ANTIFUNGAL EFFECT OF ROSEMARY (ROSMARIANUS OFFICINALIS) EUCALYPTUS (EUCALYPTUS GLOBULUS) AND LEMON (CITRUS LIMON) ESSENTIAL OILS ON FUNGI ISOLATED FROM SPOILT BREAD

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.

Buyers has the right to create dispute within seven (7) days of purchase for 100% refund request when you experience issue with the file received. 

Dispute can only be created when you receive a corrupt file, a wrong file or irregularities in the table of contents and content of the file you received. 

ProjectShelve.com shall either provide the appropriate file within 48hrs or send refund excluding your bank transaction charges. Term and Conditions are applied.

Buyers are expected to confirm that the material you are paying for is available on our website ProjectShelve.com and you have selected the right material, you have also gone through the preliminary pages and it interests you before payment. DO NOT MAKE BANK PAYMENT IF YOUR TOPIC IS NOT ON THE WEBSITE.

In case of payment for a material not available on ProjectShelve.com, the management of ProjectShelve.com has the right to keep your money until you send a topic that is available on our website within 48 hours.

You cannot change topic after receiving material of the topic you ordered and paid for.