ABTRACT
Two Lactobacillus specie isolates designated L1 and L2, isolated from fermented maize (Akamu) and identified as Lactobacillus plantarum and Lactobacillus brevis respectively. Each isolate was used at three different treatment concentrations (0.5ml, 1.0ml and 2.0ml) to treat and preserve locally produced watermelon juice stored at ambient temperature for seven days. Bacteriocin extracted from each isolate was also used in the preservation of watermelon juice. The watermelon juice was divided into nine samples (control, L1 0.5ml, L1 1.0ml, L1 2.0ml, L2 0.5ml, L2 1.0ml, L2 2.0ml, L1Bacteriocin and L2Bactriocin). After inoculation, the juice samples were subjected to chemical and microbial analyses on 24 hour-interval for seven days. The microbial analysis showed that the control, L1 0.5ml and L2 0.5ml had higher bacterial load of 5.9 x 105cfu/ml 4.4 x105cfu/ml and 4.6 x 105cfu/ml respectively for four storage days, and decreased later, and fungal load of 5.9 x 103cfu/ml 5.2 x 103cfu/ml and 5.2 x 103cfu/ml respectively. The samples preserved with L1 2.0ml and L2 2.0ml had lower bacterial load of 2.8 x 105cfu/ml and 2.9 x 105cfu/ml respectively and fungal load of 2.3 x 103cfu/ml for each. The results of this study revealed that the concentration of preservative affects the nature and microbial load of micro-organisms found in fruit juices during storage.
TABLE OF CONTENTS
Title i
Certification ii
Dedication iii
Acknowledge iv
Table of contents v
List of tables viii
List of figures ix
Abstract x
Chapter One
1.1 Introduction 1
1.2 Aim and objectives of the study 6
Chapter Two
2.1 Origin and distribution of watermelon 7
2.2 History Review of watermelon 7
2.3 Watermelon varieties 9
2.4 Nutritional and medicinal value of
watermelon 12
2.5 Economic importance and uses of
watermelon 16
2.6 Lactic acid bacteria 17
2.7 Lactobacillus
19
2.7.1 Classification of Lactobacillus species 19
2.7.2 Metabolites from Lactobacillus 21
2.7.3 Antimicrobial activity of Lactobacillus 23
Chapter Three
3.1 Procurement of materials 25
3.2 Isolation of Lactobacillus species 25
3.2.1 Characterization of isolates 25
3.2.2 Biochemical tests 26
3.2.3 Sugar utilization test 26
3.3 Identification of Lactobacillus isolates 26
3.4 Production of watermelon juice 26
3.5 Treatment of juice with Lactobacillus 27
3.6 Shelf life studies 28
3.6.1 Determination of total solid content 28
3.6.2 Determination of total titratable acidity 29
3.6.3 Determination of pH 30
3.6.4 Determination of microbial load 30
3.6.5 Sensory evaluation 31
3.7 Statistical analyses 32
Chapter Four
Results 33
Chapter Five
5.1 Discussion 48
5.6 Conclusion 50
5.2 Recommendation 50
References 51
LIST OF TABLES
Tables Title page
1 Summary of the different varieties of
watermelon 11
2 Phytoconstituents of Citrullus lanatus 15
3 Metabolic products of Lactobacillus
species with antimicrobial properties 21
4 Morphological, physiological and
biochemical characteristics of isolates 35
5 The bacterial count of the preserved
watermelon juice 45
6 The fungal count of the preserved
watermelon juice 46
7 Mean scores of the sensory evaluation
of the watermelon juice on day 4 47
LIST OF FIGURES
Figures Title page
1 The damage caused by an inhibitor
agent (Enterocin AS-48)
produced
by a LAB strain on L. Monocytogenes cell 18
2 The pH, total titratable acidity,
total solid and sugar level tests for control 36
3 The pH, total titratable acidity,
total solid and sugar level tests for L1 0.5ml 37
4 The pH, total titratable acidity,
total solid and sugar level tests for L1 1.0ml 38
5 The pH, total titratable acidity,
total solid and sugar level tests for L1 2.0ml 39
6 The pH, total titratable acidity,
total solid and sugar level tests for L2 0.5ml 40
7 The pH, total titratable acidity,
total solid and sugar level tests for L2 1.0ml 41
8 The pH, total titratable acidity,
total solid and sugar level tests for L2 2.0ml 42
9 The pH, total titratable acidity,
total solid and sugar level tests for L1 bacteriocin 43
10 The pH, total titratable acidity, total
solid and sugar level tests for L2 bacteriocin 44
CHAPTER ONE
1.1 INTRODUCTION
The
increasing demand for fresh-tasting, healthy, nutritious and ready-to-eat foods
has stimulated the expansion of minimally processed fruit and vegetable markets
worldwide (Abadias et al., 2008 and
Oms-Oliu et al., 2010). Processing of
the products resulting from natural fruits and vegetables has been observed to
increase certain reactions leading to susceptibility to microbes. From a
consumer perspective, increasing scientific evidence for consumption of fresh
fruits for prevention of biological problems, demand for low-calorie diet and
increasing microbiological and pesticide content in processed food has
increased the consumption of ready-to-eat vegetables and fruits (Rico et al., 2007). This health based option
for customers has been short lived due many inappropriate or manipulative storage
conditions that again lead to microbiological spoilage and disease (Abadias et al., 2008).
Fruits and vegetables are an
essential part of human nutrition. Unfortunately, the daily intake of fruits
and vegetables is estimated to be lower than the recommendation of the World
Health Organization (WHO), who suggest a dietary intake of 450g and 500g of
fruits and vegetables, respectively. Vegetables and fruits are strongly
recommended in the human diet because they are rich in antioxidants, vitamins,
dietary fibres and minerals. The majority of vegetables and fruits consumed in
the human diet are fresh, minimally processed, pasteurized or cooked by boiling
in water or microwaving, and vegetables can be canned, dried, or juiced or made
into paste, salads, sauces, or soups (Dalia et
al., 2015).
Fresh vegetables or those that
have been minimally processed have a particularly short shelf-life because they
are subjected to rapid microbial spoilage. In addition, the above cooking
processes can cause a number of potentially undesirable changes in physical
characteristics and chemical composition. Therefore, these drawbacks could be
reduced by novel technologies, such as new packaging systems, high-hydrostatic
pressure processing, ionization radiation and pulsed electric fields. The use
of natural antimicrobial preservatives is considered to be the simplest and
most valuable biological technique to keep and/or enhance the safety,
nutrition, palatability and shelf life of fruits and vegetables (Devlieghere et al., 2004).
Modern technologies in food processing and microbiological food safety
standards have reduced but not eliminated the likelihood of food-related
illness and product spoilage in industrialized countries. Food spoilage refers
to the damage of the original nutritional value, texture and flavor of the food
that eventually renders the food harmful to people and unsuitable to eat. The
increasing consumption of precooked food, prone to temperature abuse, and the
importation of raw foods from developing countries results in outbreaks of
food-borne illness (Nath et al., 2014).
One of the concerns in food industry is the
contamination by pathogens, which are frequent causes of food borne diseases.
In order to achieve improved food safety against pathogens, food industry makes
use of chemical preservatives or physical treatments (e.g. high temperatures).
These preservation techniques have many drawbacks which includes the proven
toxicity of the chemical preservatives (e.g. nitrites), the alteration of the
organoleptic and nutritional properties of foods, and especially recent
consumer demands for safe but minimally processed products without additives. Currently,
there is a strong debate about the safety aspects of chemical preservatives due
to impairment/reduction of the nutritional value of food, episodes of adverse
food reactions, cardiovascular disease, many carcinogenic and teratogenic
attributes as well as residual toxicity (Calo-mata et al., 2008). Processing at high temperature extensively damages
the organoleptic, nutritional and physicochemical properties of the food.
Refrigerators are either expensive to maintain or means for their maintenance
(electricity) are lacking and this method of preservation makes the food prone
to microbial and other sources of contamination (Amin, 2014).
To
harmonize consumer demands with the necessary safety standards, traditional
means of controlling microbial spoilage and safety hazards in foods are being
replaced by combinations of innovative technologies that include biological
antimicrobial systems such as lactic acid bacteria (LAB) and/or their
metabolites (Nath et al., 2013). The
increasing demand for safe food has increased the interest in replacing
chemical additives by natural products, without injuring the host or the
environment. Hence, the last two decades have seen
intensive investigation on Lactic acid bacteria (LAB) and their antimicrobial
products to discover new bacteriocinogenic LAB strains that can be used in food
preservation.
Biopreservation
refers to extended storage life and enhanced safety of foods using the natural microflora
and (or) their antibacterial products. It can be defined as the extension of
shelf life and food safety by the use of natural or controlled microbiota
and/or their antimicrobial compounds (Ananou et al., 2007). One of the most common forms of food biopreservation
is fermentation, a process based on the growth of microorganisms in foods,
whether natural or added. It employs the breakdown of complex compounds,
production of acids and alcohols, synthesis of Vitamin-B12, riboflavin and
Vitamin-C precursor, ensures antifungal activity and improvement of
organoleptic qualities such as, production of flavor and aroma compounds. The organisms involved mainly comprise lactic acid bacteria,
which produce organic acids and other compounds that, in addition to
antimicrobial properties, also confer unique flavours and textures to food
products. Compounds such as organic acids, bacteriocins, diacetyl and
acetaldehyde, enzymes, CO2, hydrogen peroxide etc. contribute to antimicrobial
activity by microbiota (Nath et al.,
2014 and Ananou et al., 2007).
Traditionally, a great number of foods have been protected against
spoiling by natural processes of fermentation. Currently, fermented foods are increasing
in popularity (60% of the diet in industrialized countries) (Holzapfel et al., 1995) and, to assure the
homogeneity, quality, and safety of products, they are produced by the
intentional application in raw foods of different microbial systems
(starter/protective cultures). The starter cultures can be defined as preparations
of one or several systems of microorganisms that are applied to initiate the
process of fermentation during food manufacture (Wigley, 1999), fundamentally
in the dairy industry and, currently, extended to other fermented foods such as
meat, vegetable products, and juices. The bacteria used are selected depending
on food type with the aim of positively affecting the physical, chemical, and
biological composition of foods, providing attractive flavour properties for
the consumer. To be used as starter cultures, microorganisms must fulfill the
standards of Generally Recognized As Safe (GRAS) status by people and the
scientific community and present no pathogenic nor toxigenic potential (Dass,
1999).
For the starter cultures, generally LAB, metabolic activity, such
as acid production in cheese, is of great technological importance, whereas
antimicrobial activity is secondary. However, for the protective culture,
generally LAB also, the objectives are the opposite and must always take into
account an additional factor for safety as its implantation must reduce the
risk of growth and survival of pathogenic microorganisms (Holzapfel et al., 1995). An ideal strain would
fulfill both the metabolic and antimicrobial traits.
The hurdle concept stated that the microbial safety, stability, sensorial,
and nutritional qualities of foods are based on the application of combined
preservative factors (called hurdles) that microorganisms present in the food
are unable to overcome. Thus, hurdle technology refers to the combination of
different preservation methods and processes to inhibit microbial growth (Leistner,
1978). An intelligent application of this technology requires a better
understanding of the occurrence and interaction of different hurdles in foods
as well as the physiological responses of microorganisms during food
preservation.
Using an adequate mix of hurdles is not only economically
attractive; it also serves to improve not only microbial stability and safety,
but also the sensory and nutritional qualities of a food. In the past and often
still today, hurdle technology has been applied empirically without knowledge
of the governing principles in the preservation of a particular food. In
industrialized countries, hurdle technology is of great interest in the food
industry for extending the shelf life and safety of minimally processed foods,
such as those that display low fat contents and/or salt (Leistner, 2000).
Similarly, it is applied in fermented or refrigerated foods in which low
temperature is often the only hurdle to be overcome (e.g. during distribution),
which can lead to the alteration and intoxication of the foods. In developing countries,
most foods are stored without refrigeration and are stabilized by the empiric
use of hurdle technology. Several traditional foods have already been optimized
by the intentional application of hurdles
for safety and stability enhancement (Ananou, 2007).
The need to incorporate novel and effective combinations has
spurred interest for natural and biological preservatives such as Lactic Acid Bacteria
and their antimicrobial compounds.
1.2 AIM AND OBJECTIVES OF THE STUDY
The aim of this study was to evaluate potential use of some
species of Lactobacillus as biopreservative
agents in fruit (watermelon) juice. Therefore, the work has been developed with
the following objective:
1. To isolate, identify and characterize potentially antagonistic Lactobacillus species found in locally
prepared fermented maize (Akamu).
2. To use some of the Lactobacillus
species isolated from the fermented maize (Akamu) to extend the shelf life of
locally prepared watermelon juice.
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