliquots of the standard tannic acid solution
into 100mls flasks containing 75ml of distilled water. Five millilitres of Folin-Denis reagent
together with 10ml sodium carbonate solution were added. The solution was diluted to volume
with distilled water and mixed thoroughly. Optical densities were determined at 760nm after
30min and absorbance plotted against mg tannic acid/100ml, to obtain a standard curve.
A ground sample of 0.5g was extracted in a mortar and pestle with 50ml distilled water, and
filtered. One millilitre of the filtrate was pipetted into 100ml flask containing 75ml distilled
water. Five millilitres of Folin-Denis reagent and 10ml sodium carbonate solution were then
added. The solution was made to volume, mixed thoroughly and then absorbance determined at
760nm after 30min incubation. Milligrams of tannic acid per 100g of sample were calculated
from the standard curve.
44
3.4.13. Determination of Chlorophyll
One gram of the dried vegetable sample was weighed and ground in 16ml of acetone in a mortar
and pestle in the presence of some acid-washed sand. The homogenate was filtered, the residue
rewashed with 80% acetone until the filtrate was colourless and, then volume made to 100ml.
The absorbance of an aliquot of the crude extract was measured at 645 and 663 nm. Total
chlorophyll concentration in the crude extract was calculated from absorbance at 645 and 663nm
in 80% acetone. Using the formula:-
Chlorophyll a (µg/g) = (12.7 A663 – 2.69 A645 ) × 100
Chlorophyll b (µg/g) = (22.9 A645 – 4.68 A 663) × 100
Total chlorophyll (µg/g) = Chlorophyll a + Chlorophyll b
3.4.14. Determination of pH and Total Titratable Acidity
The pH of the fermenting liquors was determined at specified intervals by a pH meter (Model
290 Mk 2 PYE UNICAM) after standardization with an appropriate buffer solution (Lee, 1975).
The total titratable acidity (TTA) of fermenting liquor was determined at specific intervals by
addition of 10ml of distilled water to 2ml of the liquor, followed by boiling to drive off carbon
dioxide. Five drops of 1% phenolphthalein solution were added and sample titrated with 0.01N
sodium hydroxide. Percent lactic acid was calculated as below:
Percent lactic acid = ml alkali × alkali normality × 9 / weight of sample in grams.
NB: One millilitre is equivalent to 1g.
45
3.4.15. Microbiological Analyses
Microbiological analyses were performed on duplicate samples of the fermenting liquor.
Samples of the top layer and bottom layer of the liquor were drawn asceptically on 1, 4, 7, 13
and 21 days and analyzed for standard plate count, slime-forming bacteria, lactic acid bacteria,
gram negative bacteria, and yeast and mold counts. Serial decimal dilutions were prepared using
physiological saline (0.85% NaCl).
Standard plate count was determined by pour plate method using plate count agar (Biotec, U.K).
Appropriate dilutions of 1ml were prepared from the fermenting liquor, plated and then
incubated at 30oC for 2 – 4 days and colonies counted.
Slime forming organisms were determined using sucrose-gelatin Agar. The Sucrose-Gelatin
Agar was prepared as follows: 10g of Tryptone (Oxoid), 5g of Yeast extract (Oxoid), 5g of NaCl
(Howse & McGeorge Ltd), 5g of potassium hydrogen phosphate (Kobian, Kenya Ltd), 1g of
Glucose (Howse & McGeorge Ltd), 50g of Sucrose (Howse & McGeorge Ltd) and 0.2g of
Sodium Azide (Aldrich Chemicals) were dissolved in 500mls distilled water, the pH was
adjusted to 7.2 and 50g of Gelatin (BDH Chemicals) and 500mls of 3% agar solution (Oxoid),
added. Appropriate dilutions of 0.1ml were surface plated onto pre-poured sucrose-gelatin agar
plates and incubated for 2 days at 30oC then observed for the presence of large, thick and slimy
colonies, which were then counted.
46
Lactic acid bacteria were determined using MRS agar (Oxoid). Appropriate dilutions of 1ml
were transferred onto plates and pour plated with MRS agar tempered at 50o – 55o C. The plates
were then incubated at 30oC for 2 – 4 days and colonies counted.
Gram-negative bacteria were determined using Plate Count Agar (Biotec) with 1% Crystal Violet
(BDH Chemicals). Appropriate dilutions of 0.1ml were surface plated onto pre-poured agar
plates and incubated at 30oC for 2 days, then plates observed and colonies counted.
Yeasts and molds counts were determined using acidified Potato Dextrose Agar (Oxoid) (to pH
3.5 with 10% tartaric acid). Appropriate dilutions of 1ml were mixed with molten tempered
Potato Dectrose Agar. The plates were incubated at 23o – 25oC for 5 days and the colonies
counted.
Morphological and biochemical tests
Primary classification of bacteria isolates was based on the results of gram staining, cell
morphology, and catalase tests (Harrigan and McCance, 1976). Catalase negative bacteria were
identified using the Gibson media for hetero-fermenters. A loopful of growth was emulsified in a
drop of hydrogen peroxide (10% v/v) on a slide. Effervescence, due to liberation of free oxygen
indicated the presence of catalase enzyme in the culture under test.
47
The heterofermenters and homofermenters were differentiated by the ability of heterofermenters
to produce carbon dioxide gas from glucose in Gibson’s semi-solid Tomato Juice Medium
prepared by mixing 2.5g of Yeast extract, 50g of D-Glucose, 100ml of tomato juice, 800ml of
reconstituted skim milk and 200ml of Nutrient Agar. The tomato juice was mixed with the
reconstituted skim milk. The yeast extract and glucose were added and heated. While still hot,
the molten nutrient agar was added and mixed well. The pH was adjusted to 6.5. The medium
was distributed in test-tubes to a depth of 5 – 6 cm, and sterilized for 30 minutes (Gibson and
Abd-el-Malek, 1945). Approximately 0.5 ml of a young broth isolate culture, was inoculated,
mixed well and cooled in tap water. After setting, molten nutrient agar at 50oC was poured into
the tube to give a layer 2 – 3 cm deep above the surface of the medium. This was incubated at
30oC for up to 14 days. The agar seal trapped any carbon dioxide gas produced in the medium.
This was indicated by disruption of the agar seal and by the presence of gas bubbles in the
medium (Harrigan and McCance, 1976).
The gram positive lactobacillus sp. was identified by growth at 15oC and at 45oC in MRS broth,
which was prepared by dissolving 10g of peptone (Oxoid), 10g of Lab-Lemco meat extract
(Oxoid), 5g of Yeast extract, 20g of D-Glucose, 1g of Tween 80 (Atlas), 2g of Dipotassium
hydrogen phosphate, 5g of Sodium acetate, 2g of triammonium citrate, 0.2g of hydrated
magnesium sulphate and 0.05g of hydrated manganese sulphate in 1 litre distilled water and
heated to dissolve. The pH was adjusted to 6.2 – 6.6, and sterilized at 121oC for 15 min.
(Harrigan and McCance, 1976). The tubes were inoculated with isolates and incubated at 15o and
45oC for 5 days. Growth were observed and recorded.
48
Acid producers were identified by growth in Litmus milk, indicated by colour change and action
on casein. Sufficient litmus solution was added to reconstituted skim milk (1g powder in 10 ml
distilled water) to give a pale mauve colour [10 ml of 4% litmus solution (BDH Chemicals Ltd.)
per litre of milk]. The medium was distributed in test-tubes to a depth of 5 – 6 cm and sterilized
at 121oC for 5 min, followed by heating for 30 min. It was inoculated as for a broth culture and
incubated at 30oC for up to 14 days. The tubes were examined daily and any colour changes in
the medium recorded.
3.4.16. Sensory Evaluation
Sensory analyses were performed on the fermented vegetables during the determination of
optimal level of sugar; and on fermented-, acidified- and control-dried samples. The vegetable
samples were evaluated for appearance, colour, flavour, texture and overall acceptability using
untrained panelists, familiar with the taste of cooked cowpea leaves. The vegetables, prior to
presentation to panelists were prepared as follows: Ten grams of finely chopped onions were
weighed into an aluminium pot with 10g shortening (Kimbo, Bidco Oil Refinaries Ltd, Kenya).
The container was then heated on an electric plate at medium heat setting until the onions turned
golden brown in colour. 100g of the fermented drained leaves and 1g salt (Kensalt, Salt
Manufacturers Kenya Ltd) were added. The ingredients were thoroughly mixed and 150ml of
water added. The pot was covered and heating continued for 10 minutes with occasional mixing
of the vegetables. The heating setting was changed to low and vegetables simmered for another
10 minutes. The vegetables were then served to the panelists as an accompaniment for ugali (a
maize meal paste). (The way the vegetable is commonly served in the community).Each panelist
had twelve samples, six samples for testing each day. The panelists were then asked to score the
49
sensory attributes of the samples on a seven point hedonic rating scale with 1 = dislike very
much and 7 = like very much. A sample of the questionnaire is presented in appendix A.
3.4.17. Determination of Rehydration Properties.
This was performed to determine the rehydration properties of the freshly dried and stored dried
products. Samples of the dried vegetable were rehydrated in either cold water (20o – 22oC) or hot
water (80o – 90o C) for different durations i.e. 5, 10, 15, 20, 30 or 40 minutes. The weights of the
samples were taken at 5 minutes intervals, until constant values were obtained (when no more
water could be absorbed). Percent rehydration was calculated as follows:
Weight after rehydration X 100
Weight before drying
3.4.18. STORAGE STABILITY EVALUATION
Fermented-, acidified- and control-dried samples were randomly divided into three batches. One
batch was stored at 18oC, second at ambient temperatures (22o – 26oC) and the third at 32oC for
three (3) months. The samples were either packaged in kraft paper or polyethylene bags prior to
storage. Assessment of the effect of the packaging material, storage temperature and time on the
product quality was determined. Ascorbic acid, beta-carotene and rehydration properties were
determined at the beginning of the storage period and subsequently after every month of storage.
Sensory evaluation and chlorophyll analyses were carried out after the second and third month of
storage. Minerals and anti-nutrients were analyzed at the beginning of the storage period and at
the end of the third month.
50
3.5. EXPERIMENTAL DESIGN AND STATISTICAL DATA ANALYSIS
All the experiments were arranged in a completely randomized factorial design with three main
treatments of fermented-soalr dried vegetables, acidified-solar dried vegetables and control-solar
dried vegetables. The sub-treatments were two types of packaging material, (i.e kraft paper and
polyethylene), three different storage temperatures, (i.e. 18oC, 22o – 26oC and 32oC) and three
different durationss of storage, (i.e. 1, 2 and 3 months). The experiments were replicated twice.
All data were then subjected to analysis of variance (ANOVA) and means were separated by
Duncan Multiple Range Test using Genstat 6th Edition and Costat Statistical Software
Programmes.
51
CHAPTER 4
RESULTS AND DISCUSSION
4.1. PROXIMATE COMPOSITION OF RAW COWPEA LEAVES
The proximate composition of raw cowpea leaves obtained from a retail market at Kangemi,
Nairobi (Source: Nyanza province) was as presented in Table 5. The values were comparable
with those reported from other studies, Table 3 and 4 (Bubenheim et al., 1990; Maundu et al.,
1999).
Table 5: Proximate composition of raw cowpea leaves expressed as percent of edible portion on
dry matter basisa.
Moisture
content
(%)
Dry
matter
(%)
Crude Protein
(N x 6.25)
(%)
Total Ash
(%)
Crude fibre
(%)
Lipid
(%)
Sugar
(%)
87.0 ± 0.6 13.2 ± 0.9 31.8 ± 1.5 12.1± 1.5 18.2 ± 2.3 3.0 ± 1.5 6.4 ± 0.4
a Mean ± Standard Deviation (n =6)
4.2. OPTIMAL LEVELS OF SALT AND SUGAR
4.2.1. Determination of the optimal Salt levels.
Figure 2 shows the development of total titratable acidity (TTA) expressed as percent lactic acid
for the first preliminary trial. The development of lactic acid was low for all the levels of salt
added as it averaged 0.1% lactic acid on the first day and 0.2% lactic acid on the last day (Day
24). However, 3%-salt-concentration sample attained the highest levels of TTA, but this was still
52
far below the target of 1.5% lactic acid. This salt-concentration was chosen for all later
fermentations. The inability to ferment to the required acid values could be attributed to low
levels of fermentable sugars in the vegetable leaves. Due to insufficient acid production during
the early phase of the fermentation, organisms responsible for putrefaction were favoured, thus
rendering the products unacceptable (Carr et al., 1975). Consequently, the sugar level in the
cowpea leaves was determined, to check whether they were comparable with those in cabbage,
raw material for sauerkraut making. The results were as given in Table 6.
Figure 2: Development of acidity during fermentation of the vegetables with varying levels of added salt.
Acid Development
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
1 5 10 17 24
Time In Days
Per
cent
Lac
tic A
cid 2
2.533.544.55
53
Table 6: Moisture, dry matter and sugar levels expressed as percent of edible portion of fresh
cowpea leaves from three sources.
Source Moisture % Dry matter % Sugar %
Eastern 85.6 ± 0.6b 14.4 ± 0.6a 0.84 ± 0.02a
Nyanza 89 ± 1.4a 11.0 ± 1.4b 0.75 ± 0.04b
Western 85 ± 2.0b 15.0 ± 2.0a 0.69 ± 0.01c
L.s.d 2.54 2.04 0.049 a Mean ± Standard Deviation (n = 3) :
NB: Values within a column followed by the same superscript are not significantly different.
The moisture levels of samples from Nyanza province were significantly higher (P<0.05) than
for the other provinces. The sugar levels in samples obtained from the three provinces were very
highly significant (P<0.01) (Appendix B:1), although all were far below the sugar levels in
cabbage. On average, raw cabbage has been reported to contain 4% sugar (McCance and
Widdowson, 1991) and as a general rule, approximately half as much acid is produced as there is
sugar present in the cabbage (Desrosier and Desrosier, 1987) during spontaneous fermentation.
The second preliminary experiments were then performed by adding fermentable sugars (glucose
or sucrose).
4.2.2. Determination of the optimal Sugar Levels
Figure 3 gives the TTA results for the second preliminary experiment. Unlike the low acidity
levels attained during the first preliminary experiment (Fig. 2), the acidity levels during the
second preliminary experiment (Fig. 3) increased steadily up to 1.5%, for all the samples.
54
Samples with 3.0% and 3.5% glucose attained upto 1.9% lactic acid. Desrosier and Desrosier
(1987) reported that 1.5% acidity was sufficient for inhibition of spoilage microorganisms hence
preservation of sauercraut. It shows that the addition of sugar contributed positively towards the
development of acidity during the fermentation. Therefore, it was necessary to carry out sensory
analysis to determine the acceptability of the samples to which sugar was added, as this is
unusual during preparation of vegetables. Table 7 gives a summary of the sensory evaluation
results obtained during the second preliminary experiment. All the samples attained above
average score on a seven-scale hedonic rating in all the attributes. However, the sample
containing 2.5% glucose scored highest in overall acceptability, but it was not significantly
different (P<0.05) from that of 3% and 3.5% sucrose (Appendix B: 2). Since sucrose (common
Figure 3: Development of acidity during fermentation of the vegetables with 3% added salt and varying levels of sucrose (S) and glucose (G).
Acid Development
0.00
0.50
1.00
1.50
2.00
2.50
1 4 7 10 13 16
Time In Days
Perc
ent
Lact
ic A
cid 2.5S
3.0S3.5S2.5G3.0G3.5G
55
sugar) is readily available, 3% sucrose level was recommended as the sugar level to be added to
the vegetables prior to fermentations, to increase the level of fermentable sugars present in the
vegetables.
Table 7: Mean ranking scores for the sensory attributes of fermented cowpea leaves treated with
different levels of sugars.
% SUGAR FLAVOUR TEXTURE APPEARANCE COLOUR OVERALL
ACCEPTABILITY
2.5 S 4.3b 4.7b 5.2c 4.9c 4.6c
3.0 S 4.9ab 5.5a 5.9a 5.1bc 5.3ab
3.5 S 5.4a 5.3ab 5.5bc 5.6ab 5.5ab
2.5 G 5.4a 5.5a 6.0a 5.7a 5.7a
3.0 G 4.9ab 4.8b 5.6abc 5.6a 5.1bc
3.5 G 4.4b 5.5a 5.8ab 5.2abc 4.9bc
L.s.d 0.83 0.58 0.47 0.54 0.58
S – Sucrose G – Glucose
NB: Values within a column followed by the same superscript are not significantly different from each other
(P<0.05).
4.3. NUTRIENT LEVELS IN COWPEA LEAVES
The levels of beta-carotene, ascorbic acid, iron, calcium and chlorophyll for raw, fermented-,
acidified- and control-dried cowpea leaves are given in Table 8. The levels of these nutrients for
raw cowpea leaves in this study were comparable to values reported by Onyango et al. (2000),
Bubenheim et al. (1990) and Maundu et al. (1999). The levels for ascorbic acid, calcium and
chlorophyll in the fermented, acidified and control dried samples were significantly higher
56
(P<0.05) from those of the raw cowpea leaves (Appendix C). These results indicate that
blanching and sun drying of cowpea leaves under shade provision can result in significant
reduction in levels of ascorbic acid, calcium and chlorophyll. The fermented-dried samples were
not significantly different (P<0.05) in iron from the raw cowpea leaves, but the acidified- and the
control-dried samples were significantly different (P<0.05). There was apparent difference
Table 8: Levels of vitamins, minerals and chlorophyll of raw, fermented- acidified- and control-
dried cowpea leaves expressed in mg/100g edible portion on dry matter basis.
Sample Ascorbic acid Beta-carotene Calcium Iron Chlorophyll
Raw 308 ± 14a 33 ± 12 a 1736 ± 43a 64.4 ± 16.7a 1663 ± 96a
Fermented-dried 45 ± 9b 30 ± 4.5a 1217 ± 29b 47 ± 11ab 1136 ± 42b
Acidified-dried 52 ± 6.8b 20 ± 1.5a 1040 ± 50b 38 ± 9b 1009 ± 78b
Control-dried 42 ± 7.6b 19 ± 1.5a 1171 ± 64b 38 ± 8b 806 ± 62b
L. s. d. 163.3 17.12 436.7 20.8 444.4 Mean ± Standard Deviation (n =4). Values within a column followed by the same superscript are not significantly
different from each other (P<0.05).
between the raw and the fermented-dried samples from the acidified- and the control-dried
samples in beta-carotene content. Such losses in ascorbic acid during drying have been reported
for other vegetables (Mziray et al., 2000). It is generally recognized that dehydration of leafy
vegetables results in losses of vitamins, the extent of loss depending on the type of vegetable
(Belitz 1987; Gareth et al., 1998). It has been reported that dehydrated vegetables lose colour
appeal as a result of alteration of chlorophyll due to pheophytinization reaction occuring during
heat-treatment and oxidation during the drying process (Cruess, 1958; Steel and Tong, 1996).
There was significant destruction of both beta-carotene and ascorbic acid during heat-treatment
57
and drying. Tannenbaum (1976) reported that at high temperatures, the long chain
polyunsaturated carbons undergo isomerization from the trans to the cis form, leading to loss of
the vitamin activity. The loss in ascorbic acid could have resulted from leaching during
blanching, effects of the processing temperatures or due to enzymatic and chemical degradation
Table 9. Recommended daily intakes by World Health Organization (WHO) for some nutrients.
Age Vit C (mg) Retinol eq. (µg) Calcium (mg) Iron (mg)
1 20 300 500-600 5-10
1-3 20 250 400-500 5-10
Children
3 -5 20 300 400-500 5-10
5 -7 20 300 400-500 5-10
7 -10 20 400 400-500 5-10
10 -12 20 575 600-700 5-10
12 -14 27.5 725 600-700 8-16
14 -16 30 750 600-700 8-16
Boys
16 -18 30 750 500-600 5-9
5-7 20 300 400-500 5-10
7-10 20 400 400-500 5-10
10-12 20 575 600-700 5-10
12-14 27.5 725 600-700 10-20
14-16 30 750 600-700 13-26
Girls
16-18 30 750 500-600 14-28
Men 18 + 30 750 400-500 5-9
Women 18 + 30 750 400-500 14-28
Pregnancy
Last 3 mths
30 750 14-28
Lactation
first 6 mths
30 1200 14-28
Source: Kowtaluk and Kopan (1986)
58
especially in the presence of traces of heavy metal ions (Labuza, 1972; Ana and Lia, 1997; FAO,
1995; Belitz, 1987). Nevertheless, the destruction would have been more pronounced if the
drying was done without shade provision as has been reported by Mudambi (1977), Gomez
(1981), Maeda and Salunkhe (1981). It has been reported that the main route of loss of minerals
during blanching is leaching into the heat-treatment water.
The Recommended Dietary Allowance (RDA) of ascorbic acid for adults is 30mg/day and
20mg/day for children (Table 9). Therefore, consumption of 10g of fermented-dried cowpea per
day can provide approximately 15% and 22.5% of RDA of ascorbic acid for adult and children
respectively. Consumption of 10g of fermented-dried cowpea leaves per day would also provide
more than 100% of RDA of beta-carotene for adult and children, approximately 20% of RDA of
calcium for teenagers, who need the highest amounts, and more than 20% of RDA of iron for
teenage girls and women who need the highest amounts. The levels of vitamin C and beta-
carotene have been reported to decrease during cooking of vegetables, however, the effect of
cooking on levels of vitamin C and beta-carotene in fermented-dried vegetables was not carried
out in this study.
4.4 LEVELS OF ANTI-NUTRIENTS IN COWPEA LEAVES
Table 10 shows the levels of nitrates, oxalates and phenolics in raw, fermented-, acidified- and
control-dried cowpea leaves. The levels of nitrates in raw cowpea leaves were significantly
higher (P<0.05) than those in the fermented-, acidified- and control-dried samples, which
indicate that much of the nitrate had leached into the blanching water. Leaching of nitrates has
59
Table 10: Anti-nutrients levels in raw, fermented-, acidified- and control-dried cowpea leaves
expressed in mg/100g edible portion on dry matter basisa.
Sample Nitrates Oxalates Phenolics
Raw 771 ± 36a 1889 ± 98a 2783 ± 88a
Fermented- dried 217 ± 27b 1679 ± 84a 1992 ± 115a
Acidified -dried 166 ± 13b 1859 ± 67a 2119 ± 89a
Control - dried 352 ± 34b 1830 ± 103a 1959 ± 96a
L. s. d. 376.1 536.2 871.1 a Mean ± Standard Deviation (n =4) Values within a column followed by the same superscript are not significantly
different.
been reported by Varoquax et al., (1986), Barbara and Ken (1987) and Mziray (1999). It seems
blanching; fermentation, acidification and dehydration had insignificant effect on the levels of
oxalates and phenols for the three samples. There was no significant difference among the raw
cowpea leaves and the fermented-, acidified- and control-dried samples (P<0.05) in the levels of
oxalates and phenolics. It has been reported that oxalates and phenolics could change in form
during food processing. However, the methods used for their determination in this study could
not differentiate these forms, hence their levels did not change with the treatments. Mbugua et al.
(1992) when working with fermented Uji, reported that drum drying directly, or in combination
with fermentation with or without boiling, did not affect the content of phenolic compounds.
4.5. MICROBIOLOGICAL RESULTS
4.5.1. Physical properties of the fermented cowpea leaves
The titratable acidity increased steadily during fermentation, from 0.3% on the first day to 1.3%
at the end of the fermentation [Fig. 4 (a)], while the pH level dropped slightly during
60
Initial stage Intermediate stage Final stage
Figure 4 (a): Acid development during spontaneous fermentation of cowpea leaves.
Initial stage Intermediate stage Final stage
Figure 4 (b): pH changes during spontaneous fermentation of cowpea leaves.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1 4 7 10 13 16 21
Time in Days
% A
cid
as L
actic
2.5
3
3.5
4
4.5
1 4 7 10 13 16 21
Time in Days
pH le
vels
61
fermentation, from 3.9 on the first day to 3.2 at the end of fermentation [Figure 4 (b)]. Such
acidity compares well with 1.2% produced at the end of fermentation of kales (Mutegi, 2002).
However, this level is slightly lower than 1.5 to 2.0% lactic acid, which has been reported to be
produced during fermentation of cabbage (Kordylas, 1990). The Kales have been reported to
have an initial pH of 5.6 which dropped to 4.0 during fermentation (Mutegi, 2002), while
cabbage has been reported to have an initial pH of 6.2 which drops to 3.4 -3.7 during
fermentation (Hang et al., 1972; Stamer et al., 1969).
Although the cowpea leaves’ fermentation resulted in a lower content of lactic acid (i.e. 1.3%),
pH level (i.e. 3.2) was in the range that could suppress the growth of spoilage microorganisms
(Stamer et al., 1969; Carr et al., 1975). These differences could be due to the type of vegetable,
its buffering capacity and the specific microorganisms involved in the fermentation (Stamer et
al., 1969). Figure 4 shows the development of acidity and changes in pH during fermentation of
the cowpea leaves. There was no significant difference in temperature inside and outside the
fermenting vessel, the fermentation was carried out at 22o – 24oC, the range depended on natural
changes in the room temperature.
4.5.2. Development of microorganisms during fermentation
The microbial sequence in spontaneous fermentations may be divided into three stages: Initial,
intermediate and final. During the initial stage (0 - 4 days), the total number of microorganisms
was significantly higher (P<0.05) than in the other two stages (Table 11). The microbial load
increases initially (0 – 4 days) due to low initial levels of acidity (0.6% lactic acid or pH 3.5) but
as the fermentation progresses, the developed acidity increases to levels that inhibit growth of
62
microorganisms, hence decrease in total microbial load. The total number of microorganisms
during the intermediate stage (7 - 13 days) was significantly higher (P<0.05) than in the final
stage (21 days).
Table 11: Meana number (log10 cfu/ml) of total microorganisms at different days during
spontaneous fermentation of cowpea leaves.
Time in Days
0 4 7 13 21 L.s.d
Mean (No. log10
cfu/ml).
5.72a 5.91a 5.02b 4.75c 3.25d 0.23
aMean ± Standard deviation (n = 4) Values followed by the same superscript are not significantly different.
Anderson (1984) reported that during fermentation of vegetables, many different bacteria are
present during the initial stage. As fermentation progresses, most are eliminated leaving, only
those that can withstand low pH levels or high acidity levels. The salt level and other anti-
bacterial metabolic products (bacteriocins) are also inhibitory to microorganisms. The total
number of microorganisms from the bottom layer of the liquor was significantly higher (P<0.05)
than that from the top layer. This was evident from each category of the microorganisms (Figure
5). This could have resulted from differences in the liquors of favorable environmental
conditions required by the microorganisms (Pederson, 1971). Pederson (1971) reported that
yeasts and molds are less exacting in their nutritional requirements, unlike lactic acid bacteria.
For lactic acid bacteria, besides an energy source, a variety of essential growth factors and
microaerophilic conditions must be made available as provided for in the fermenting vessel in
this study. This could explain why the numbers of yeasts and molds for top and for bottom layers
63
were more or less the same [Fig. 5 (e)]. The slime-formers, yeasts, molds and gram-negative
bacteria are undesirable during fermentation; hence cause spoilage effects in the fermented
vegetables.
The results of standard plate count are shown in Fig. 5 (a): This was carried out on the top layer
of the liquor and on the bottom layer. The count on the top layer decreased steadily from 7.4 log
bacterial number initially to 4.7 log bacterial number on day 21, this was due to increased acidity
as the fermentation progressed, which inhibited most of the bacteria growth in the top layer.
However, the count on the bottom layer increased from 6.3 log bacterial number initially to 8.1
log bacterial number by the seventh day. Later, it started dropping steadily up to 5.8 log bacterial
number on day 21. These numbers were influenced by the increase and/or decrease of the various
microflora present during fermentation. Lactic acid bacteria are microaerophilic hence growth is
favoured due to reduced oxygen tension in the bottom layer hence increase in number of
standard plate count.
The results of slime-formers are shown in Fig.5 (b): The high initial load of slime-formers
increased moderately up to a maximum within the initial four days for the bottom layer of the
liquor but slightly for the top layer. This corresponded to the titratable acidity of about 0.65%
lactic acid or a pH of 3.4 (Fig. 4). It has been reported that the terminal pH values and acidities
tolerated by leuconostoc spp involved in vegetable fermentations were 1.04% lactic acid and a
pH of 3.7 for cabbage, and 0.23% lactic acid and a pH of 3.7 for cucumbers (Stamer et al., 1969;
Carr et al., 1975). These values could vary slightly for cowpea leaves, due to the fact that it was
a different fermentation process and also a different vegetable with variable sugar composition.
64
Fig. 5. (a): Standard Plate Count During Fermentation.
Fig. 5 (b): Slime Formers Development
0
1
2
3
4
5
6
7
8
9
0 4 7 13 21
Time in Days
Log 1
0 of N
o.,c
fu/m
l
Top liquor Bottom liquor
-1
0
1
2
3
4
5
6
7
8
0 4 7 13 21
Time in Days
Log 1
0 No.
cfu/
ml
Top liquor Bottom liquor
65
Thereafter, the slime formers decreased drastically to zero by the seventh day. Stamer et al.
(1969) reported that the short life span of leuconostoc spp apparently is not caused by the lack of
essential nutrients. The rapid death rate may result from the inability of the microorganism to
survive at the lower pH values or its greater sensitivity to the more undissociated forms of lactic
and /or acetic acids.
The results of gram-negative bacteria are shown in Fig. 5 (c): The high initial gram-negative
load decreased evenly as the fermentation progressed upto 4 log bacterial number. Gram-
negative bacteria are undesirable in fermented vegetables since they cause spoilage due to
development of odours and consistency defects. The initial decrease can be attributed to the
inhibition of the gram-negative bacteria by 3% (w/w) salt added, while later decline can be
attributed to the inhibition of the gram-negative bacteria by a combined effect of developed
acidity and salt concentration (Fulde and Fabian, 1953).
The results of lactic acid bacteria are shown in Fig. 5 (d): The lactic-acid bacteria load was high
from the beginning. The numbers increased to a maximum of 8 log bacterial number by day 7 for
both top and bottom layers of the liquor and thereafter, started decreasing. The higher recovery
of lactic acid bacteria could be due to the high sensitivity of the MRS Agar medium used. The
total count values were however low as the Plate Count Agar medium used has low recovery
sensitivity due to variability in the general requirements of the total flora. The increase in acidity
favoured growth of lactic-acid bacteria, which produced more of desiarable acid that completed
the fermentation (Pederson and Albury, 1954). Lactic-acid bacteria grow in the brine until the
fermentable sugars are exhausted or low pH values are attained, the resulting lactic and acetic
66
Fig. 5. (c): Gram-Negative Bacteria Development
Fig. 5 (d): Lactic -acid Forming Bacteria Development
1
2
3
4
5
6
7
8
0 4 7 13 21
Time in Days
log 1
0 of
No.
,cfu
/ml
Top liquor Bottom liquor
0
1
2
3
4
5
6
7
8
9
0 4 7 13 21
Time in days
Log 1
0 of
No.
,cfu
/ml
Top liquor Bottom liquor
67
acids inhibit their growth. Buffering capacity and the fermentable carbohydrate content of the
plant material are important factors, which govern the extent of fermentation by lactic-acid
bacteria (Fleming, 1982).
The results of yeasts and moulds are shown in Fig. 5 (e): The levels of yeasts and moulds
remained relatively low compared to other microflora. This could be due to low concentrations
of salt and the microaerophilic conditions in the fermenting liquor, which favour a primary
fermentation, predominated by lactic-acid bacteria (Fleming, 1982). The load decreased slightly
during the initial 4 days then increased slightly upto the seventh day and thereafter, it decreased
upto the end of the fermentation. This is comparable to the trends in the lactic acid bacteria
growth which also decreased after the seventh day. Fleming (1982) reported that fermentative
yeasts may become established during the primary fermentation, as they are acid tolerant and if
Fig. 5 (e): Yeasts and Molds Development
1
2
3
4
5
6
7
8
0 4 7 13 21
Time in Days
log 1
0 of N
o., c
fu/m
l
Top liquor Bottom liquor
68
fermentable sugars remain after the lactic-acid bacteria are inhibited at low pH values, they can
continue to grow until the fermentable sugars are exhausted. In this study, the continual decrease
could have resulted from reduction of nutrients and development of microaerophilic conditions
that developed as the fermentation progressed. Also, the reduced oxygen tension
(microaerophilic conditions) in the fermenting liquor was ensured by pressing down the
vegetables with a weight (water in polyethylene bag); this discouraged growth of yeasts and
moulds which are undesirable in fermented vegetables.
4 .6. STORAGE RESULTS
4.6.1.Retention of Vitamins and Chlorophyll
The retention of beta-carotene, ascorbic acid and chlorophyll in fermented-, acidified- and
control-dried cowpea leaves stored at 18oC, 21o – 26oC and 32oC, and packaged in either Kraft-
paper or polyethylene bags for three months are presented in Figures 6, 7 and 8, respectively.
4.6.1.1. Retention of Beta-Carotene
The retention of beta-carotene during storage of fermented-, acidified- and control-dried cowpea
leaves is presented in Figure 6 (a,b,c) respectively. The beta-carotene content of raw cowpea
leaves was 33.3mg/100g (dry weight basis). For the fermented-dried sample 100% represents
29.5mg of beta-carotene, the quantity present in 100g (dry weight basis) of fermented-dried
cowpea leaves before storage [Fig. 6 (a)]. This represented 11% loss compared to the raw
cowpea leaves. Fermentation resulted in better retention of beta-carotene, compared to
acidification or drying. During storage, the loss was higher during the first and second months
than in the third month for samples stored in Kraft-paper bags, whilst the loss was higher during
69
the first month and slight during the second and third months for samples stored in polyethylene
bags. This could have resulted from oxidation due to oxygen retained in the package, which was
more during the first month than in the second and third months and light due to transparent
polyethylene bags used. The higher rate of oxidation of beta-carotene can be attributed to
reaction kinetics, where there was more beta-carotene initially (as reactant) which decreased with
oxidation thus lowering the reaction rate during the later months of storage. Gareth et al., 1998,
reported that losses of beta-carotene in stored dehydrated vegetables are usually due to oxidation
mainly by the oxygen retained in the package and catalyzed by light.
The loss in beta-carotene was highest for the samples stored at 32oC and decreased with decrease
in storage temperature. Similar results were obtained by Mutegi (2002), who showed that an
increase in temperature had a significant reduction effect on the retention of beta-carotene. The
higher the temperature of storage, the higher was the loss in beta-carotene. At the end of three
months storage, the retention ranged between 7.6mg (sample stored at 32o C and packaged in
Kraft- paper bag) and 17.4mg/100g on dry weight basis (sample stored at 18oC and packaged in
polyethylene bag). At each storage temperature, the retention of beta-carotene was higher in
samples stored in polyethylene bags than in samples stored in Kraft-paper bags. It has been
reported that losses of beta-carotene in stored dehydrated vegetables are usually due to oxidation.
Hence, Kraft-paper being permeable to air, could have contributed to the higher losses of beta-
carotene compared to polyethylene-paper which is less permeable. Kraft paper also can allow
moisture uptake, hence not the best material (although clean and easily available) for packaging
and storage of dehydrated products. However, in this study moisture content of the dried
products were not monitored during the storage period.
70
For acidified-dried sample, 100% represents 19.7mg/100g (dry weight basis) of beta-carotene
[Fig. 6 (b)]. This represents 40.8% loss compared to the raw cowpea leaves. This loss was higher
than that of the fermented sample. Loss of beta-carotene during the first month of storage was
higher than in the second and third months. The loss in beta-carotene was highest for samples
stored at 32oC and the losses decreased with decrease in storage temperature. At the end of three
months, the retention ranged between 0.5 for sample stored at 32oC and packaged in
polyethylene bag and 1.4mg/100g (dry weight basis) for sample stored at 18oC and packaged in
polyethylene bag.
For the control-dried sample, 100% represents 18.9mg/100g (dry weight basis) beta-carotene
[Fig. 6 (c)]. This represents 43% loss compared to the raw cowpea leaves. Among the three
samples, the control sample had the greatest loss. It can be concluded that both acidification and
fermentation had a positive effect on the retention of beta-carotene (Fig. 6a, 6b and 6c). Percent
loss of beta-carotene was highest during the first month but decreased as storage period increased
for all the samples. At each temperature of storage, the loss was higher for samples packaged in
polyethylene bags than for those packaged in kraft-paper bags. Gareth et al. (1998) reported that
light and oxidants catalyze the oxidation of beta-carotene in stored dehydrated vegetables
causing great losses. It is therefore recommended that dehydrated vegetables be stored away
from direct sunlight. Kraft-paper being opaque whereas polyethylene-paper is transparent could
have contributed to these differences. However, it is difficult to compare the two packaging
materials, as Kraft paper is permeable to oxygen unlike polyethylene bag that is less permeable
to oxygen. At the end of three months of storage, the retentions ranged between 1.5mg for
sample stored at 32oC and packaged in polyethylene bag and 6.8mg/100g (dry weight basis) for
71
Fig. 6 (a): Retention of beta-carotene in fermented-dried cowpea leaves during storage for three months.
0
20
40
60
80
100
120
0 1 2 3
Storage time (months)
Perc
ent r
eten
tion
of b
eta-
caro
tene
18CK 25CK 32CK 18CP 25CP 32CP
72
Fig. 6 (b): Retention of beta-carotene in acidified-dried cowpea leaves during storage for three months.
0
20
40
60
80
100
120
0 1 2 3
Storage time (months)
Per
cent
rete
ntio
n of
bet
a-ca
rote
ne
18CK 25CK 32CK 18CP 25CP 32CP
73
Fig. 6 (c): Retention of beta-carotene in control-dried cowpea leaves during storage for three months.
0
20
40
60
80
100
120
1 2 3 4Storage time (months)
Per
cent
rete
ntio
n of
bet
a-ca
rote
ne
18CK 25CK 32CK 18CP25CP 32CP
74
sample stored at 18oC and packaged in Kraft-paper bag. Gomez (1981) also reported similar
losses of beta-carotene in dried leafy vegetables.
4. 6.1.2. Retention of Ascorbic Acid:
The retention of ascorbic acid during storage of fermented-, acidified- and control-dried cowpea
leaves is presented in Figure 7a, b, c; respectively. The ascorbic acid content of raw cowpea
leaves was 308.3mg/100g (dry weight basis). For fermented-dried sample, 100% represents
44.7mg ascorbic acid per 100g (dry weight basis) of fermented-dried cowpea leaves before
storage [Fig. 7 (a)]. There was 85.5% loss compared to the raw cowpea leaves due to procesing.
During storage, percent loss was highest during the first month, and was least during the third
month for all the samples. The high rate of ascorbic acid loss during the first month of storage as
compared to the second and the third months was probably due to the effect of the residual
oxygen retained in the packaging material during the initial packaging and light due to
transparent polyethylene bag (Mziray et al., 2000). As storage progressed, the residual oxygen in
the package decreased and therefore the rate of oxidation of ascorbic acid also decreased. Such
trends in the loss of ascorbic acid during storage of fruits and vegetables have been reported by
Mutegi (2002), Smooth and Nagy (1979), and Philip and Manuel (1991). The total loss in
ascorbic acid was highest for samples stored at 32oC and decreased with decrease in storage
temperature. Barth et al., (1990) observed that the ascorbic acid content of stored products
generally decreases more rapidly at higher storage temperatures. The samples stored in Kraft-
paper bags had higher loss compared to those stored in polyethylene bags at each storage
temperature. This was probably due to air permeability of the Kraft-paper, leading to more
oxidation of the ascorbic acid compared to the polyethylene bags. At the end of three months, the
75
retentions ranged between 11.4mg for sample stored at 32oC and packaged in Kraft-paper bag
and 22mg/100g (dry weight basis) for sample stored at 18oC and packaged in polyethylene bag.
For acidified-dried sample, 100% represents 52.3mg/100g (dry weight basis) ascorbic acid
before storage [Fig.7 (b)]. This represents 83% loss compared to that present in the raw cowpea
leaves. Acidification contributed to better retention capacities of ascorbic acid during processing
when compared to either fermented sample or the control sample. During storage, the highest
percent loss was in the first month for all the samples. The total loss in ascorbic acid was highest
for the samples stored at 32oC and decreased with decrease in storage temperature. At the end of
the storage period, the retentions ranged between 12.1mg for sample stored at 32oC and
packaged in Kraft-paper bag and 22.7mg/100g (dry weight basis) for sample stored at 18oC and
packaged in polyethylene bag.
For control-dried sample, 100% represents 42.4mg/100g (dry weight basis) ascorbic acid before
storage [Fig. 7 (c)]. There was 86.2% loss compared to that present in the raw cowpea leaves.
The control-dried samples had the highest percent loss in ascorbic acid during processing; hence
we can conclude that fermentation and acidification resulted in better retention of ascorbic acid.
In the first month of storage the samples experienced the highest loss in ascorbic acid, and the
third month showed least loss. The loss in ascorbic acid was highest for samples stored at 32oC
and decreased with decrease in storage temperature. At the end of the third month, the retention
ranged between 13.6mg for sample stored at 32oC and packaged in Kraft-paper bag and
18.2mg/100g (dry weight basis) for sample stored at 18oC and packaged in polyethylene bag.
76
Fig. 7 (a): Retention of ascorbic acid in fermented-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 1 2 3
Storage time (months)
Perc
ent r
eten
tion
of a
scor
bic
acid
18CK 25CK 32CK 18CP 25CP 32CP
77
Fig. 7 (b): Retention of ascorbic acid in acidified-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 1 2 3
Storage time (months)
Per
cent
rete
ntio
n of
asc
orbi
c ac
id
18CK 25CK 32CK 18CP25CP 32CP
78
Fig. 7 (c): Retention of ascorbic acid in control-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 1 2 3
Storage time (months)
Perc
ent r
eten
tion
of a
scor
bic
acid
18CK 25CK 32CK 18CP25CP 32CP
79
4. 6. 1. 3. Retention of Chlorophyll:
Figure 8a, b and c, shows the retention of chlorophyll during storage of fermented-, acidified-
and control- dried cowpea leaves, respectively. Raw cowpea leaves contained 1663mg/100g (dry
weight basis) chlorophyll. For fermented-dried sample, 100% represents 1136mg/100g (dry
weight basis). There was 32% loss of chlorophyll during in the fermented-dried samples during
processing [Fig. 8 (a)]. During storage, the chlorophyll lost was highest during the third month.
Samples stored in Kraft-paper bags had a higher retention than samples stored in polyethylene
bags at each temperature of storage. Chlorophyll loss can be through oxidation due to light and
oxygen and conversion to pheophytin due to acidity. However, the effect of this was not
separated in this study. It has been reported that light intensity accelerates browning (Pederson
and Robinson, 1952), therefore, the packaging material should preferably be opaque. The loss in
chlorophyll was highest for samples stored at 32oC and decreased with decrease in storage
temperature. Total chlorophyll content of stored leaves has been found to decrease with increase
in storage temperature. Therefore, storage temperature is of vital importance in relation to the
maintenance of green colour in dried foods (Salunkhe et al., 1974; Negi and Roy, 2004). At the
end of three months of storage, the retentions ranged between 200mg for sample stored at 32oC
and packaged in polyethylene bag and 376mg/100g (dry weight basis) for sample stored at 18oC
and packaged in Kraft-paper bag. However, loss of chlorophyll through light-induced oxidation
was also more determinant as shown by loss in polyethylene bags versus Kraft bags above.
For acidified-dried sample, 100% represents 1008mg/100g (dry weight basis), which was 61% of
the chlorophyll present in the raw cowpea leaves [Fig. 8 (b)]. The percent loss in chlorophyll was
highest during the third month compared to the first two months. The loss in chlorophyll was
80
highest for the samples stored at 32oC and decreased with decrease in storage temperature. At the
end of the third month, the retentions ranged between 198mg for sample stored at 32oC and
packaged in Kraft–paper bag and 280mg/100g (dry weight basis) for sample stored at 18oC and
packaged in polyethylene bag.
For control-dried sample, 100% represents 826mg/100g (dry weight basis), which was 50% of
the original chlorophyll present in raw cowpea leaves [Fig. 8 (c)]. The loss in chlorophyll was
highest for the samples stored at 32oC and decreased with decrease in storage temperature. At the
end of three months of storage, the retentions ranged between 152mg for sample stored at 32oC
and packaged in Kraft-paper bag and 377mg/100g for sample stored at 18oC and packaged in
polyethylene bag. The retention of chlorophyll is important in dried vegetables, as it has been
reported that chlorophyll in green leafy vegetables may provide useful protection against liver
cancer caused by aflatoxin (Galvano et al., 2001). Overall there was no specific trend for
samples packaged in polyethylene bags neither those packaged in Kraft paper regarding
chlorophyll retention during storage in the three samples.
81
Fig. 8 (a): Retention of chlorophyll in fermented-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 2 3
Storage time (months)
Perc
ent r
eten
tion
of c
hlor
ophy
ll
18CK 25CK 32CK 18CP 25CP 32CP
82
Fig. 8 (b): Retention of chlorophyll in acidified-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 2 3
Storage time (months)
Perc
ent r
eten
tion
of c
hlor
ophy
ll
18CK 25CK 32CK 18CP25CP 32CP
83
Fig. 8 (c): Retention of chlorophyll in control-dried cowpea leaves during storage for 3 months.
0
20
40
60
80
100
120
0 2 3
Storage time (months)
Per
cent
rete
ntio
n of
chl
orop
hyll
18CK 25CK 32CK 18CP 25CP 32CP
84
4. 6. 2. Retention of Minerals and Anti-nutrients during Storage.
The effect of fermentation and acidification; storage temperature and packaging material on the
retention of iron, calcium, nitrates, oxalates and phenolic compounds during the three months of
storage is given in Tables 12, 13 and 14 respectively. There were apparent losses in iron and
calcium and nitrates, oxalates and phenolic compounds during storage. Fermentation and
acidification did not significantly affect the retention of iron, calcium, and oxalates during
storage (Table 12). Fermented-dried sample had a significantly (P<0.05) lower nitrate level
compared to the acidified- and control-dried samples. Acidified-dried sample had a significantly
higher level of phenolics compared to the fermented- and control-dried samples (Table 12).
Table 12: Effect of fermentation and acidification on the minerals and anti-nutrients during
storage (mg/100g solids).
Mean Values retained during storage for 3 months
Samples Calcium Iron Nitrates Oxalates Phenolics
Fermented-dried
378.0a 37.1a 96.2b
729.5a 1438b
Acidified-dried
302.3a
30.3a
205.3a
847.0a
1712a
Control- dried
328.0 a
34.1a
227.3a
819.7a
1485b
L. s. d. 107.3 7.2 51.2 276.5 167.6 Means in the same column followed by the same superscript are not significantly different (P < 0.05).
The temperature of storage did not have a significant effect on the levels of calcium and nitrates
during storage. Samples stored at 18oC had a significantly higher level of oxalates than those
85
stored at either 22o - 26oC or 32oC. Level of phenolic compounds was significantly lower for
samples stored at 32oC compared to those stored at 18oC and 22o - 26oC (Table 13). Generally,
oxalates and phenolic compounds are easily vaporised organic compounds. Possibly low storage
temperature (18oC) hindered the vaporization of both oxalates and phenolic compounds
compared to the higher temperatures of 22o – 26o C and 32oC. There was no significant effect on
iron, calcium, nitrates, oxalates and phenolic compounds due to the packaging material during
storage (Table 14).
Table 13: Effect of storage temperature on the minerals and anti-nutrients during storage
(mg/100g solids).
Mean Values retained during storage for 3 months
Storage Temperature Calcium Iron Nitrates Oxalates Phenolics
18oC 302.3a 29.5 b 161.4 a 1035 a 1616 a
22o – 26oC 355.3 a 40.2 a 174.2 a 702 b 1596 a
32oC 350.8 a 32.6 b 193.2 a 659 b 1424b
L. s. d. 107.6 7.2 51.5 276.5 167.6
Means in the same column followed by the same superscript are not significantly different (P < 0.05).
Table 14: Effect of packaging material on the minerals and anti-nutrients during storage
(mg/100g solids).
Mean Values retained during storage for 3 months
Packaging Material Calcium Iron Nitrates Oxalates Phenolics
Kraft-paper 351.5a 34.1 a 173.5 a 769 a 1496 a
Polyethylene 320.5 a 34.1 a 178.8 a 829 a 1595 a
L. s. d. 87.9 6.1 41.7 225.8 137.1 Means in the same column followed by the same superscript are not significantly different (P < 0.05).
86
4.7. REHYDRATION PROPERTY
Rehydration of the fermented-, acidified- and control-dried samples were carried out
immediately after drying. These results are shown in Table 15. The effect due to the temperature
of water used for rehydration (hot and cold water) was very highly significant (P<0.001).
Table 15: Rehydration of fermented-, acidified- and control-dried samples soon after drying.
Percent water uptake
Hot watera Cold waterb
Samples
5 min 10 min 15 min 5 min 10 min 15 min
Fermented-
drieda
84.7aa 89.8 aa 92.0aa 55.6ba 61.1ba 62.4ba
Acidified-
driedb
69.8ab 73.6ab 74.9ab 53.5bb 57.8bb 58.6bb
Control-
driedb
66.5ab 67.8ab 70.8ab 50.0bb 54.4bb 59.0bb
Means within a row followed by the same superscript are not significantly different (P < 0.05)
Rehydration using hot water was significantly faster than cold water, as hot water has been
reported to have high rehydration or dissolving power. There was highly significant difference
(P<0.01) in rehydration capacity between the samples. The fermented-dried sample rehydrated
significantly faster than acidified- or control-dried samples, however there was no significant
difference in rehydration durations i.e. 5, 10 and 15 minutes; though the percentage rehydration
(expressed as % water uptake) increased as the duration of rehydration increased. Consequently,
rehydration for fermented-dried stored samples (for the three months of storage), were carried
out, using hot water and extended rehydration duration rehydrations i. e. 5, 10, 15, 20, 30 or 40
minutes.
87
The percent rehydration increased with the increase in duration taken to rehydrate the fermented
dried samples (Table 16). Rehydration for 40 minutes was significantly different (P<0.05) from
15, 10 and 5 minutes rehydration duration. Thirty minutes rehydration was significantly different
from 10 and 5 minutes rehydration durations. Twenty minutes rehydration was significantly
different from 5 minutes rehydration duration. There was no significant difference in the
rehydration between the 15, 10 and 5 minutes durations. This was similar to the rehydration
results obtained for 5, 10 and 15 minutes durations, carried out immediately after drying (Table
15). Rehydration for 20 minutes is recommended, as it is not significantly different from that of
40 minutes, yet it saves on time used for rehydration during preparation of the dried vegetables.
Table 16: Effect of duration on rehydration for fermented-dried stored samples using hot water.
Duration
(in minutes)
5 10 15 20 30 40
Water uptake
(%)
64.84d 67.30cd 68.91bcd 70.67abc 72.22ab 74.37a
L.s.d. = 4.55: Means within a row followed by the same superscript are not significantly different (P <
0.05).
There was no significant difference in the rehydration due to the length of storage, temperature
of storage or the packaging material used (Table 17, 18 and 19). Samples stored for three months
rehydrated better than those stored for either two months or one month, but were not
significantly different. Samples stored at 18oC rehydrated better than those stored at either 25oC
or 32oC, but were not significantly different. Samples stored in polyethylene bags rehydrated
better than those stored in Kraft-paper bags, but were not significantly different.
88
Table 17: Effect of length of storage on rehydration for fermented-dried stored samples using
hot water.
Length of storage in
months
1 2 3
Water uptake (%) 68.84a 69.68a 70.63a
L.s.d. = 3.22
Table 18: Effect of storage temperature on rehydration for fermented-dried stored samples using
hot water.
Storage temperature 18oC 22o –26oC 32oC
Water uptake (%) 71.60a 68.41a 69.14a
L. s.d. = 3.22
Table 19: Effect of packaging material on rehydration for fermented-dried stored samples using
hot water.
Packaging material Kraft-paper Polyethylene
Water uptake (%) 68.64a 70.80a
L.s.d. = 2.63
4.8. SENSORY EVALUATION
Sensory evaluation was carried out immediately after drying and after the third month of storage
The panelists mean scores for appearance, colour, flavour, texture and overall acceptability of the
fermented, acidified and control samples immediately after drying are presented in Table 20. The
89
acidified-dried sample had significantly higher (P<0.05) scores for appearance, flavour and
colour than the fermented-dried sample. The scores for texture and overall acceptability were not
significantly different. However, all the scores were above 4.0 (neither like nor dislike category).
Table 20: Mean scores for sensory attributes for freshly processed cowpea leaves.
Sensory
attributes
Fermented-
dried sample
Acidified-dried
sample
Fresh-dried
sample
L. s.d
Appearance
4.7b 5.3a 5.2ab 0.54
Colour
4.7b 5.3a 5.3a 0.49
Flavour
4.3b 5.2a 5.1ab 0.74
Texture
(Mouthfeel)
4.6a 4.4a 4.7a 0.80
Acceptability
4.8a 5.3a 5.3a 0.58
Values within a row followed by the same superscript are not significantly different (P<0.05)
Table 21 gives a summary of sensory evaluation mean scores at the end of storage. The control-
dried sample stored at 18oC in polyethylene bag had significantly higher (P<0.05) scores for
appearance than fermented-dried samples stored at 18oC or 25oC in Kraft-paper or 32oC in
polyethylene bag and acidified-dried sample stored at 18oC in Kraft-paper. The control-dried
sample stored at 32oC in polyethylene bag had significantly higher (P<0.05) scores for flavour
than control-dried stored at 25oC in polyethylene bag, acidified-dried samples stored at 18oC and
90
Table 21: Mean scores for sensory attributes after three months of storage Attribute Storage condition Fermented-dried Acidified-dried Control-dried sample sample sample Appearance 18oC (Kr) 4.3d 4.8bcd 5.8ab (L.s.d. 0.92) 18oC (Po) 4.8bcd 5.3abcd 6.1a 25oC (Kr) 4.6cd 5.0abcd 5.8ab 25oC (Po) 5.3abcd 5.1abcd 5.3abcd 32oC (Kr) 5.5abc 5.6abc 5.3abcd 32oC (Po) 4.9bcd 5.6abc 5.8ab Flavour 18oC (Kr) 3.6e 3.7de 5.0abc (L.s.d. 1.06) 18oC (Po) 4.5abcde 4.3cde 4.8abcd 25oC (Kr) 5.1abc 4.6abcde 5.6ab 25oC (Po) 5.3abc 5.2abc 4.3bcde 32oC (Kr) 5.5abc 5.5abc 4.9abc 32oC (Po) 5.1abc 5.3abc 5.7a Texture 18oC (Kr) 4.7abc 3.9bc 5.0ab (Mouthfeel) 18oC (Po) 4.3abc 3.5c 4.5abc (L.s.d. 1.04) 25oC (Kr) 4.8ab 4.3abc 4.7abc 25oC (Po) 4.9ab 4.8ab 3.9bc 32oC (Kr) 5.5a 4.9ab 4.3abc 32oC (Po) 5.0ab 4.1bc 5.3ab Overall 18oC (Kr) 4.6cde 4.2e 5.3ab Acceptability 18oC (Po) 4.7cde 4.8cde 5.5abc (L.s.d. 0.82) 25oC (Kr) 5.2abcd 4.9bcde 5.9a 25oC (Po) 5.4abc 5.3abc 4.3de 32oC (Kr) 5.8ab 5.4abc 4.9bcde 32oC (Po) 5.1abcde 5.3abc 5.9a Values for an attribute followed by the same superscript are not significantly different. Kr : Kraft-paper bags Po: Polyethylene bags
fermented-dried stored at 18oC in Kraft-paper. The fermented-dried sample stored at 32oC in
Kraft-paper had significantly higher (P<0.05) scores for texture than acidified-dried samples
stored at 18oC or 32oC in polyethylene and control-dried stored at 25oC in polyethylene bag. The
control-dried samples stored at 32oC in polyethylene bag and at in Kraft-paper, fermented
91
samples stored at 18oC and acidified-dried sample stored at 18oC in polyethylene bagand at 25oC
in Kraft-paper.
From these results, we can conclude that packaging in either Kraft-paper bags or polyethylene
bags does not have significant effect on the sensory attributes. The temperature of storage also,
had no significant effect on the sensory attributes. Fermentation or acidification did not
significantly affect the sensory attributes of the samples. Therefore, the consumers could easily
accept the fermented product, as its sensory attributes do not significantly differ from those of
the control sample. The length of storage of the dried cowpea leaves did not significantly affect
their sensory attributes (Table 20). Thus, storage for upto three would not affect the sensory
attributes of the dried vegetables, but would ensure they are available for consumption for longer
periods, thu s solve the issue of seasonality of the cowpea leaf vegetable.
92
CHAPTER 5
CONCLUSION AND RECOMMENDATION
5.1. CONCLUSION
This study shows that cowpea leaves can be fermented at 22o – 26oC (room temperature) for 21
days into an acceptable product after addition of 3% sucrose and 3% salt. Fermentation,
blanching and solar-drying of vegetables retained substantial levels of beta-carotene, ascorbic
acid, calcium, iron and chlorophyll. It also effected substantial reductions in the anti-nutrients:
nitrates, oxalates and phenolic compounds. Although there were no statistical significant
differences due to fermentation or acidification, there were apparent differences in values
obtained.
Storage of the fermented-dried, acidified-dried and control-dried vegetables led to loss in beta-
carotene, ascorbic acid and chlorophyll. The fermented-dried samples retained higher levels of
beta-carotene and chlorophyll than the acidified-dried and control-dried samples, while the
acidified-dried sample retained the highest levels of ascorbic acid. Fermented-dried samples
retained significantly lower levels of nitrates, while acidified-dried sample retained significantly
higher levels of phenolic compounds. Samples stored at 18oC retained significantly higher levels
of oxalates, while those stored at 32oC retained significantly lower levels of phenolic compounds
than those stored at 18oC and at 22o – 26oC. There was no significant difference in iron, calcium,
nitrates, oxalates and phenolic compounds due to the packaging material.
Rehydration of the samples in hot water was significantly higher than in cold water. The
fermented samples rehydrated significantly better than the acidified and control samples.
93
Rehydration for 40 minutes was significantly higher than for 15, 10 and 5 minutes rehydration
durations. Rehydration for 30 minutes was significantly higher than for 10 and 5 minutes
rehydration durations. Rehydration for 20 minutes was significantly higher than for 5 minutes.
Rehydration for 20 minutes is recommended. Fermentation or acidification, temperature of
storage, type of packaging material and storage period of the dried cowpea leaves did not
significantly affect their sensory attributes.
5. 2. RECOMMENDATIONS
In view of the results of this study, the following recommendations are made:
1. A microbiological study should be carried out to ascertain which specific species of
microorganisms are involved in fermentation of cowpea leaf vegetables to give a
uniform product and for large-scale production.
2. The cooked vegetables should be analyzed for their nutritional composition unlike in this
study where only sensory evaluation was carried out on the cooked samples.
3. A study should be carried out on the storage of the dried vegetables in other types of
packaging material, especially those that are conventionally used by communities to store dry
foods.
4. Storage of the dried vegetables should be carried out for longer periods than the three months
in this study.
5. Similar trials should be carried out on other popular traditional vegetables.
6. Lastly, this technology being cheap and effective should be transferred to the local
communities and women groups for preservation of seasonal vegetables like cowpeas.
Together with it, the promotion for increased acceptability and consumption of the fermented
94
and dehydrated vegetables should be done among the rural communities, where the
deficiency of vitamin A and iron is likely to be rampant during the period of drought.
95
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APPENDICES Appendix A: QUESTIONNAIRE GIVEN TO THE PANELISTS DURING SENSORY
EVALUATION HEDONIC SCALE SCORING
NAME ____________________________________________________________________
DATE______________________________________________________________________
PRODUCT__________________________________________________________________
INSTRUCTIONS
Please, observe and taste each sample in order from left to right. Use the scale provided below to indicate
how much you like or dislike the sample you have tasted. Please, comment on your attitude. Remember
you are the only one who can tell what you like. An honest expression of your personal feeling will help
us.
DEGREE OF PREFERENCE SCALE
Like very much 7
Like moderately 6
Like slightly 5
Neither like nor dislike 4
Dislike slightly 3
Dislike moderately 2
Dislike very much 1
ATTRIBUTES
Sample Code Appearance Colour Flavour
(aroma &taste)
Texture
(Mouthfeel)
Overall
Acceptability
Comment______________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
109
Appendix B: ANOVAS FOR PRELIMINARY TESTS
B.1: ANOVAS for proximate results for cowpea leaves from different sources.
Variable: Dry matter
Source of variation d.f. s.s. m.s. v.r. F pr.
Samples 2 27.920 13.960 13.42 0.006
Residual 6 6.240 1.040
Total 8 34.160
Variable: Moisture
Source of variation d.f. s.s. m.s. v.r. F pr.
Samples 2 25.076 12.538 7.75 0.022
Residual 6 9.707 1.618
Total 8 34.782
Variable: Sugar
Source of variation d.f. s.s. m.s. v.r. F pr.
Samples 2 0.0342 0.0171 28.50 <.001
Residual 6 0.0036 0.0006
Total 8 0.0378
110
B.2: Two way ANOVAS for sensory analysis results during determination of optimal sugar
levels
Variable: Flavour
Source of variation d. f. s. s. m. s. v. r. F pr.
Panelists 10 33.08 3.31 1.70 0.090
Sample 5 27.95 5.59 2.87 0.018
Replicate 1 1.71 1.71 0.87 0.352
Residual 115 224.27 1.95
Total 131 286.99
Variable: Texture (Mouthfeel)
Source of variation d. f. s. s. m. s. v. r. F pr.
Panelists 10 35.56 3.56 3.72 <.001
Sample 5 13.07 2.61 2.73 0.023
Replicate 1 0.92 0.92 0.96 0.330
Residual 115 109.93 0.96
Total 131 159.48
Variable: Appearance
Source of variation d. f. s. s. m. s. v. r. F pr.
Panelists 10 28.30 2.83 4.54 <.001
Sample 5 14.42 2.89 4.62 <.001
Replicate 1 0.48 0.48 0.78 0.380
Residual 115 71.76 0.62
Total 131 114.97
111
Variable: Colour
Source of variation d. f. s. s. m. s. v. r. F pr.
Panelists 10 37.33 3.73 4.65 <.001
Sample 5 11.15 2.23 2.78 0.021
Replicate 1 0.48 0.48 0.6 0.439
Residual 115 92.36 0.80
Total 131 141.33
Variable: Overall acceptability
Source of variation d. f. s. s. m. s. v. r. F pr.
Panelists 10 25.67 2.56 2.72 0.005
Sample 5 18.06 3.61 3.83 0.003
Replicate 1 0.27 0.27 0.29 0.592
Residual 115 108.33 0.94
Total 131 152.33
112
Appendix C: ANOVAS FOR NUTRIENTS AND ANTI-NUTRIENTS IN RAW AND IN
DIFFERENTLY TREATED AND DRIED COWPEA LEAVES
Variable: Ascorbic_acid
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 206722. 68907. 6.13 0.009
Residual 12 134897. 11241.
Total 15 341618.
Variable: Beta-carotene
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 631.6 210.5 1.70 0.220
Residual 12 1488.0 124.0
Total 15 2119.6
Variable: Calcium
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 1122893. 374298. 4.66 0.022
Residual 12 963694. 80308.
Total 15 2086587.
Variable: Chlorophyll
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 1556898. 518966. 6.91 0.006
Residual 12 900598. 75050.
Total 15 2457496.
113
Variable: Iron
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 1912.5 637.5 3.51 0.049
Residual 12 2182.4 181.9
Total 15 4094.9
Variable: Nitrates
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 711490. 237163. 4.02 0.034
Residual 12 707192. 58933.
Total 15 1418683.
Variable: Oxalates
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 158644. 52881. 0.05 0.983
Residual 12 11930959. 994247.
Total 15 12089603.
Variable: Phenolics
Source of variation d.f. s.s. m.s. v.r. F pr.
sample 3 1788383. 596128. 1.86 0.189
Residual 12 3836282. 319690.
Total 15 5624665.
114
Appendix D: ANOVA FOR MICROBIAL ANALYSIS
Variable: Numbers (Log10 cfu/ml) of microorganisms during
spontaneous fermentation
Source df ss MS F P
---------------------------------------------------------------
Main Effects
type 4 319.6 79.9 586.2 .0000 ***
date 4 89.2 22.3 163.7 .0000 ***
posi 1 2.3 2.3 17.2 .0001 ***
Interaction
type x date 16 160.0 10.0 73.4 .0000 ***
type x posi 4 4.2 1.0 7.7 .0001 ***
date x posi 4 7.2 1.9 13.6 .0000 ***
ty x da x po 16 13.6 0.9 6.2 .0000 ***
Error 50 6.8 0.1
----------------------------------------------------------------
Total 99 603.2
115
Appendix E: ANOVAS FOR NUTRIENTS AND ANTI-NUTRIENTS DURING
STORAGE
E.1: ANOVAS for vitamins and chlorophyll during storage for three months
Variable: Beta-carotene
Source of variation d. f. s. s. m. s. v. r. F pr.
Month 2 69.78 34.89 76.60 <.001
Pack 1 4.47 4.47 9.81 0.002
Product 2 188.01 94.00 206.39 <.001
Temperature 2 83.55 41.78 91.72 <.001
Residual 208 94.73 0.46
Total 215 440.54
Variable: Ascorbic acid
Source of variation d. f. s. s. m. s. v. r. F pr.
Month 2 190.61 95.31 267.60 <.001
Pack 1 14.16 14.16 39.76 <.001
Product 2 57.54 28.77 80.78 <.001
Temperature 2 52.87 26.43 74.23 <.001
Residual 208 74.08 0.36
Total 215 389.26
Variable: Chlorophyll
Source of variation d. f. s. s. m. s. v. r. F pr.
Month 1 392363 392363 960.6 <.001
Pack 1 101.0 101.0 0.25 0.620
Product 2 149596 74798 183.13 <.001
Temperature 2 36716 18358 44.95 <.001
Residual 137 55957 408.4
Total 143 572666
116
E.2: ANOVAS for minerals and anti-nutrients after three months of storage
Variable: Calcium
Source of variation d.f. s.s. m.s. v.r. F pr.
pack 1 17042. 17042. 0.50 0.485
sample 2 71449. 35725. 1.04 0.361
temp 2 41344. 20672. 0.60 0.552
pack.sample 2 56580. 28290. 0.82 0.445
pack.temp 2 12345. 6173. 0.18 0.836
sample.temp 4 23653. 5913. 0.17 0.952
pack.sample.temp 4 13908. 3477. 0.10 0.982
Residual 54 1858923. 34424.
Total 71 2095245.
Variable: Iron
Source of variation d.f. s.s. m.s. v.r. F pr.
pack 1 0.5 0.5 0.00 0.957
sample 2 609.0 304.5 1.95 0.152
temp 2 1410.9 705.4 4.52 0.015
pack.sample 2 404.5 202.2 1.30 0.282
pack.temp 2 218.7 109.3 0.70 0.501
sample.temp 4 1384.0 346.0 2.22 0.079
pack.sample.temp 4 112.1 28.0 0.18 0.948
Residual 54 8428.7 156.1
Total 71 12568.3
117
Variable: Nitrates
Source of variation d.f. s.s. m.s. v.r. F pr.
pack 1 448. 448. 0.06 0.812
sample 2 235233. 117616. 15.01 <.001
temp 2 12722. 6361. 0.81 0.449
pack.sample 2 18732. 9366. 1.19 0.311
pack.temp 2 8636. 4318. 0.55 0.580
sample.temp 4 13259. 3315. 0.42 0.791
pack.sample.temp 4 9511. 2378. 0.30 0.874
Residual 54 423248. 7838.
Total 71 721789.
Variable: Oxalates
Source of variation d.f. s.s. m.s. v.r. F pr.
pack 1 64823. 64823. 0.28 0.596
sample 2 181096. 90548. 0.40 0.674
temp 2 2032826. 1016413. 4.45 0.016
pack.sample 2 26676. 13338. 0.06 0.943
pack.temp 2 389813. 194906. 0.85 0.431
sample.temp 4 157171. 39293. 0.17 0.952
pack.sample.temp 4 162800. 40700. 0.18 0.949
Residual 54 12323903. 228220.
Total 71 15339107.
118
Variable: Phenols
Source of variation d.f. s.s. m.s. v.r. F pr.
pack 1 179924. 179924. 2.15 0.149
sample 2 1033555. 516778. 6.16 0.004
temp 2 530937. 265469. 3.17 0.050
pack.sample 2 68222. 34111. 0.41 0.668
pack.temp 2 296373. 148187. 1.77 0.180
sample.temp 4 586769. 146692. 1.75 0.153
pack.sample.temp 4 145063. 36266. 0.43 0.784
Residual 54 4527217. 83837.
Total 71 7368061.
119
Appendix F: ANOVAS FOR REHYDRATION PROPERTIES
F.1: ANOVAS for rehydration properties before storage.
Variable: Percent rehydration
Source df ss MS F P
---------------------------------------------------------------
Main Effects
Water type 1 3501.88 3501.88 56.33 .000 ***
Duration 2 243.50 121.75 1.96 .170 ns
Product 2 1067.60 533.80 8.59 .002 **
Interaction
w-t x dura 2 3.60 1.80 0.02 .972 ns
w-t x prod 2 406.61 203.30 3.27 .061 ns
dura x prod 4 12.33 3.08 0.05 .995 ns
w-t x dura x prod 4 8.45 2.11 0.03 .998 ns
Error 18 1118.96 62.16
----------------------------------------------------------------
Total 35 6362.93
F.2: ANOVAS for rehydration properties during storage for three months
Variable: Percent rehydration
Source of variation d.f. s.s. m.s. v.r. F pr.
duration 5 2128.99 425.80 4.44 <.001
month 2 116.44 58.22 0.61 0.546
pack 1 252.18 252.18 2.63 0.106
temp 2 401.70 200.85 2.09 0.126
Residual 205 19654.74 95.88
Total 215 22554.06
120
Appendix G: ANOVAS FOR SENSORY EVALUATION
G.1: Two way ANOVAS for sensory evaluation for dried samples before storage
Variable: Appearance
Source df SS MS F P
---------------------------------------------------------------
Main Effects
Sample 2 8.12 4.06 2.72 .0716 ns
Panelist 9 36.8 4.09 2.74 .0072 **
Interaction
Samp x Pane 18 36.05 2.00 1.34 .1830 ns
Error 90 134.5 1.49
----------------------------------------------------------------
Total 119 215.47
Variable: Colour
Source df ss MS F P
---------------------------------------------------------------
Main Effects
Sample 2 8.07 4.03 3.31 .0411 *
Panelist 9 22.74 2.52 2.07 .0402 *
Interaction
Samp x Pane 18 37.43 2.08 1.71 .0527ns
Error 90 109.75 1.22
----------------------------------------------------------------
Total 119 177.99
121
Variable: Flavour
Source df ss MS F P
---------------------------------------------------------------
Main Effects
Sample 2 18.22 9.11 3.31 .0412 *
Panelist 9 20.03 2.23 0.81 .6102 ns
Interaction
Samp x Pane 18 29.62 1.65 0.60 .8930 ns
Error 90 248 2.76
----------------------------------------------------------------
Total 119 315.87
Variable: Texture
Source df ss MS F P
---------------------------------------------------------------
Main Effects
Sample 2 1.72 0.86 0.27 .7653 ns
Panelist 9 56.97 6.33 1.98 .0510 ns
Interaction
Samp x Pane 18 12.78 0.71 0.22 .9996 ns
Error 90 288 3.2
----------------------------------------------------------------
Total 119 359.47
122
Variable: Overall acceptability
Source df SS MS F P
---------------------------------------------------------------
Main Effects
Sample 2 5.72 2.86 1.66 .197 ns
Panelist 9 34.01 3.78 2.19 .030 *
Interaction
Samp x Pane 18 14.62 0.81 0.47 .964 ns
Error 90 155.25 1.73
----------------------------------------------------------------
Total 119 209.59
G.2: ANOVAS for sensory evaluation after three months of storage.
Variable: Appearance
Source df SS MS F P
------------------------------------------------------------------------------------------------------------------
Main Effects
Sample 17 52.92 3.11 2.17 .0059 **
Panelist 5 65.67 13.13 9.15 .0000 ***
Time 1 24.08 24.08 16.78 .0001 ***
Interaction
Samp x pane 85 77.08 0.91 0.63 .9921 ns
Samp x time 17 34.33 2.02 1.41 .1348 ns
Pane x time 5 20.03 4.01 2.79 .0182 *
Sam x pan x tim 85 93.56 1.10 0.77 .9203 ns
Error 216 310 1.43
----------------------------------------------------------------
Total 431 677.67
123
Variable: Colour Source df SS MS F P
---------------------------------------------------------------
Main Effects
Sample 17 74.43 4.38 3.04 .0001 ***
Panelist 5 44.32 8.86 6.15 .0000 ***
Time 1 4.69 4.69 3.25 .0728 ns
Interaction
Samp x pane 85 77.39 0.91 0.63 .9922 ns
Samp x time 17 45.02 2.64 1.84 .0253 *
Pane x time 5 19.47 3.89 2.70 .0217 *
Sam x pan x tim 85 82.08 0.97 0.67 .9829 ns
Error 216 311.5 1.44
----------------------------------------------------------------
Total 431 658.89
Variable: Flavour
Source df SS MS F P
---------------------------------------------------------------------------------------------------------------------
Main Effects
Sample 17 82.88 4.88 2.46 .0015 **
Panelist 5 125.02 25.00 12.62 .0000 ***
Time 1 1.56 1.56 0.79 .3752 ns
Interaction
Samp x pane 85 91.73 1.08 0.54 .9992 ns
Samp x time 17 83.35 4.90 2.47 .0014 **
Pane x time 5 29.49 5.90 2.98 .0128 *
Sam x pan x tim 85 98.59 1.16 0.59 .9975 ns
Error 216 428 1.98
----------------------------------------------------------------
Total 431 940.63
124
Variable: Texture (mouthfeel)
Source df SS MS F P -----------------------------------------------------------------------------------------------------------------------------------------
Main Effects
Sample 17 27.17 1.60 0.72 .7785 ns
Panelist 5 74.70 14.94 6.75 .0000 ***
Time 1 0.23 0.23 0.10 .7467 ns
Interaction
Samp x pane 85 97.17 1.14 0.52 .9997 ns
Samp x time 17 69.27 4.07 1.84 .0248 *
Pane x time 5 27.85 5.57 2.52 .0307 *
Sam x pan x tim 85 84.64 1.00 0.45 1.000 ns
Error 216 478 2.21
----------------------------------------------------------------
Total 431 859
Variable: Overall acceptability
Source df SS MS F P
---------------------------------------------------------------------------------------------------------------------
Main Effects
Sample 17 53.51 3.15 2.13 .0068 **
Panelist 5 54.48 10.90 7.39 .0000 ***
Time 1 5.56 5.56 3.77 .0535 ns
Interaction
Samp x pane 85 64.97 0.76 0.52 .9997 ns
Samp x time 17 65.77 3.87 2.62 .0007 ***
Pane x time 5 14.93 2.99 2.02 .0763 ns
Sam x pan x tim 85 71.03 0.84 0.57 .9985 ns
Error 216 318.5 1.47
-------------------------------------------------------------
Total 431 648.72