ABSTRACT
This work present information on the storage stability of zobo drink blended with leaf extracts with local names; “Nchanwu” (Ocimum gratissimum), “Uziza” (P. guineense) and “Moringa” (Moringa oleifera). This study aimed at improving microbial quality of zobo drink thereby increasing its shelf life using natural spices. Zobo drink was prepared using hot water extraction method and the juice obtained was blended separately with aqueous extracts of M.oleifera, O.gratissimum and P. guineense leaves. Microbiological and physicochemical properties of the various blends of zobo were investigated at day 0, day 1, day 3, day 5 and day 7 during the one week duration of the study. The results obtained from this study showed that zobo samples contained different concentrations of ascorbic acid which ranged from 8.21 mg/100g to 28.75 mg/100g. The pH range was between 2.3 to 6.1 while the titratable acidity was between 0.25% to 0.89%. The bacterial counts of the different samples during storage showed that there was an increase in bacterial growth from day 0 up to day 5 but decreases at the 7th day of storage. In the control samples (Unblended zobo) values obtained throughout the period of storage ranged from 24.7 x 104 to 42.0 x 103 cfu/ml. M25% values ranged from 24.7 x 104 – 21.3 x 103 cfu/ml, M50% values ranged from 24.3 x 103 – 4.3 x 103 cfu/ml. In the O25%, values obtained ranged from 24.3 x 103 – 26.3 x 10 cfu/ml, O50% values ranged from 24.7 x 104 – 11.3 x 10 cfu/ml and in P25% values ranged from 24.3 x 103 – 11.3 x 103, P50% values ranged from 24-0 x 103 – 2.7 x 104 cfu/ml. The sample containing P.guineense extract had the lowest bacterial count of 2.7 x 104 cfu/ml at the 7th day of the storage while the sample containing no extract (control) had the highest bacterial count of 42.0 x 103 cfu/ml within the same period of storage. Fungal count were also seen to be high in the unblended (control) samples than in the blended samples. The highest count of fungi was 24.7 x 103 cfu/ml which was seen in the unblended (control) samples and the lowest value was 16.7 x 103 cfu/ml obtained from P50% samples. Also, the microorganisms present in the samples were identified using staining and biochemical techniques. The bacterial organisms identified include Staphylococcus aureus, Pseudomonas sp, Escherichia coli, Bacillus sp, Lactobacillus sp and Proteus sp while the fungal organisms identified were Aspergillus sp and Saccharomyces sp. However, the samples containing P.guineense were better rated in terms of parameters tested. The results of this study indicate that incorporation of P.guineense, O.gratissimum and M.oleifera leaves into zobo drink could be an effective means of improving quality (microbial) and extending the shelf-life of zobo drinks. The study shows the possibility of large scale production of zobo using treatment with these leaves as a safe, low cost means of preservative.
TABLE OF CONTENTS
Cover page
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of contents v
List of Figures ix
List of Tables x
Abstract xi
CHAPTER ONE
1.0 Introduction 1
1.1 Aims and Objective 4
CHAPTER TWO
2.0 Literature review 5
2.1 An Overview of Hibiscus sabdariffa
(Zobo) Drink 5
2.2 Bacteriological Quality of Beverages 6
2.2.1 Control of
Microbiological Quality of Beverages 7
2.2.2 Hazard
Analysis and Critical Control Point (HACCP) 9
2.3
Uses of Hibiscus sabdariffa 11
2.3.1
Traditional culinary use 11
2.3.2
Use in Local and Traditional Food and Medicine 11
2.3.3
Animal Feed 12
2.3.4
Cosmetic 12
2.4
Phytochemistry of Hibiscus sabdariffa 12
2.4.1
Nutritional Value 12
2.4.2
Bioactive Constituents 12
2.5
Biological and Pharmacological Activities of Hibiscus sabdariffa 13
2.5.1
Antibacterial, Antifungal and Antiparasite Activity 13
2.5.2
Antioxidant Activity 13
2.5.3
Hepatoprotective Activity 14
2.5.4
Cancer-Preventive Activity 15
2.5.5
Anti-Diabetic Activity 15
2.6
Overview of Some Important Leaves with Antimicrobial Properties 15
2.6.1 Piper guineense 15
2.6.2 Moringa oleifera 17
2.6.3 Ocimum gratissimum 20
CHAPTER THREE
3.0
Materials and Methods 24
3.1
Collection of Samples 24
3.2
Preparation of Zobo drink 24
3.2.1
Blending of Zobo drink with leaf extracts 25
3.3
Storage of samples 26
3.4
Sterilization of materials 26
3.5
Microbiological analysis 26
3.5.1
Bacterial isolates 26
3.5.2
Fungal isolates 27
3.6
Biochemical tests 27
3.6.1
Catalase test 27
3.6.2
Coagulase test 27
3.6.3
Oxidase test 28
3.6.4
Sugar fermentation 28
3.6.5
Spore staining test 29
3.6.6
Gram staining 29
3.7
Lactophenol cotton blue stain reaction 30
3.8
Physicochemical analysis 30
3.8.1
pH determination 30
3.8.2
Determination of titratable acidity (%) 30
3.8.3
Determination of ascorbic acid (vitamin C) 31
CHAPTER FOUR
4.0
Results 32
CHAPTER FIVE
5.0
Discussion, conclusion and recommendation. 42
5.1
Discussion 42
5.2
Conclusion 45
5.3
Recommendations 45
REFFERENCES
LIST OF FIGURES
Fig
1: Diagram
for the preparation of zobo drink
LIST OF TABLES
Table
4.1:
Bacterial counts of the different zobo blends during storage
Table
4.2:
Fungal counts of different zobo blends during storage
Table
3: Changes
in titratable acidity of zobo drinks during storage
Table
4: Changes
in pH of stored zobo drinks
Table
5: Changes
in vitamin C content of stored zobo drinks
Table
6: Identification
of isolated bacteria: Result of cultural, morphological and biochemical test
Table
7: Identification
of fungal isolates
CHAPTER ONE
1.0 INTRODUCTION
Zobo drink, a
non-alcoholic local beverage, it is produced from the dried petals of Hibiscus sabdariffa. Zobo drink has been
shown to be a good source of natural carbohydrate, protein and vitamin C
(Ogiehor et al, 2007). It is locally
called “zobo rodo” (Hausa), the Yoruba call the leaves “Amukan”, while the
flower is called “Isapa”. The Igbos call it names like “Ojo”, “Akwaroazo” and
sorrel in English and is a delicacy in many parts of Nigeria (Adebayo-Tayo and
Samuel, 2008). The H.sabdariffa plant, commonly called Roselle,
while native to India and Malaysia is now found in many tropical and
subtropical countries of Africa, Asia and the Americas (Bola and Aboaba, 2004).
It is a dicotyledonous plant belonging to the subclass Archichlamydea, order
Malvale and family Malvaceae (Shivali and Kamboj, 2009).
Zobo drink is
prepared first by boiling the dried leaves of the Roselle, followed by cooling
and filtration. The filtrate, which is red in colour maybe taken hot as tea or
sometimes sweetened to taste with pineapple, apple, orange or sugar and spiced
up with ginger depending on choice. It is further allowed to cool and is best
served chilled (Egbere et al, 2007).
Due to Economic,
increased religious and health campaigns against alcoholic beverages in Nigeria
and the consequent increase in the consumption of alcoholic beverages in
certain areas; zobo drink has great potential as a local alternative to
imported red wines in particular and alcoholic beverages in general (Egbere et al, 2007). Moreover, production of
this and similar local beverages has become the main source of income in many
homes in the rural communities and more recently in the urban areas where these
have grown to cottage business properties due to support from the government
through the poverty alleviation schemes, thereby alleviating poverty among the
people (Essien et al, 2011).
Phytochemical
analyses of extracts of various parts of the Roselle plant reveal the high
nutritive and medicinal value of this plant. The flower is reported to contain
carbohydrates and sugars like sucrose and mannose. It also contains proteins,
fats and vitamins with other acids. The seeds contain starch, cholesterol,
cellulose, some acids (Oleic acid, Formic acid) and alcohols. The leaves
contain alcohols, malic acid, fibre and ash. The fruit contain acids (formic
acid, acetic acid), alcohols, pectin and minerals. The roots contain tartaric
acid and saponin (Shivali and Kamboji, 2009).
Medicinal value of
aqueous extracts from the Roselle plant has been reported to include
anti-hypersensitive, antiseptic, astringent , diuretic and purgative
activities, remedy for cancer, abscesses, cough, dysuria, laxative, scurvy and
fever (Osueke and Ehirim, 2004). In spite of its health and nutritional
benefit, zobo drink is often contaminated with enteropathogenic microorganisms
with as much as 2.49 x 104cfu, which could be harmful to persons who
consume large quantities of the drink (Bukar et al, 2009). Major points of contamination of the zobo drink
include: the packaging material, as most retailers package the drink in already
used plastic bottles and polythene bags, which are not properly disinfected
prior to packaging (Nwafor and Ikenebomeh, 2009). The dried calyces are also a
major point of contamination as they harbor spoilage organisms such as Penicillum and Aspergillus sp. (Amusa et al,
2005) and the retailers, who seldom prepare the drink under aseptic conditions
and often do not do enough boiling to reduce the microbial load in the
preparation of the beverage. Some of the microbes commonly found in the drink
include Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Aspergillus sp. amongst a host of other (Nwachukwu et al, 2007). The shelf life of the zobo
drink depend on various factors such as the packaging material, contamination
during preparation and refrigeration to mention a few, however, it has an
average shelf life of 24 to 48 hours after which spoilage organisms may begin
to reduce the quality of the zobo (Nwafor and Ikenebomeh, 2009).
Despite the fact
that the popularity of zobo juice is increasing, one of its greatest limitation
for large scale production is that it has very short shelf life if not
refrigerated (Omemu et al, 2006).
Therefore, there is urgent need to explore various preservation methods that
could be employed to extend the shelf life of this product. Dougheri et al. (2007) employed some chemical
preservatives to improve the shelf life of zobo drink. They reported that only
samples treated with benzoic acid remained organoleptically attractive after 14
days of storage. However, the problem with the use of chemical preservatives in
food is that they tend to have adverse effects on the health of consumers
(Adesokan et al, 2010). Therefore,
naturally occurring plants with proven antimicrobial properties will be
preferred in food preservation (Kolapo et
al, 2007). The use of natural preservatives has become more popular as compared
to synthetic antimicrobials and antioxidants (Aliu et al, 2007). Moreover, the extracts of traditional natural spices
have been shown to have a broad spectrum antibacterial activity, including
effects on Escherichia, Salmonella,
Staphylococcus, Streptococcus, Klebsiella, Proteus, Clostridium, Mycobacterium
and Helicobacter species (Groppe et al., 2002: Sagdic, 2003; Shan et al., 2007). These magnitude of
success observed in vitro have not
been reported in vivo particularly in
food items that must be cooked before eating. For example, Bello and Osho (2012) reported some measure
of antimicrobial activity of spices on spoilage organisms isolated from
moin-moin in vitro, Ayoade et al., (2012) however observed no
effects of these spices on the progression of spoilage in this food item in vivo. The absence of antimicrobial
effect in vivo was ascribed to the
sustained heat used in cooking the moin-moin which is thought to have a
suppressing effect on the antimicrobial properties of these spices. This kind of
wide differences observed in vivo and
in vitro is not expected in food
items like zobo where no cooking is required. Transitioning zobo from a locally
marketed to commercial product status is hampered due to its poor shelf life of
24 to 48 hours, which would require little inventory.
1.1
AIMS
AND OBJECTIVE
This work is aimed
at improving the microbial and physicochemical quality of zobo drinks, thereby
increasing the shelf life using leaf extracts namely Moringa oleifera, Ocimum gratissimum, and Piper guineense.
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