MICROBIAL ASSESSMENT OF POWDERED TOBACCO SNUFF SOLD IN UMUAHIA METROPOLIS

ABSTRACT

A total of fifteen (15) snuff samples were collected from five different markets in Umuahia, Abia State and the samples were transported to the laboratory and analyzed for bacterial contamination. One gram of each snuff sample was measured out, 10 fold serial dilutions was made and spread-plated on MacConkey, Nutrient agar, Blood agar and Saboraud dextrose agar and was incubated at 370C for 24hours. The isolated bacteria species after microbial analysis were Pseudomonas aeruginosa, Escherichia coli, Staphyloccoccus aureus, Klebsiella spp, Bacillus spp while the fungal isolates include Penicillum, Mucor and Fusarium. The total Viable Bacteria Counts from the snuff samples used in this study ranged from 3.7x104 to 1.0x102. While the Total Fungal Counts from the snuff samples ranged from 3.5x103 to 1.3x103.  The percentage occurrence of the isolates showed that Pseudomonas aeruginosa (33.3%) had the highest occurrence followed by Staphylococcus aureus (25%), Klebsiella spp (16.7%), Bacillus sp (13.9%) while Escherichia coli (11.1%) had the lowest occurrence. The fungal isolates shows that Penicillum  (38.5%), showed the highest occurrence followed by Mucor (34.6%) , while Fusarium (26.9%) recorded the least occurrence among the isolates. From this study it could be concluded that most snuff samples sold in markets in Abia State were contaminated by potentially pathogenic microbes. Good manufacturing process and improvement in the sanitary conditions of the markets will help in reducing such contamination.

TABLE OF CONTENTS

Title Page                                                                                                                    i

Certification                                                                                                                ii

Dedication                                                                                                                  iii

Acknowledgements                                                                                                    iv

Table of contents                                                                                                        v

Lists of Tables                                                                                                             vii

Abstract                                                                                                                      viii

CHAPTER ONE

1.0       INTRODUCTION                                                                                         1

1.1       CLASSIFICATION OF TOBACCO PLANT                                              2

1.1.1    Family Salanaceae                                                                                           2

1.1.2    Nicatiana. rustica                                                                                            3

1.2        CHEMICAL COMPOSITION OF TOBACCO                                           3

1.3       AIMS AND OBJECTIVES                                                                           3

1.4        OBJECTIVES                                                                                                3

CHAPTER TWO

2.0       LITERATURE REVIEW                                                                              4

2.1       BACTERIAL CONTAMINATION OF SNUFF                                         8

CHAPTER THREE

3.0       MATERIALS AND METHODS                                                                  11

3.1       SAMPLE COLLECTION                                                                             11

3.2       MEDIA USED                                                                                               12

3.3       STERILIZATION                                                                                          12

3.4       ISOLATION OF MICROORGANISM                                                       12

3.5       CHARACTERIZATION AND IDENTIFICATION

OF THE BACTERIAL ISOLATES                                                              12

3.5.1    Grams Staining                                                                                               12

3.6       BIOCHEMICAL  CHARACTERISTICS OF THE ISOLATES                 13

3.6.1    Motility Test                                                                                                  13

3.6.2    Indole Test                                                                                                      13

3.6.3    Citrate Utilization                                                                                           14

3.6.4    Sugar Fermentation                                                                                         14

3.6.5    Oxidase Test                                                                                                   15

3.6.6    Coagulase Test                                                                                                15

3.6.7    Urease Test                                                                                                     15

3.7       CHARACTERIZATION AND IDENTIFICATION

OF THE FUNGAL ISOLATES                                                                    16

3.7.1    SPORE STAINING                                                                                       16

3.7.2    LACTOPHENOL COTTON BLUE STAINING                                         16

3.7.3    SLIDE CULTURE TEST                                                                              17

CHAPTER FOUR

4.0        RESULTS                                                                                                      17

CHAPTER FIVE

5.0       DISCUSSION, CONCLUSION AND RECOMMENDATION                 27

5.1       DISCUSSION                                                                                                            27

5.2       CONCLUSION                                                                                             28

5.3       RECOMMENDATION                                                                                 28

REFERENCES                                                                                              29

LIST OF TABLES

Table 1:           Total Viable Bacterial Count (Cfu/g)                                                             18

Table 2:           Total Fungal Count                                                                                         19

Table 3:           Cultural Characteristics of Bacterial Isolates.                                                 20

Table 4:           Morphological Identification and Characterization of Fungal Isolates          21

Table 5:           Total percentage occurrence of bacteria                                                         22

Table 6:           Percentage occurrence of fungal                                                                     23

Table 7:           Biochemical Characteristics of Bacterial Isolates                                           24

LIST OF FIGURES

Fig 1:   Percentage Occurrence of the Bacterial                                                                     25

Fig 2    Percentage occurrence of fungal                                                                                 26

                                                               CHAPTER ONE

1.0       INTRODUCTION

Tobacco plant (Nicotiana tabacum L.) is one of the ancient consumer goods. It was named by Linné and its cultivation and preparation have been a state monopoly since 1867 in Hungary. Tobacco is popular because of its alkaloids, among them nicotine that can produce both calmative and exciting effects (Ehiri et al., 2001).

In the last decades the technology of tobacco processing has markedly changed. The period of the tobacco fermentation was shortened because of new technologies, and their environmental conditions became better controlled and regulated than they were earlier. The appearance of new varieties of tobacco cultivars and the changes mentioned above in the tobacco industry required the study of the properties of the quality of these varieties, and their changes during different steps of tobacco leaf processing before the industrial process (Rubinstein  et al.,2001).

These examinations can be useful not only in the tobacco industry but also in other uses of tobacco plants. Nicotine in isolated form is a component of sprays in plant protection and this compound is an important agent in the study of different nerve-cell receptors. During smoking active carcinogenic agents can be produced in tobacco products, therefore smoking is considered a dangerous health risk agent. Because of these facts the importance of tobacco industry could be decreased, therefore other uses of tobacco plants could come to the fore, for example the use of their protein content (Rubinstein et al., 2001).

Tobacco leaves as well as other higher plant leaves contain soluble and insoluble proteins which are usually equal in quantity. It was found that the water soluble protein fraction of tobacco leaves is well balanced containing high levels of essential amino acids, Nowadays high protein content, especially soluble protein fraction (F1 and F2 proteins) of the tobacco plant is considered a protein source in nutrition as a supplement. F1 protein is an enzyme, called RUBISCO (ribulose 1,5-bisphosphate carboxylase-oxygenase) and F2 protein is a mixture of soluble proteins of both cytoplasmic and chloroplast origin. After isolation of soluble proteins the residual leaf material could be used in the tobacco industry (Broun and Massey., 2009).

1.1       CLASSIFICATION OF TOBACCO PLANT

The genus Nicotiana is classified among the family Solanaceae which comprises about 100 species. The most famous species is the largely cultivated Varginia. tobacco, Nicotiana. tobacum, Turkish tobacco and Nicotina rustica (Broun and Massey,2009).

Tobacco is believed to be native of tropical America and was cultivated and used by native inhabitants before the discovery of America. It is the one of the few major contributions to civilization which the new world can claim.

The first who used tobacco were the Indian of north and South America and spread to other countries France 1556, England 1565and from these countries to the different parts of the world (Hussain, 2008).The ancestry of Nicotiana tobacumis not known. Nicotine is the main alkaloid of the genus and is prepared commercially from waste material of the tobacco industry.

1.1.1    Family Salanaceae

Herbaceous or woody plant Leaves are without stipules, alternate, simple flowers, hermaphrodites or very rarely unisexual. Usually actionomorphic, calyx 4.6 lobed persistent corolla gomopetalous usually five lobed folder, contributed or valuate stamens inserted on the corolla lobes rarely two anther loculi paralley, ovary usually two locular. The loculi sometimes divided by a false septum style terminals .ovule very numerous exiles, Fruity capsule or berry (Andreus, 2001).

1.1.2    Nicatiana. Rustica

It is semi desert plant, grows in different areas in the Sudan but mainly in Dar four at the western region (Hiday – talla, 2003). Herb is up to four feet high. Leaves petiulate ovate obtuse at the apex, sometimes subcordate at the base, up to one feet high long glandular pubescent. Flowers greenish yellow, in terminal subpaniculate .Racemes with or without bract .Capsule subgloose slight longer than the calyx (Broun and Massey, 2009).

1.2       CHEMICAL COMPOSITION OF TOBACCO

The active ingredient in tobacco is alkaloids of naturally occurring compound containing nitrogen and having the properties of an amine base, they have dramatic effects on the human system (Hommond, 2002).

It was first isolate from genus nicotiana in 1828, nicotine is a colorless oily liquid alkaloid, and it considers of the most toxic drugs known to human, a dose of 60 mg is lethal in a few minutes (Pavaiaet al.,2006). Hussain, (2004) reported that nicotine constitutes 0, 9 to 3, 8% of Nicotiana. Tobacum and between 7-12% plant of Nicotiana rustica.

1.3       AIMS AND OBJECTIVES

The aim of this study is to assess the microbial quality of powdered tobacco (Snuff) sold in umuahia, Abia State.

1.5        OBJECTIVES

1. To isolate microorganisms associated with powdered tobacco.

2. To identify and characterize bacteria isolated from powdered tobacco.

3. To identify fungi isolated from powdered tobacco.

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