ABSTRACT
This study determined the effect of local plant preservatives on the microbiology and shelf life stability of palm wine. Fresh palm wine samples collected from traditional palm wine tappers in Umuahia were used for this analysis. The microbiological analysis of the palm wine samples was carried out and this showed high viable counts for both bacteria (TAPC = 6.9 × 106 and 6.4 × 106, TCPC = 5.2 × 106 and 4.6 × 106, TLABC = 3.6 × 106 and 6.1 × 106) for samples A and B respectively fungi (TFPC = 4.0 × 106 and 5.3 × 106) also for samples A and B. Also pathogenic microorganisms were isolated from the palm wine samples. The microorganisms isolated in this study were Bacillus subtilis, Escherichia coli, Enterobacter species, Lactobacillus species, Klebsiella species, Saccharomyces cerevisae, Aspergillus flavus and Rhodotorula species of which Saccharomyces cerevisae was the most predominant. The extracts of local plants such as lime, bitter leaf, scent leaf and black pepper were tested against the bacteria and fungi isolates. The lime juice extracts had the highest inhibitory activity producing a zone of inhibition of 14 mm while the black pepper extract exhibited the lowest inhibitory activity with an inhibition zone of 6 mm. This signified that the plant extracts had inhibitory effects on the microorganisms present in palm wine. Furthermore, the shelf life of the palm wine samples was studied and the results showed a reduction in cell counts when palm wine was treated with different concentrations (0.5, 1.0, 1.5 and 2.0 mL) of the plant extracts. The cell counts were observed to decrease considerably as the concentration of the plant extracts increased. The ethanolic extract of scent leaf and aqueous extract of lime juice had the lowest counts of 40 cfu/ml and 120 cfu/ml after 72 hrs. This showed that scent leaf and lime juice were the best plant preservatives amongst the studied plants to be used for preservation of palm wine. From this study, it can be deduced that these plant extracts studied can be used to prolong the shelf life of palm wine as it significantly reduces the number of microorganisms present in palm wine.
TABLE
OF CONTENTS
Title
page i
Certification ii
Dedication iii
Acknowledgments iv
Table
of contents v
List
of tables viii
List
of Figures ix
Abstract x
CHAPTER ONE
Introduction 1
1.1 Aim and Objectives 3
1.1.1 Objectives
3
CHAPTER TWO
Literature
Review 4
2.1 Description of Palm Tree 4
2.2 The African Oil Palm Tree (Elaeis guineensis) 5
2.3 Description of the Palm Sap 6
2.4 Palm Wine 6
2.4.1 Nutritional Content of Palm Wine 7
2.4.2 Tapping of Palm Wine 8
2.4.3 Palm Wine Fermentation 8
2.4.4 Microorganisms Involved in the Fermentation
of Palm Wine 9
2.4.5 Uses of Palm Wine 10
2.5 Bitter Leaf Plant (Vernonia amygdalina) 11
2.5.1 Uses 12
2.5.2 Antimicrobial Properties 12
2.6 Basil Plant (Scent leaf) (Ocimum gratissimum) 13
2.6.1 Importance 13
2.6.2 Antimicrobial Properties 13
2.7 Lime (Citrus
aurantifolia) 14
2.7.1 Uses 14
2.7.2 Antimicrobial Properties 16
2.8 Black Pepper Plant (Piper nigrum) 17
2.8.1 Importance 18
2.8.2 Antimicrobial Properties 19
2.9 Empirical Studies 19
CHAPTER THREE
Materials and Methods 26
3.1 Sample
Collection 26
3.2 Materials 26
3.3 Plant Materials 27
3.3.1 Extraction
of Plant Materials 27
3.4 Microbiological
Analysis 28
3.4.1 Serial
Dilution 28
3.4.2 Inoculation
of Samples 28
3.5 Colony
Count of Isolates 28
3.6 Characterization
and Identification of Isolates 29
3.7 Biochemical
Analysis of the Isolates 29
3.7.1 Gram
Staining 29
3.7.2 Catalase
Test 30
3.7.3 Coagulase test 30
3.7.4 Oxidase Test 31
3.7.5 Citrate Test 31
3.7.6 Motility Test 31
3.7.7 Indole Test 32
3.7.8 Methyl Red Test 32
3.7.9 Vogues Proskauer Test 32
3.7.10 Carbohydrate Fermentation Test 32
3.8 Isolation, Characterization and
Identification of Fungi from Fermented
Palm Wine 33
3.9 Antimicrobial Screening of Plant Extracts 33
3.10 Effects of Plant Extracts on the Shelf Life
of Palm Wine 34
CHAPTER FOUR
Results 35
CHAPTER FIVE
Discussion,
Conclusion and Recommendation 53
5.1 Discussion 53
5.1 Conclusion
and Recommendation 57
References 58
LIST OF TABLES
|
TABLE
|
TITLE
|
PAGE
|
|
4.1
|
Identification and Characterization of Bacteria Isolated from Palm Wine
Samples
|
38
|
|
4.2
|
Identification and Characterization of Fungi Isolated from Palm Wine
Samples
|
39
|
|
4.3
|
Total Viable Microbial Count of Palm Wine Samples
|
40
|
|
4.4a
|
Percentage Occurrence of Bacterial Isolates in
the Palm Wine Samples
|
41
|
|
4.4b
|
Percentage Occurrence of Fungal Isolates in the
Palm Wine Samples
|
42
|
|
4.5a
|
Antimicrobial Screening of Plant Ethanol Extract against
Isolates
|
43
|
|
4.5b
|
Antimicrobial Screening of Plant Water Extract against Isolates
|
45
|
|
4.6
|
Cell Counts after Treatment of Palm Wine
with Ethanolic Extracts of Different Plants
|
47
|
|
4.7
|
Cell Counts after Treatment of Palm Wine
with Ethanolic Extracts of Different Plants
|
48
|
|
| | |
LIST OF FIGURES
|
FIGURE
|
TITLE
|
PAGE
|
|
4.1
|
Effects of Ethanolic Extract of Different Plants on the pH of
the Palm Wine Sample
|
49
|
|
4.2
|
Effects of Aqueous Extract of Different Plants on the pH of the
Palm Wine Sample
|
50
|
|
4.3
|
Effects of Ethanolic Extract of
Different Plants on the Temperature of the Palm Wine Sample
|
51
|
|
4.4
|
Effects of Aqueous Extract of Different Plants on the
Temperature of the Palm Wine Sample
|
52
|
CHAPTER
ONE
INTRODUCTION
Palm wine is a fermented
traditional beverage consumed in many parts of the world and well known as a
white coloured alcoholic drink. The drink is known by different names around
the world and is regarded as a heavy suspension of yeasts in fermenting palm
sap (Nwaiwu et al., 2016). Palm wine
is a generic name for a group of alcoholic beverages obtained from the
fermentation of the sap of palm trees. Nigerian palm wine is produced from Elaeis guineensis (oil palm tree) and Raphia species. The palm wine is very
popular amongst the inhabitants of West Africa (Morah and Robinson, 2015). It
is an alcoholic beverage produced by the spontaneous yeast/lactic fermentation
of the sugary sap of palms or may be referred to as a popular traditional
alcohol consumed by more than ten million people in West Africa. Even though
palm wine is consumed around the world and plays an important role in the
economic and social life of the people, palm wine has not been comprehensively
evaluated for quality improvement and possible exploitation of the biological
and chemical constituents or byproducts (Nwaiwu et al., 2016).
The unfermented sap is
clean, sweet, colourless syrup containing above 10-12% sugar, which is mainly
sucrose. Upon fermentation by the natural microbial flora, the sugar level
decreases rapidly as it is converted to alcohol and other products whereas, the
sap becomes milky-white due to the increased microbial suspension resulting
from the prolific growth of the fermenting organism (Ogbulie et al., 2007). Yeasts, lactic acid
bacteria (LAB) and acetic acid bacteria (AAB) are the most reported microbial
constituent in literature and the species Saccharomyces
cerevisae is normally the main organism of interest because it is mainly
responsible for converting the sugary sap to alcohol. Palm wine drinkers know
that the drink tastes differently at different stages of fermentation as a
result of yeast fermentation and accumulation of organic acids especially
acetic acid from fermentation by AAB as fermentation progresses each day
(Nwaiwu et al., 2016). Previous
studies on the microbiology of E.
guineensis and R. hookeri have
incriminated several bacterial and yeast flora to be involved in the
fermentation process. These organisms have also been reported to originate from
several sources which include tapping equipment, containers and the environment
(Ogbulie et al., 2007).
Plants and spices are
excellent sources of biologically active compounds with potential antimicrobial
activity. Essential oils, secondary metabolites produced by plants, have
valuable capability of suppressing growth of wide variety of food-spoilage and
food-borne microorganisms including bacteria, yeasts and molds (Khaled, 2017).
Occurrence of bioactive compounds in plants or spices/herb extracts is the
basis to the antimicrobial effects which at time protects the plant themselves
against microorganisms, fungus and viral infectivity (Tagoe et al., 2010). They can be extracted
from different parts of plants including flowers, roots, bark, leaves, seeds,
peel, fruits, wood, buds and the entire plant (Sanchez et al., 2010; Khaled, 2017).
Compilation of data
proves that natural antimicrobial/antioxidants, fruits and vegetable extracts
have significant consideration to be used in food as food additives or natural
preservatives with intention for multidimensional quality (flavour, aroma) and
nutritional level enhancement as well as antimicrobial property (Velu et al., 2014). In Nigeria, for example,
it has been estimated that over 40% of known plants serve as food whereas about
30% serve as spices and medicinal plants (Nwobegu, 2002). The spices are used
to give aroma and flavour to food and at the same time they can serve as food
preservatives because they possess active ingredients which are either
microbistatic or microbicidal (White, 2006; Okigbo and Igwe, 2007). Velu et al. (2014) also suggested that the
main advantages of natural antimicrobial agents were focused in the field of
food safety, quality and preservation; as a controlling agent for microbial
contamination in food and in lengthening food products shelf life by removal of
unpleasant food borne pathogens or spoilage bacteria. Some of the most
important bioactive constituents are alkaloids, tannins, flavonoids and
phenolic compounds which can be found in plants. Some plants have been studied
and have been noted to have antimicrobial properties and have been successfully
used as preservatives in food. Among these plants are Vernonia amygdalina (bitter leaf plant), Ocimum gratissimum L. (basil plant also known as scent leaf),
Citrus aurantifolia (lime plant) and Piper nigrum (black pepper plant).
1.1 Aim and Objectives
The aim of this research
is to determine the microbiological flora of palm wine and understanding how
the presence of different plant preservatives affect the microbiology and shelf
life stability of palm wine.
1.1.1 Objectives
1. To
isolate, characterize and identify the microorganisms present in palm wine.
2. To
prepare the ethanolic and water (aqueous) extracts of the various local plants.
3. To
determine the effect of different local plant extracts on the isolates from
palm wine.
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