MICROBIAL ASSESSMENT OF POWDERED TOBACCO USED SNUFF

ABSTRACT

The study assessed the microbiological quality of tobacco powder used as snuff sold in Umuahia metropolis. A total of ten (10) powdered tobacco samples were purchased randomly from five different markets (designated as Gate 6 Market, Ahieke Market, Umuariga, Nndoru and Orieugba Market). The major bacteria species isolated include; Pseudomonas aeruginosa, Proteus species, Klebsiella species and Bacillus cereus, while the fungal species are; Aspergillus niger, and Aspergillus flavus. The total bacterial mean count recorded in this study was found to be higher in samples obtained from Ndioru 1.32 x 105 (cfu/g), while the least bacterial count was recorded for Orieuga at 1.03 x 105 (cfu/g). Gate six recorded the highest fungal counts at 1.11 x 105 (cfu/g), while least fungal count was recorded for Gate six at 0.31 x 105 (cfu/g). From the findings in this study, it was observed that Bacillus cereus was the most frequently occurred isolate with a percentage occurrence of 5(38.5%), followed by Pseudomonas aeruginosa 3(23.1%), Klebsiella species 3(23.1%), and Proteus species 2(15.4%). However, there were wide variations of fungi population in the tobacco powder samples, with Aspergillus flavus 4(57.1%), being the most predominant and frequently occurring isolates on the powdered tobacco samples, followed by Aspergillus niger 3(42.9%). Conclusively, powdered tobacco (snuff) must therefore be processed and handled hygienically to minimize the incidence of these organisms thereby reducing the health risk they pose to its users. The powdered tobacco (snuff) samples studied contained some bacteria and fungi which are known to be pathogenic to man. It is therefore important that powdered tobacco sample (snuff) should be processed, packaged and handled hygienically.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          ix

List of Figures                                                                                                                         x

Abstract                                                                                                                                  xi

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim of Study                                                                                                              3

CHAPTER TWO

2.0       Literature Review                                                                                                       4

2.1       Powdered Tobacco                                                                                                     4

2.2       Brief History and Classification of Tobacco Plant                                                     5

2.2.1    Family Salanaceae                                                                                                     5

2.2.2    Nicatiana rustica                                                                                                        5

2.3       Prevalence of Powderless Powdered Tobacco in the World                                      6

2.4       Pathogenic Bacteria Associated with Tobacco                                                          7

2.5       Mycotoxins Associated with Powdered Tobacco                                                      8

2.5.1    Aflatoxins                                                                                                                   8

2.5.2    Ochratoxins                                                                                                                8

2.5.3    Fumonisins                                                                                                                 9

2.5.4    Trichothecenes                                                                                                           9

2.5.5    Zearalenone                                                                                                                9

2.6       Chemical and Biological Components of Tobacco Powder                                       9

2.7       Tobacco and Harm Associated with Microorganisms                                               11

2.8       Health Risks of Tobacco-Associated Microorganisms                                              11

2.9       Disease Association to Powderless-Tobacco Use                                                      13

2.9.1    Cancer                                                                                                                         13

2.9.2    Oral Mucosal Lesions Found in Powderless Tobacco Users                                     15

2.10     Bacterial Infections Associated with Tobacco                                                           15

2.10.1  Cystic Fibrosis                                                                                                            15

2.10.2  Pneumonia, Legionnaires' disease and bronchitis                                                      16

2.10.3  Tuberculosis                                                                                                               17

2.10.4  Bacterial meningitis                                                                                                   17

2.11     Composition of Tobacco                                                                                            18

CHAPTER THREE

3.0       Materials and Methods                                                                                               20

3.1       Sample Collection                                                                                                      20

3.2       Materials and Media Used                                                                                          20

3.2.1    Sterilization of Materials                                                                                            20

3.2.2    Preparation of Culture Media                                                                                     21

3.2.2.1 Mannitol Salt Agar (MSA)                                                                                         21

3.2.2.2 Nutrient Agar (NA)                                                                                                    21

3.2.2.3 Salmonella and shigella Agar (SSA)                                                                          21

3.2.2.4 MacConkey Agar (MA)                                                                                              21

3.2.2.5 Sabouraud Dextrose Agar (SDA)                                                                               22

3.3       Microbiological Assessment                                                                                      22

3.3.1    Inoculation and Enumeration of Microorganisms                                                      22

3.3.1    Purification of Isolates                                                                                               22

3.4       Microbial Characterization and Identification                                                           23

3.4.1    Gram Staining                                                                                                            23

3.5       Identification of the Bacterial Isolates                                                                        23

3.6       Colonial Morphology of Fungal Isolates                                                                   23

3.7       Biochemical Test                                                                                                        24

3.7.1    Catalase Test                                                                                                               24

3.7.2    Indole Test                                                                                                                  24

3.7.3    Citrate Utilization Test                                                                                               24

3.7.4    Motility Test                                                                                                               24

3.7.5    Voges-Proskauer Test                                                                                                 25

3.7.6    Urease Test                                                                                                                 25

3.7.7    Methyl Red Test                                                                                                         25

3.7.8    Coagulase Test                                                                                                           25

3.7.9    Oxidase Test                                                                                                               26

3.7.10  Sugar Fermentation                                                                                                    26

3.8       Identification of Fungal Isolates                                                                                 26

3.8.1    Wet Preparation                                                                                                          26

CHAPTER FOUR

4.0       Results                                                                                                                        27

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendations                                                        41

5.1       Discussion                                                                                                                   41

5.2       Conclusion and Recommendation                                                                             44

LIST OF TABLES

LIST OF FIGURES

CHAPTER ONE

1.0       INTRODUCTION

            Snuff is a tobacco product which contains the chemical stimulant, nicotine. It is a product made from ground tobacco leaves and is an example of powderless tobacco. Snuff is tobacco in the form of powder that can be inhaled or placed against the gums (Donatelle and Davis, 2009). It is one of the oldest tobacco products known. There are two types of snuff, the dry snuff and moist snuff (Russell et al., 2001). Dry snuff is produced as a dry powder while moist snuff is usually fine-cut, rather than ground and maintains high moisture content. Snuff is generally inhaled or snuffed through the nose either directly from the fingers or by using specially made snuffing devices. When snuff is taken through the mouth, the tobacco releases its nicotine into the saliva, which is then absorbed through the mucous membrane in the mouth (Donatelle and Davis, 2009). Users of powderless products including snuff, face no known cancer risk in the oral region than powderrs, and have a greater cancer risk than people who do not use any tobacco products (Boffetta et al., 2008). As the primary harm from smoking comes from the powder itself, snuff has been recommended as a way of reducing harm from tobacco (Phillips and Heavner, 2009).

            Snuff is usually scented or flavoured. Tropical flavours are floral, mentholated (medicated), fruit and spice, either pure or in blends, camphor, cinnamon, rose, spearmint, bourbon, cherry, cola and whisky. Snuff comes in the range of texture and moistness, from very fine to coarse and from very dry to very moist. It has been found to be beneficial in some cases of hay fever because it may prevent allergens from getting to the mucous membrane within the nose (Elferink, 2003). It is also useful in opening the nasal cavities in those suffering from common cold. Medicated snuffs, flavoured with mentholated crystals, eucalyptus oils or camphor are recognized as being a great cure for a stuffy head (Porter et al., 2007).

            Snuffing has become quite popular as a medication of long grief, pains and aches. It has been reported that 12.6% of students between 14 and 19 years of age in England use snuff in a studied population (Poulson et al., 2004). The widespread use of tobacco in Nigeria is well known as a result of the high demand for snuff. There is corresponding maintenance of high supply by the snuff producing industries and importers. In most markets in the south eastern part of Nigeria especially Onitsha, Owerri, Enugu, Aba, Awka and Umuahia, snuff retailers abound. The effect of occupational exposure to local powdered tobacco on pulmonary function was studied on snuff industry workers in Onitsha and Enugu Markets. The dust sampling result showed that chronic exposure to Nigeria snuff dust impairs lung function and the effect is progressive with time (Donatelle and Davis, 2009).

            Local snuff powders can be contaminated from the production to the consumption by the consumers through exposure to soil and dust during the curing of the tobacco leaves (Pauly and Paszkiewicz, 2011) the underlying microorganisms which contributed to the fermentation of the tobacco leaves; exposure to soil and dust while grinding locally re-use of unwashed storage containers and the powdery dust generated from the snuff (Maduka et al., 2009). Microbiological assessment of snuff involve the isolation of both bacteria and fungi from snuff,

            There are many microorganisms that are associated with snuff mostly transferred during production. Snuff can be traditional home made and commercial or individualized snuff product. According to Ogundero, (2000), the local way of producing snuff involves pounding or crushing of the cured leaves with locally made mortars, also depending on the scale of the operation, grinding stones may be used. People take snuff for different reasons: for medicinal purposes, cultural and traditional purposes, for smoking cessation program among others (Ureme et al., 2007). Some of the health effects of snuff use include: increase heart beat and raised blood pressure, development of oropharyngeal and upper respiratory tract cancer (gingival recession, caries, staining and abression), cardiovascular diseases and adverse reproductive outcome (England et al., 2003).

            The principal content of tobacco is nicotine and has no use in medicine, though it is of value chiefly as an insecticide. Snuff use is wide spread worldwide. According to Smith, (2000), teen use of powderless tobacco has increased, while use of all tobacco products by teen has decreased in the United States. Snuff is particularly popular amongst South African black women, with prevalence of 13.2% as compared to smoking prevalence of 5.4% in the same population group during 1998 (Ayo-Yusuf et al., 2000).

In Igbo communities of Nigeria, where tobacco is utilized for cultural and traditional purposes, nicotine has been associated with addiction in regular powderrs and snuffers as noted by Ureme et al., (2007). In Nigeria, cured tobacco is processed into snuff by small scale millers at various markets, using traditional milling or grinding stones, mortar and grinding machines (Ogundero, 2000). These milling methods and poor sanitary conditions prevailing around the snuff mills and the markets can expose the snuff to high levels of microbial contamination. Snuff is taken raw without further processing to reduce the microbial loads, mostly by elderly people with lower immunity and other health conditions.

1.1       AIM OF STUDY

The main aim of this study is to carry out a microbial assessment on powdered tobacco (Nicotiana tobacum) used as snuff sold in Umuahia Metropolis, while the specific objectives are;

·       To determine the microbial load of the powdered tobacco samples.

·       To isolate and identify microorganisms present in the powdered tobacco samples.

·       To determine the percentage occurrence of various isolate present in the powdered tobacco samples.

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