CHARACTERISATION OF MICROORGANISMS ASSOCIATED WITH THE SPOILAGE OF BREAD

ABSTRACT

This study was aimed at isolating and identifying different microorganisms associated with the spoilage of bread and this was achieved by using the standard Spread Plate method and also by carrying out different biochemical tests which include; Coagulase test, Catalase test, Voges Proskauer test, Methyl red test, Indole test, Urease test, Hydrogen sulphide test etc. The total of 5 samples used in this study were purchased from different shops in local markets and was exposed to the environment for 7 days. The media used in this were MacConkey agar, Nutrient agar, Sabouraud dextrose agar, Mannitol salt agar. The bacteria and fungi isolated in this study include; Bacillus species, Lactobacillus plantarum, Leuconostoc mesantreroids, Penicillium species, Fusarium species, Aspergillus niger and Aspergillus flavus. The bacterial organisms were identified using colonial morphologies, Gram staining and Biochemical tests while the fungi organisms were identified using colonial morphologies and Staining with Lactophenol cotton blue. The mean counts of organisms identified ranged from 1.07×10`5 cfu/g to 1.96×10`5 cfu/g, the fifth bread sample has the highest mean count of 1.96×10`5 cfu/g while second bread sample had the least mean count of 1.07×10`5 cfu/g.

TABLE OF CONTENT

Title page

Declaration

Certification

Dedication

Acknowledgements

Table of contents

List of Tables

Abstract

CHAPTER ONE: INTRODUCTION

1.1   Objective of the study

1.2.  Aim of the study

CHAPTER TWO: LITERATURE REVIEW

2.1.   Bacterial Rope Spoilage

2.2.   Fungal Spoilage

2.3.   Yeast Spoilage

2.4.    Mold Spoilage

2.5.    Physical Factors Influencing Microbial Growth

2.5.1. Effect of Temperature, pH and Water Activity

2.6.    Early Detection and Differentiation of Bread Spoilage

2.6.1  Colony Forming Units

2.6.2. DNA Probes

2.6.3  Polymerase Chain Reaction

2.6.4. Latex Agglutination Test

2.6.5. Adenosine Triphosphate (ATP) Bioluminescence

2.6.6. Enzyme Linked Immunusorbant Assay (ELISA)

2.7.    Control of Microbial Growth in Bakery Products

2.7.1. Reformulation to reduce product aw

2.7.2. Freezing

2.8     Sodium Chloride and Magnesium Chloride in Bread Preservation

2.8.1. Lactic Acid Bacteria and Shelf-life of Bread

2.8.2. Effect of Chemical Preservatives

2.8.3. Sorbic Acid and Sorbates

2.8.4. Propionic Acids and its Salts

2.9.    Economic Importance of Bakery Products

CHAPTER THREE: MATERIALS AND METHODS

3.1.     Study Area and time

3.2.     Sample collection

3.2.1   Preparation of sample

3.2.2.  Inoculation

3.2.3.  Incubation

3.2.4.  Counting of colonies

3.2.5.  Subculture

3.3.     Identification of isolates

3.3.1.  Gram staining method

3.3.2.  Fungal characterisation

3.4.     Biochemical Analysis

3.4.1.     Catalase test

3.4.2.     Hydrogen sulphide test

3.4.3.     Indole test

3.4.4.     Urease test

3.4.5      Methyl Red test

3.4.6.     Voges Proskauer test

3.4.7.     Coagulase test

3.4.8.     Citrate test

3.4.9.     Motility test

3.4.10   Sugar fermentation test

CHAPTER FOUR: RESULTS

4.1.    Total viable counts of microorganisms from bread samples

4.2.     Identification of bacteria isolated from the samples

4.3.    Identification of fungi isolated from the samples

4.4.    Microorganisms isolated during the spoilage assessment period

4.5.    Percentage occurrence of the isolates from the samples

CHAPTER FIVE: DISCUSSION AND CONCLUSION

5.1      Discussion

5.2.     Conclusion

5.3.     Recommendations

References

LIST OF TABLES

TITLE

Tables

1: Shows viable counts of microorganisms in the bread samples

 2: Shows the identification of the bacteria isolates

 3: Shows identification of the fungi isolated from the samples

 4: Shows microorganisms isolated during spoilage period

 5: shows percentage occurrence of the isolates from the samples

CHAPTER ONE

1.0    INTRODUCTION

Bread is a food product that is universally accepted as a very convenient form of food that has desirability to all population rich and poor, rural and urban. Its origin dates back to the Neolithic era and is still one of the most consumed and acceptable staple food in all parts of the world. It is a good source of nutrients, such as macronutrients and micronutrients that are all essential for human health .Bread and other bakery products are subjected to various spoilage problems, viz.,physical, chemical and microbial; The latter is the most serious one particularly bacteria (Bacillus sp.) and mold growth. Various molds involved in spoilage of bread includes Rhizopus, Mucor, Penicillium, Eurotium, Aspergillus and Monilia (Saranraj and Geetha, 2012). Likewise, yeast spoilage known as “Chalk mold” is caused by Pichia butonii.  In the past, control of the safety of foods has been mainly carried out by product testing of both raw materials and end products. The main problem with performing end-product testing is the high number of samples to be examined before one can decide on the safety of the product batch, especially when pathogens are expected to be hetergenously distributed in the batch (de Boer & Beumer, 1999).  Also end product testing detects only failures and does not identify causes. The Hazard Analysis Critical control Points (HACCP) system is now generally considered the method of choice for ensuring the safety of foods (Jay, 1996; de Boer & Beumer, 1999). HACCP involves identifying places in the production process where hazards could occur (critical control points, CCPs) and putting monitoring procedures in place to prevent these hazards from occurring. Even with a HACCP system in place, samples still need to be tested for the presence of microorganisms. Traditional cultural detection of most microorganisms requires growth of the organism on selective media, which can take a number of days from isolation to identification. These methods are sensitive, inexpensive and give qualitative information on the number and the nature of the microorganisms present in a food sample. However, conventional methods require several days to produce results because they rely on the ability of microorganisms to multiply to visible colonies (de boer & Beumer, 1999). Moreover, culture medium preparation, inoculation of plates, colony counting and biochemical characterization make these methods labour intensive (de Boer & Beumer, 1999). Traditional methods are of limited value especially for the analysis of perishable foods since the foods are sold and eaten before the results of the test are known. Rapid methods can reduce the time taken to achieve results from days to a few hours or even minutes.

Rapid methods commercially available includes: DNA probes, the Polymerase Chain Direct epifluorescent filter techniques (DEFT), Latex agglutination test (LAT), Enzyme Linked Immunusorbant Assay (ELISA), Conductance and impedance methods, Colony Forming Units (CFU). Methods currently available for measuring mould contamination in food includes: Microscopic examination, Culture on agar, Electrical Measurements of Conductance and other changes in the electrical properties of the contaminated food substratum and detection of fungal metabolites such as Chitin, Ergosterol, or ATP. (Girardin, 1997).

1.1        Objective of the Study

The objective of this study is to isolate and identify different organisms involved in the microbial spoilage of bread.

1.2        Aim of the Study

Isolation and identification of microorganisms associated with the spoilage of bread.

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