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TITLE PAGE

PHYSICO CHEMICAL AND MICROBIOLOGICAL

PROPERTIES OF ZOBO SOLD IN ENUGU


A PROJECT SUBMITTED IN PARTIAL

FULFILLMENT OF THE REQUIREMENT FOR

THE AWARD OF BACHELOR OF SCIENCE (B. SC)

DEGREE IN MICROBIOLOGY AND BIOTECHNOLOGY


PRESENTED BY

ONUORAH, S.
PEACE CHIAMAKA

MB/2006/138

DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY

FACULTY OF NATURAL SCIENCES

CARITAS UNIVERSITY, AMORJI
-
NIKE

EMENE, ENUGU STATE



AUGUST, 2010

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CERTIFICATION PAGE

This is to certify that this project work “Physico
-
chemical and
microbi
ological properties of Zobo sold in Enugu” was carried out
by Onuorah S. Peace Chiamaka (MB/2006/138) under the
supervision of Dr. Orji Michael Uchenna. It is found worthy of
acceptance in partial fulfillment of the requirement for the award
of Bachelor of

Science (B.Sc) Degree in Microbiology and
Biotechnology.

_________________




____________________

Onuorah Peace Chiamaka





Date

Student

_________________




____________________


Dr Orji M. Uchenna







Date

Supervisor












________________
___



______________________

Dr, Amadi,

E. C







Date

Head of department



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DEDICATION




This work is dedicated to my parents, late Mr. Samuel
Onuorah for his moral and financial support towards my
upbringing and my mother, Mrs. Dorathy Oluchi Onuorah fo
r her
great care and encouragement. To my sisters, Miss. Lindamary
Onyinye and Miss. Jennifer Nkechi Onuorah. Also to chief Mr. and
Mrs. Titus Okonkwo Igweani for their guidance and financial
support towards my educational back ground and welfare.


A
bove all, to God almighty, who is my rock, refuge and
stronghold for his divine love and mercies towards my academic
journey.








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ACKNOWLEDGEMENT


My profound gratitude goes to my parents late Mr. Samuel
Onuorah for his love, care and support towar
ds my education
and upbringing, my his gentle soul rest in perfect peace. To my
mother Mrs. Dorathy Oluchi Onuorah for her motherly care and
her assistance. To my Sisters Miss Onyinye Lindamary and
Nkechi Jennifer Onuorah, my brother Master Chukwuemezie
Saviour’swill Onuorah for their encouragement and assistance.
Special thanks to Chief and Mrs. Titus Okonkwo Igweani for their
financial support and help towards my education and welfare.


I would also like to say a word of thanks to my friends an
d
course mates, Mrs. Ifunanya Nnodu, Ifeyinwa Ejike, Okpoko
Ogechi Patricia, Omotor blessing, Iheme Victoria Onyinyechi,
Nweke Emily Amarachukwu,

Onyinye Hilda Oforegbu, Anibuogu
Nkechi, Eze Tochi Nwanneka, Ibeme
-

Bede Lawrencita Uzoamaka
etc for their
encouragement, advice and assistance towards this
work. love you all.


I am not forgetting my Anuty, Blessing Okoroude and my
Uncle Thompson Okoroude for their efforts made to accomplish
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this work. I also want to acknowledge and show appreciation to

my supervisor Dr. Orji Michael Uchenna for his wonderful
contributions and assistance thereby exposing me more
educationally. Thank you sir,


I also want to appreciate the Dean, faculty of Natural
Sciences, Prof. Nduka okafor, Head of Department of
microbiology department, Dr Amadi, my lecturers Mr. Humphrey
Nwobodo, Mrs. Iloghalu, B. U. Mrs. Nwagu, Mrs Nnedimma. Prof.
Ogeneh Bryan


Above all persons, My God Almighty who brought me to this
world with love for a successful completion of this wor
k. All
praise and honour be unto his name.








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TABLE OF CONTENTS

Title page

.

.

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.

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.

.

.

i

Certification

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.

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.

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.

ii

Dedication .

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iii

Acknowledgement

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iv

Table of contents .

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vi

List of tables .

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x

Abstract

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xi

CHAPTER ONE: INTRODUCTION

1.1

Objective of Study

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5

CHAPTER TWO: LITERATURE REVIEW

Preparation of ‘Zobo’ made from Roselle

(Hibiscus sabdariffa

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6

2.2

Characteristics of Roselle

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7

2.2.1. Beta (
-

carotene in calyces of Roselle

(Hibiscus sabdariffa)


.

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9

2.2.2. Ascorbic acid (Vitamin C) in calyces

.

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9

2.2.3. Effect on beta
-

carotene and ascorbic

acid( Vitamin C)

.

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.

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10

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2.3. Types of Roselle

.

.

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10

2.3.1. Sabdariffa Var altissima


.

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10

2.3.2


Hibiscus sabdariffa


.

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10

2.4 Uses of Roselle

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11

2.4.1 Leafy Vegetables/ Green

.

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13

2.4.2 Roselle Tea

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13

2.4.3

Roselle beverages

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14

2.4.4 Jam and preserves

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15

2.4.5 Medicinal uses

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15

2.5 Phytochemicals

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16

2.6 Production of Roselle

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16

2.7 Crop research

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17

2.7.1 Crop genetic resources and improvement

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18

2.7.2 Mutation breeding

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18

2.7.3 Natural outcrossing under local conditions

.

19

2.8 How Roselle is obtained

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20

2.9 Scie
ntific classification of Roselle

.

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20

2.10


Names of Roselle Plant

.

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21

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CHAPTER THREE: MATERIALS AND METHODS

Materials

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22

Sample collection

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22

Microbiological analysis of the sample

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22

Isolation of pure cul
ture

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23

Identification of Isolates

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23

Bacteria Identification

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24

Gram’s staining

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24

Motility Test

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24

Biochemical tests

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25

Urease test

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25

Catalase test .

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26

Methyl red test

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26

Voges


proskeur Test (V.R)

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27

Indole test

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28


Citrate Utilization test

.

..

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29

Sugar fermentation test

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30

Spore stain

..

..

..

..

..

..

..

..

31

Lactophenol cotton blu
e test

.

..

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32

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Physico chemical(Biochemical) properties of Zobo

.

32


CHAPTER FOUR

Results

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34

CHAPTER FIVE: Discussion, conclusion, Recommendation.

5.1

Discussion

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39

5.2

Conclusion .

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5.3

Recomme
ndation .

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40


References

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42


Appendix I

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48


Appendix II


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56










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LIST OF TABLES

Table 1:

The identities of microorganisms isolated from

‘Zobo’ sample. .

.

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35

Table 2:

Characteristics o
f the fungal isolates. .

.

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36

Table 3:

Micro organisms isolated from Zobo collected

From different locations.

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37

Table 4:

Frequency occurrence of the isolates.

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38

Table 5:

Physico chemical properties of Zobo.

.

.

39












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ABSTRACT

Micr
obiological and physico
-
chemical properties of Zobo sold in
Ogbete market, Enugu State were evaluated using standard
micro
-
biological and analytical methods. Five bacterial genera
comprising Klebsiella, Escherichia, Bacillus, Staphylococcus,
Lactobacillus
and two fungal genera; Aspergillus and Rhizopus
were isolated. Of the 9 samples of Zobo examined, Lactobacillus
were isolated from 8 (88.8%), Bacillus from 7 (77.78%),
Klebsiella 5 (55.5%), Staphylococcus 4(44.4%), Escherichia coli
from 4(44.4%) and Rhizo
pus from 2(22.2%). The values of the
physico
-

chemical properties of the samples ranged: pH (5.03
-

5
-
10), Titrable acidity (0.30%
-

0.34) specific gravity (1.076


1.86), Total sugar (0.82%
-

0.86) and protein % (4.10


4.31).







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CHAPTER ONE

INTRODU
CTION


Zobo drink, a non alcoholic local beverage is produced from
the dried petals of Hibiscus sabdariffa (Linn Roselle) by boiling
and filtration. Hibiscus sabdariffa (Roselle) is a Vegetable plant of
West Africa origin. (Tindal, 1983) and (Omemu,

et al, 2006)
however, noted that the crop is a native to India but was
introduced to other part of the world such as central Africa, West
Indies, Australia, Africa and many tropical countries. It is best
grown in tropical and subtropical regions. It has t
he most
widespread acceptance in the Roselle producing areas of the
Nigerian savanna region where it is grown as a vegetable crop
(Omemu, et al, 2006). The Roselle is an annual herbaceous,
upright plant growing up to two metres belonging to the family
Mal
vaceae. It’s habitat is variable and the leaves also vary in
shape and size. The flowers are usually yellowish, sometimes
occuring with dark red pigmentation at the center (Rice, et al,
1990).


The fruits are up to 2
-
5cm in length and are surrounde
d by
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enlarged fleshy calyces containing 22
-
34 seeds per capsule. The
seed is dark brown in colour, 4
-
6cm long and about 0.025g in
weight (Tindal, 1983). Two botanical types of Roselle are
recognized. Sabdariffa var altissima which is a tall vigorous un
-
branched plant with fibrous spicy and inedible calyces mainly
cultivated for fiber and Hibiscus sabdariffa which is bushy,
branches sub
-
shrub with red or green stand red to yellow inflate
edible calyce. (Kocchar, 1986). The young shoots and leaves of
the

latter Roselle variety are usually cooked and eaten as
vegetables while the fleshy, swollen red calyces and the flowers
are used to colour and season other food as well as in the
preparation of a fruit drink called ‘Zobo’ in Nigeria.


The name ‘Zobo’
is derived from the local Hausa (Nothern
Nigeria) name for the Roselle plant that is ‘Zoborodo’. The calyce
contains about 8.3% moisture, 4% citric acid, 1.5% pigment
(mainly anthocyanin), 6.9% protein and about 9% soluble solids
with pH of about 2.7% (Om
emu, 2006) . ‘Zobo’ is an indigenous
non


alcoholic drink made from a hot water extract of Roselle
calyces. It is usually sweetened with sugar and may be flavoured
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with other materials such a ginger, pineapple and strawberry. It
is quite popular in North
ern Nigeria, enjoying patronage at
various social gatherings and its popularity has recently spread
across the entire country because of its purported medicinal value
as well as the increasing cost of other available soft drinks whose
concentrates are most
ly imported constituting a drain on the
economy.


Inspite of the increasing popularity of Zobo juice one of its
greatest limitations for large scale production is that it
deteriorates rapidly. Infact, it’s shelf
-
life is approximately
twenty
-
four hour
s following production if not refrigerated. There
are searity information on the micro
-
flora associated with both
the dried calyce and resulting zobo juice, which in addition to
other factors could contribute to its spoilage.



The physico
-
chemical charac
teristics of Roselle was studied and it
was characterized as a highly acidic fruits with low sugar content
source. Succinic acid and oxalic acid was quantified as two
predominant organic acids in Roselle. Roselle was found to
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contain higher amount of ascor
bic acid. (Wong, et al, 2002)
compared to orange and mango.

It was found to be fair
























































sources of vitamin A (Holden, et al, 1999). It is also rich in
riboflavin, niacni, calcium and iron (Qi, et al, 2005); (Baba lola, et
al, 2000). It also
contains antioxidants including flavonoids,
gossy petine, hibiscentine and sadderetine,. Some of
anthocyanins of Roselle identified by chromatographic process
include delphinidin
-
3 glucose (Hong, et al, 1990).


The seeds of Roselle are pounded int
o meal which is used as
oily soup or sauce after roasting. Oil extracted from the seed is a
substitute for castor oil while the residue is used in a fermented
form as soup or cake. (Aliyu, 2000). In countries like India,
Roselle calyces are utilized in
producing refreshing beverages,
jellies, jam, sauce and food preserves. (Clydescale, et al, 1979).


Zobo beverage has been shown to be good source of natural
carbohydrate, protein and vitamin C. (Okoro, 2003); (Ogiehor, et
al, 2004). These componen
ts tend to increase with increase in
storage period, possibly potentiated by the activities of the
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associated micro organism like Bacillus, Streptococcus,
Staphylococcus, Leuconostoc, Lactobacillus, Aspergillus
Penicillum, Geotrichum, Fusanum and Alternana

have been
reported to be associated with Zobo beverage during storage
(Akinyosoye, et al, 2000); (Ogiehor, et al, 2004). The
proliferation of the associated micro

organism potentiates
spoilage and the short shelf life (24 hours


48 hours) with this
bev
erage. The use of local spices to control the activities of
micro
-
organisms in food has been reported. (Akpomedoye, et al,
1998); (Ogeihor, et al, 2003). Apart from the antimicrobial
properties, spices are believed to have medicinal values
(especially i
n Africa settings) and have desirable determinative
influences on the overall organoleptic quality of food when used.
In addition, the use of low temperature storage to retard and
stabilize microbial growth in food is well documented (Jay, 1978).
Thus the

application of extracts of spices alone or in combination
with low temperature storage could possibly control microbial
activities associated with zobo drink while retaining the native
and economic quality.

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1.1.


OBJECTIVE OF STUDY


The objectiv
e of this study is to determine the physico
chemical (biochemical) and microbiological properties of Zobo
produced from dried calyces of Hibiscus sabdariffa (Linn Roselle).
The biochemical content of the zobo has undesirable effects on
human health when c
onsumed.













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CHAPTER TWO

LITERATURE REVIEW


The Roselle (Hibiscus sabdariffa) also known as sorrel (English)
is a specie of the Hibiscus native to the old world tropics.

It is a herbaceous plant and a member of the family malvaceace
(Gibbon, et
al, 1988) which are consumed as vegetable or dried
calyces and made into food products such as jam and drink.



PREPARATION OF ‘ZOBO’ MADE FROM

ROSELLE (Hibiscus Sabdariffa).



The Zobo drink is prepared by boiling the dry calyces of
Hibiscus sabdariffa in

water for about 10
-
15 mins from which the
pigment or flavour embedded is extracted. After extracting the
filterate may be taken as hot tea or allowed to cool and packaged
in plastic sachet containers then taken as a refreshing drink when
chilled. The shar
p taste of the raw extract is usually sweetened
with sugar cane or granulated sugar, pineapple, orange or other
fruits depending on choice. The sweetness of Zobo drink does not
last long due to spoilage by microbial activities. It life shelf is
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approximate
ly

twenty
-
four hours following production if not refrigerated.

(Omemu, et al, 2006).


2.2.

CHARACTERISTICS OF ROSELLE



It is an annual or perennial herb or woody based sub
-
shrub
growing to 2
-
2.5m (7
-
8ft) tall. The leaves are deeply three
-
five
lobed

8
-
15cm (3
-
6in) long, arranged alternately on the stem.

The flower are 8
-
10cm (3
-
4in) in diameter. White to pale Yellow
with a red dark spot at the base centre of each petal and have a
stout fleshy calyces at the base, 1
-
2cm (0.39
-
0.79 in).

The fruits ar
e up to 2.5cm in length and are surrounded by
enlarged fleshy calyces containing 22
-
34 seeds per capsule. The
seed is dark brown in colour, 4
-
6cm long and about 0.025g in
weight

(Rice, 1990) grows up to two meters and leaves vary in shape
and size. It tak
es about six months to mature.



The water extracts in calyces is rich in carotenoids
(especially beta
-
carotene) and ascorbic acid (Ibrahim, et
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al,1971). The calyces of Hibiscus sabdariffa have also been found
to be rich in vitamins and other antioxidants
(Wong,et al,2002)
and also minerals

(Babalola, s.o. et al,2000).



The physico
-
chemical characteristics of Roselle was studied
and it was characterized as a highly acidic fruit with low sugar
content. Succinic acid and oxalic acid were quantified as a two

predominant organic acids in Roselle. Roselle was found to
contain higher amount of ascorbic acid compared to orange and
mango (Wong, et al, 2002). It was found to be fair source of
vitamin A (Holden, et al, 1999). It is also rich in riboflavin, niacin,
c
alcium and iron (Qi, et al, 2005), (Babalola, et al, 2002). It also
contains antioxidants including, flavonoids, gossypetine,
hibiscetine and sadderetine. Some of the anthocyanins of Roselle
Identified by chromatographic process include delphinidin
-
3
-
sambu
bioside, cyaniding
-
3
-

sambubioside and delphinidin
-
3
-

glucose (Hong, et al, 1990). They are also known for their unique
flavour characteristics that makes them appealing to taste.

Roselle drink had been improved nutritionally by producing fruit
-
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flavoured

Roselle drinks which are richer in Vitamins and minerals
by additions of different fruits.

The calyce contains about 8.3% moisture, 4% citric acid, 1.5%
pigment (mainly anthocyanin), 6.9% protein and about 9%
soluble solids with a pH of about 2.7 (Omemu,
2006).


2.2.1

BETA
-
CAROTENE IN CALYCES OF

Roselle (Hibiscus Sabdariffa).

It is a naturally occurring fat soluble carotenoid which contains
vitamin A activity and is metabolically converted to vitamin A
after absorption by animal ( Tannenbaum, 1979). The de
ficiency
may cause impairment in bone formation, night vision and
malnutrition of epithelial tissue.


2.2.2

ASCORB
IC ACID (VITAMIN C) IN CALYCES


It is a water soluble micro nutrient required for the formation
of intercellular substances in the body i
ncluding deutrine,
cartilage and protein network of the bone. It’s deficiency causes
scurvy and impaired wound healing.

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2.2.3.


EFFECTS ON BETA


CAROTENE AND
AS
CORBIC ACID (VITAMIN C)

Processing of food material affect them in one way or the other.
App
lication of heat by blanching and dehydration destroy
enzymes and reduce ascorbic acid contents of vegetable foods. It
also adversely effects the concentration of beta carotene through
isomerization (Sweeny, et al, 1971).


2.3. TYPES OF ROSELLE

T
wo botanical types of Roselle are recognized namely; Sabdariffa
var altissima and Hibiscus sabdariffa.

2.3.1 Sabdariffa var altissima; This is a tall vigorous practically
un
-
branched plant with fibrous spicy and inedible calyces mainly
cultivated for fib
er.



Hibiscus sabdariffa;

This is a bushy branched sub
-
shrub with red or green stem and
red to yellow inflate edible calyce (Omemu, et al 2006).

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USES OF ROSELLE (Hibiscus sabdariffa).

Many parts of the result are of value. The young leaves are eat
en
as cooked vegetable especially with soup. The seeds are pounded
into meal which is used as oily soup or sauce after roasting. Oil
extracted from the seed is a substitute for castor oil while the
residue is used in a fermented form as soup or cake (Aliyu
,
2000).

In countries like Indian Roselle calyces are utilized in producing
refreshing beverages, jellies, jam sauces and food preserves.
(Clydescale, et al, 1970). The fleshy, swollen red calyces and the
flowers are used to colour and season other food

as well as in
preparation of a fruit drink called ‘Zobo’ in Northern central states
of Nigeria, which is gaining wide spread acceptance across the
country. It is also used as thickner and flavourants in soups
(Ibrahim, et al, 1971). Primarily, the plant

is also used for the
production of bast fibre from the stem of the plant and as an
infusion.

The plant is considered to have antihypertensive properties. The
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fibre may be used as a substitute for jute in making burlap
(Chau, et al, 2000). It has been us
ed in folk medicine as a
diuretic, mild laxative and treatment for cardiac and nerve
diseases and cancer (Mohammed, et al, 2002).

The red calyces of the plant are increasingly exported to America
and Europe where they are used as food colourings. It is fo
und in
market in some places in france as flowers or syrup.

The green leaves are used like a spicy version of spinach. They
give flavour to the Senegalese fish and rice dish (thi

boudieume). In myanmar, the green leaves of Roselle, are the
main ingredie
nt in making chin baung Kyaw Curry. In East African
the calyce infusion called “Sudan tea” is taken to relieve coughs.
Roselle juice with salt, pepper, arafetida and molasses, is

taken as a remedy for biliousness.

The heated leaf are applied to cracks i
n the feet and on