ABSTRACT
This
study investigates the antifungal effects of essential oils extracted from
Rosemary (Rosmarinus officinalis), Eucalyptus (Eucalyptus globulus), and Lemon
(Citrus limon) on fungi isolated from spoilt bread. The primary objectives are
to isolate and identify fungi responsible for bread spoilage using Sabouraud
Dextrose Agar (SDA), examine the antifungal sensitivity of the essential oils,
compare their efficacy, and characterize their chemical compositions. Bread
samples were obtained from Michael Okpara University of Agriculture, Umudike,
and allowed to spoil naturally over 3-4 days before fungal isolation. Essential
oils were sourced online and analyzed using Gas Chromatography-Mass
Spectrometry (GC-MS) to determine their chemical profiles. Statistical analysis
was performed using ANOVA at a 95% confidence level.
The
findings identified Rhizopus sp. and Aspergillus sp. as predominant spoilage
fungi. The antifungal sensitivity tests, conducted via agar incorporation
method, revealed that Rosemary essential oil exhibited the highest inhibitory
effect on both Aspergillus sp. (100%) and Rhizopus sp. (98.79%). Eucalyptus oil
showed moderate inhibition, more effective against Rhizopus sp. (93.98%)
compared to Aspergillus sp. (50%). Lemon oil displayed substantial inhibition
on Rhizopus sp. (96.39%) and moderate inhibition on Aspergillus sp. (86.66%).
These results suggest that essential oils are more effective against Rhizopus
sp., possibly due to the aflatoxin production by Aspergillus sp. which may
neutralize the oils' effects.
The
study concludes that essential oils, particularly Rosemary oil, can
significantly inhibit fungal growth in bread, suggesting potential for natural
preservation methods. Proper pricing strategies and exclusive distribution
policies are crucial for new product success. Promotional campaigns and
thorough product planning, considering customer needs, enhance acceptance and
longevity of new products in the market. However, the intense aroma and
potential toxicity of essential oils limit their use in food preservation,
requiring further research into encapsulation techniques and combination with
other preservation methods. The economic feasibility and regulatory standards
for essential oil use also need to be addressed to optimize their application
in food safety and quality enhancement.
TABLE OF CONTENTS
CHAPTER
ONE
INTRODUCTION
1.1 Background
1.2 Aim
of study
1.3 Objectives
of study
1.4 Hypothesis =
1.5 Statement
of problem
1.6 Significance
of study
CHAPTER
TWO
LITERATURE
REVIEW
2.1 Bread
2.2 Sources
of contamination
2.2.1 Air
and dust
2.2.2 Soil,
water and plants
2.2.3 Food
handlers
2.2.4 Food
utensils
2.2.5 Cross-contamination
2.2.6 Unsafe
temperature
2.3 Types
of spoilage in bread
2.3.1 Undesirable
high acidity
2.3.2 Ropiness
2.3.3 Bloody
bread
2.3.4 Moldiness
2.4 Mycotoxins
2.4.1 Aflatoxins
2.4.2 Ochratoxins
2.5 Microorganisms
of spoilt bread
2.5.1 Aspergillus sp
2.5.2 Rhizopus sp
2.6 Description
of the plants
2.6.1 Rosemary
– Rosmarinus officinalis
2.6.2 Eucalyptus
– Eucalyptus globulus
2.6.4 Lemon
– Citrus limon
2.7 Taxanomy
of the plants
2.7.1 Rosemary
– Rosmarinus officinalis
2.7.3 Lemon
– Citrus limon
2.8 Extraction
of essential oil
2.8.1 Distillation
(Hydrodistillation)
2.8.2 Expression
CHAPTER
THREE
MATERIALS
AND METHODS
3.1 Sample
collection of bread
3.2 Collection
of essential oils of plants
3.3 Preparation
of media
3.4 Isolation
of microorganisms from spoilt bread
3.5 Identification
of fungi
3.5.1 Staining
and viewing of growth on slide and cover slip
3.6 Antifungal
sensitivity tests
3.6.1 Agar
well diffusion assay
3.6.2 Agar
incorporation assay
3.7 Statistical
analysis
CHAPTER
FOUR
RESULTS
CHAPTER FIVE
Discussion
Conclusion and Recommendation
References
LIST
OF TABLES
Table 4.1: Colonial features of the
isolates
Table 4.2: Microscopic observations
of the isolates
Table 4.3: Zones of inhibition
observed using the agar incorporation method
Table 4.4: Zones of inhibition
observed using the agar well diffusion method
Table 4.5: Statistical analysis
Table 4.6: Levene’s test for equality
of variances (t-test for equality of means)
Table 4.7: Characterization of Rosmarinus officinalis essential oil
Table 4.8: Characterization of Eucalyptus globulus essential oil
Table 4.9: Characterization of Citrus limon essential oil
CHAPTER ONE
INTRODUCTION
1.1 Background
In
baking industry, bread occupies a unique position both in production and
utilization as compared to other bakery products. The ingredients of bread are
supportive to growth of microorganisms and multiplication at different stages
of bread production, slicing and wrapping. The main types of microbial spoilage
of bread are moldiness and ropiness which are troublesome for bakers. Ropiness
won’t be studied due to the fact that it is caused by bacterial organism which
unfortunately is not a fungal organism and as a result, not in the scope of
study for this research work. Mold growth often begins within a loaf of sliced
bread, where more moisture is available than at the surface, especially in the
crease (Liaqat, 1988).
Food
safety is a known problem worldwide, affecting hundreds of millions of people
that suffer from contaminated food. World Health Organization (WHO) defines
this issue as “one of the most widespread health problems and a major cause of
the reduction in economic productivity (WHO, 2007).
Nowadays,
consumers are continuously concerned with the growing number of illness caused
by some pathogenic and spoilage microorganisms in food and also for the safety
of foods containing synthetic preservatives. Thus, it shows a growing interest
about the replacement of synthetic preservatives with natural, effective and
nontoxic antimicrobial compounds. There is growing interest in using natural
antimicrobial compounds, such as extracts and essential oils (EOs) of species
and herbs, for food conservation. The effect of plant extracts on
microorganisms have been studied by a large number of researchers in different
parts of the world (Kumar et al.,
2006; Mathabe et al., 2006) and the
use of a variety of plant extracts and phytochemicals, both with known antimicrobial
properties, can be of great significance in therapeutic treatments. Many plants
have been used because of their antimicrobial properties, which are due to
compounds synthesized in the secondary metabolism of the plant. These products
are known by their active substances, such as, the phenolic compounds which are
part of the essential oils, as well as in tannin (Nascimento et al., 2000).
Since,
bread is an important part of our daily diet; therefore, ways and means should
be explored to increase its shelf life. The shelf life of bread can be
increased by improving the hygienic conditions of mixing and baking premises
and ensuring sterilized environment. To enhance the shelf life of bread,
several chemical antimicrobial agents have been employed but they are
considered responsible for many carcinogenic and teratogenic attributes and
residual toxicity. Due to these reasons, consumers tend to be doubtful of
chemical additives and thus the demand for natural preservatives has been
intensified (Skandamis et al., 2001).
Essential oils are volatile oily liquids
obtained from different plant parts and widely used as food flavors (Burt,
2004). They are variable mixtures of essential terpenoids, especially
monoterpenes (C10) and sesquiterpenes (C15), although diterpenes (C20) may also
be present, and of a variety a low molecular weight aliphatic hydrocarbons,
acids, alcohol, aldehydes, phenolic compounds, acyclic esters, or lactones
(Rota et al., 2004). The composition,
structure, as well as functional groups of the oils play an important role in
determining their antimicrobial activity. It has been demonstrated that the
essential oils exercises their antimicrobial activity by causing structural and
functional damages to the microbial cell membrane. It is also indicated that
the optimum range of hydrophobicity is involved in the toxicity of the
essential oils (Goni et al., 2009).
Essential
oils from spices and herbs are the most promising natural antimicrobials,
because they do not cause microbial resistance due to the diversity of
mechanisms of action. They have a Generally Recognized Asafe (GRAs) status
given by the United States Food and Drug Administration (Food and Drug
Administration, USA, 2011), meaning that they are generally recognized and safe
for human consumption without limitations on intake and commonly accepted by
consumers.
1.2 Aim of study
The
aim of this work is to isolate the fungi that cause spoilage in bread as well
as examining the antifungal sensitivity of Rosmarinus
officinalis, Eucalyptus globulus and
Citrus limon on the fungal isolates. This in order to know if the essential
oils from these plants can be used to control if not eliminate the spoilage of
bread by these fungi.
1.3 Objectives of study
The
objectives of this project work are to:
i.
Isolate and identify various fungi that cause spoilage in bread using Sabouraud
Dextrose Agar (SDA).
ii.
To examine the antifungal sensitivity of essential oils extracted from Rosmarinus officinalis, Eucalyptus globulus and
Citrus limon on the fungi isolated from spoilt bread.
iii.
Comparison of the effect of the three essential oils used on the fungal
isolates.
iv.
Characterization of the three essential oils used.
1.4 Hypothesis
=
Essential oil from Rosmarinus
officinalis, Eucalyptus globulus and Citrus
limon has no effect on fungi isolated from spoilt bread.
= Essential oil from Rosmarinus officinalis, Eucalyptus globulus and Citrus limon has an effect on fungi
isolated from spoilt bread.
1.5 Statement of problem
Problems
encountered by the society as a result of the consumption of contaminated bread
infested with fungal organisms at circulation in the market places are related
healthwise in the sense that when contaminated breads are eaten, it leads to
intestinal disorder which could be as a result of deposited toxins (aflatoxins
and ochratoxins). This can lead to an epidemic, when a batch of contaminated
bread is in circulation.
1.6 Significance of study
The
justification of this research study is to ensure that well prepared/baked
bread devoid of fungal growth is being circulated or sold and the extension in
the shelf life of the bread is achieved by the incorporation of essential oil
into it. It is also carried out on the basis of selling well baked breads that
is uncontaminated so as to avoid food poisoning associated with the ingestion
of bread.
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