ISOLATION OF SPORE FORMING BACILLUS SPECIES FROM HONEY

ABSTRACT

This research is aimed at isolation and identification of spore forming Bacillus species from honey, using the spread plate technique the honey samples were inoculated onto blood and tryptone soy agar. A total of 35 honey samples were used for this study, 5 different samples from seven different sales point. Isolate obtained from the honey samples were purified and subjected to morphological tests, and biochemical tests. Twenty-seven honey samples had microbial growth in them of which eighteen were observed to be Bacillus species isolated from the honey samples, twelve were identified as spore formers base on their biochemical characteristics. The result of the percentage occurrence of the spore forming Bacillus in the honey; Isigate and Ubani had the highest percentage of spore formers (8.5%) followed by Amawom and umuariaga (5.7%) then choice and research (2.8%). Following this research it shows that honey has the ability to support the growth of microorganism more so spore formers even as it has anti-microbial activity. As a result of the implication of the presence of spore forming Bacillus in toxin production and also on the shelf life of honey, it is necessary that honey is properly processed before final sales or final consumption.

TABLE OF CONTENTS

Title page                                                                                                                    i

Certification                                                                                                                ii

Dedication                                                                                                                  iii

Acknowledgement                                                                                                      iv

Table of Contents                                                                                                       v

Abstract                                                                                                                      viii

CHAPTER ONE

1.0 Introduction                                                                                                                      1

1.1 Aim                                                                                                                                   4

1.2 Objectives                                                                                                                         4

CHAPTER TWO

2.0 Literature review                                                                                                               5

2.1 Honey                                                                                                                               5

2.1.1 Physicochemical Composition of Honey                                                                       6

2.1.2 pH and Acidity                                                                                                              7

2.1.3 Water Content and Water Activity                                                                               7

2.1.4 Sugar                                                                                                                              8

2.2 Spores                                                                                                                               9

2.2.1 Spore germination                                                                                                          11

2.3 Spore forming bacteria and food contamination                                                              13

2.3.1 Bacillus cereus                                                                                                               14

2.3.2 B. Cereus and the food industry                                                                                               15       

2.4 Microorganisms in honey                                                                                                  16

2.5 Prevalence of spore forming bacteria in honey                                                                 19

2.6 Deterioration of honey quality                                                                                          20

2.7 Nonthermal or mild thermal technologies to eliminate bacillus spores from food           22

CHAPTER THREE

3.0 Materials and methods                                                                                                      24

3.1 Sample collection                                                                                                              24

3.2 Media preparation                                                                                                             24

3.3 Isolation of bacterial strains from honey samples                                                             24

3.4 Gram staining                                                                                                                    25

3.5 Biochemical tests                                                                                                              26

3.5.1 Catalase test                                                                                                                   26

3.5.2 Indole test                                                                                                                      26

3.5.3 Citrate utilization test                                                                                                    26

3.5.4 Hydrogen sulphide (h₂s) production test                                                                       27

3.5.5 Starch hydrolysis                                                                                                           27

3.5.6 Spore staining                                                                                                                27

CHAPTER FOUR

4.0 Results                                                                                                                              29

4.0 Summary of tables                                                                                                            29

CHAPTER FIVE

5.0 Discussion and Conclusion                                                                                               32

5.1 Conclusion                                                                                                                        33

5.2 Recommendation                                                                                                              34

      References                                                                                                                        35

      Appendix                                                                                                                          43                                                                                                       

CHAPTER ONE

1.0       INTRODUCTION

Honey is the complex substance made when nectar and sweet deposits from plants and trees are gathered, modified and stored in honeycombs by various species of honey bee (Apis spp.). Honey quality is influenced by microorganisms, particularly spore-forming bacteria and yeasts. Nevertheless, due to the natural antibacterial properties of honey and control measures used at the industrial level, it is a product with minimal microbial contamination (Snowdon and Cliver, 1996).

Honey is a natural supersaturated substance made by bees from flower nectars and has been widely recognized as a wonderful gift from God to humankind. In ancient times it was referred to as “nectar of the Gods”. Honey has been reported to possess many health benefits including agents of antioxidant, antimicrobial, anti-inflammatory, wound healing as well as an excellent dietary supplement to boost up the energy and optimizing immunity of a healthy person. Its therapeutic implications extended from superior nutritive values to preventing health disorder such as cancer, cardiovascular diseases, neurological degeneration as well as diabetic ulcers (Khalil, 2012).

In addition to its health-related advantages, honey is also useful as a preservative due to its high osmolarity that can prevent microorganism growth. While the primary sources of bacterial contamination include pollen, digestive tracts of honeybees, dust, air, earth, and nectar, secondary, post harvest sources of microbes are likely to be the same as those for other food products, and may include humans, equipment, containers, wind, dust, insects, animals, and water.

Bacterial spores are of concern to the food industry due to their ability to survive processing, the various steps designed to kill the vegetative cells, and their potential to subsequently germinate and grow in food, thereby decreasing its safety and shelf-life (Daelman et al., 2013). Spores are tough, metabolically inert structures produced by some vegetative bacterial cells as a survival strategy in response to adverse environmental conditions (pH, nutrient limitations, desiccation, and temperature). Spores cause massive problems in the food industry, because their remarkable resistance allows them to survive food processing and conservation methods. A few Gram-positive (phylum Firmicutes, classes Bacilli and Clostridia) and Gram-negative (Sporamusaovata, phylum Firmicutes, class Negativicutes) bacterial cells can produce spores (Poehlein et al., 2013). Spores can be produced by anaerobic, aerobic, or facultative aerobic bacteria, for example, by Clostridium botulinum, Thermoactinomyces vulgaris, and Bacillus cereus, respectively. Spore-forming bacteria such as C. botulinum and Bacillus cereus cause food poisoning, whereas others like Alicyclobacillus acidoterrestris, C. tyrobutyricum, and Geobacillus stearothermophilus produce enzymes which break down food components (Nevas et al., 2006).

Hence, in foods they decrease nutritional value, functionality, and sensory quality (Lindback and Granum 2006). Some spore-forming bacteria cause no apparent harm to food or consumers, but their occurrence in foods in high numbers reduces the value of a food product, for example, Anoxybacillus flavithermus or and G. stearothermophilus in milk powder (Burgess et al., 2010).

Aerobic spore-forming bacteria in honey are found only as spores, which can survive for long periods. There have been several studies on spores of Clostridium botulinum found in honey, which have been reported to cause infant botulism (Lopez and Alippi, 2007), and on related genera that cause diseases in honey bees, such as Paenibacillus (Alippi et al., 2004).

In industrial processes, the most prevalent pathogenic Bacillus is B. cereus, which is ubiquitous and commonly found in soil, plant materials, and unprocessed foods (Stenfors et al., 2008). Consumption of foods that contain more than 10⁴ spores or vegetative cells of B. cereus per gram may result in food poisoning. Bacillus cereus can produce several toxins (Granum, 2001; Stenfors et al., 2008), the most important being the emetic toxin and the HBL enterotoxin complex. Illness is caused by contamination of honey with bacterial spores, which can colonise the gut and produce toxin. The groups at highest risk are infants, as their lack of a well-established gut microflora means that the bacteria are more likely to become established.

Bacterial spores have a unique structure that endows them with unique properties, such as metabolic dormancy (Young and Setlow, 2004) and a remarkable resistance to heat, radiation, toxic chemicals, pH extremes, oxidative agents, lytic enzymes, desiccation, mechanical disruption and high pressure (Setlow, 2000). Furthermore, dormancy and resistance enable spores from a variety of species to cause serious problems in the food, medicare, paper, and space-industry.

The physicochemical properties of honey are highly dependent on its origin (geographical area, climate, season, and hive), foraging bees and floral sources. It however can be influenced by human activities during processing such as harvesting methods, storage, packaging, transportation and physical contact. Microbes like enterobacteriaceae, healthcare acquired pathogens and food spoilage organisms were reported to be susceptible to some types of honey in different degrees and effects. Generally, bacteria like Staphylococcus aureus, Bacillus subtilis, Eschericia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhimurium, Enterococcus faecalis, Listeria monocytogenes, and fungi like Penicillium expansum, Aspergillus niger and Alcaligenes faecalis were killed by honey at certain concentration (Kwakman et al., 2010, Mundo et al., 2004). Nevertheless, microbes like spore-forming organisms and yeasts were frequently reported to be major honey contaminants due to their ability to survive high osmolarity and low pH of honey. Clostridium botulinum is one of the most significant contaminants which raises concern among medical practitioners and food providers as it could cause infant botulism due to its spores in honey given to children below 2 years old (Heyndrickx, 2011).

Control of spore-formers requires an understanding of both the resistance and outgrowth characteristics of the spores.

1.1 Aim

The aim of this work was to isolate spore forming Bacillus from honey sold Umuahia.

1.2 Objectives:

1.      To isolate Bacillus species from honey samples sold in retail outlets.

2.      To determine the frequency of occurrence of spore-forming Bacillus species from non-spore formers.

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.