ANTIMICROBIAL ACTIVITIES AND PHYTOCHEMICAL SCREENING OF PIPER GUINEENSE (UZIZA)SEED AND LEAF AGAINST FOOD SPOILAGE ORGANSIMS

ABSTRACT

The antimicrobial activity of aqueous and ethanolic extract of leaf and seed of piper guineense was determined on three bacteria (Staphylococcus aureus, Escherichia coli and Salmonella spp) and three fungi (Aspergillus niger, Aspergillus flavus, and Pencillium spp) using agar well diffusion method and minimum inhibitory concentration (MIC). The ethanolic extract was found to show more activity than the aqueous extract on the isolates. The diameter zone of inhibition for the ethanolic extract (leaf and seed) range between 8 and 21mm while that of aqueous extract ranged between 8 and 17mm. The MIC of the ethanolic extract ranged from 3.125 to 100mg/ml, while for aqueous extract, the MIC was from 3.125 to 25mg/ml. Escherichia coli was found to show the greatest sensitivity of all the isolates while Penicillium spp showed the least sensitivity of all the isolates. This observed difference showed that Piper guineense extracts contains antibacterial and antifungal compounds that may be useful for the discovery of new antibiotics. The phytochemical analysis carried out on the Piper guineense leaf and seed revealed the presence of alkaloids, tannis, saponins, glycosides and flavonoids. The presence of these phytochemicals supports the use of this plants as an antimicrobial agent.

TABLE OF CONTENTS

Title Page                                                                                               i

Certification                                                                                  ii        

Dedication                                      iii                                    

Acknowledgements                                                                               iv      

Table of Contents                                                       v                

List of Tables                                               vii                                                

Abstract                                                                                              viii

CHAPTER ONE                                                               

1.0  INTRODUCTION                                                                            1   

1.1 Aims and Objectives                                                                       3                                                                                                                               

CHAPTER TWO 

2.0  LITERATURE REVIEW                                                4

2.1 Morphology and Description of Piper guineese                                                              5

2.2 Nutritional value of P. guineense                                                                                     6         

2.3 Non medicinal Uses of Piper Guineense                                                                          6

2.4 Ethnomedicinal Uses of Piper Guineense                                                                        7

2.5 Antimicrobial Activity of Piper Guineense                                                                     8

2.6 Major groups of Antimicrobial Phytochemicals from Plants                                           10

2.7 Phytochemical of Piper Gineense                                                                                    13

2.8 Description of Test Organism                                                                                          14

CHAPTER THREE                                                                                      

3.0 MATERIALS AND METHODS                                    19                  

3.1 Collection and Identification of Plant Material                          19

3.2 Sterility                                                                       19

3.3 Test Organism                                                                                                                  19

3.4 Identification of Bacteria                                                                                                 19

3.4.1 Gram reaction                                                                                                                19

3.4.2 Biochemical Test                                                                                                           20                               

3.5 Identification of isolated fungi                                                                                         22

3.6 Sample Preparation                                                                                                          23

3.7 Extraction Procedures                                                                                                       23

3.8 Media Preparation                                                                                                            23

3.8.1 Preparation of Crude Plant Extracts                                                                              23

3.8.2 Determination of Antimicrobial Activity of Extracts                                                   24

3.8.3 Determination of Minimum Inhibitory Concentration (MIC) of Crude Extracts 24

3.8.4 Phytochemical Screening of the Plant Extract (leaf and seed)      25

CHAPTER FOUR

4.0  RESULTS                                                                                      27

CHAPTER FIVE

5.0  Discussion, Conclusion, and Recommendation                             51

References                                                                                          55

LIST OF TABLES

TITLE                                                                                                 PAGE

Table 1:          Biochemical Identification of the Test Organisms                                         28

Table 2:          Cultural Morphology and Microscopic Characteristics Test Fungi                   30   

Table 3:          Antimicrobial activity of the Piper guineense (Uziza) leaf ethanol extract against test organisms                                     32 

Table 4:          Antimicrobial activity of the Piper guneense (Uziza) leaf water 4 extract against test organisms                                       35

Table 5:          Antimicrobial activity of the Piper guneense (Uziza) seed ethanol extract against test organisms                  36

Table 6:          Antimicrobial activity of the Piper guneense (Uziza) seed water extract against test organisms                                         39

Table 7:          Zone of inhibition (mm) of standard antimicrobial agent (antibiotics) against the test organism’s positive control (Streptomicin and Nystanin)                     40

Table 8:          Minimum inhibitory concentration of Piper guneense (Uziza) seed extract against test bacteria                                          42

Table 9:          Minimum inhibitory concentration of Piper guneense (Uziza) leaf extract against test bacteria                           44

Table 10:        Minimum inhibitory concentration of Piper guneense (Uziza) seed extract against test fungi                                                                                 46

Table 11:        Minimum inhibitory concentration of Piper guneense (Uziza) leaf extract against test fungi                                             48

Table 12:        Qualitative Phyto-Chemical Constituents of Piper guneense (Uziza) Plant Extract                                                    50

CHAPTER ONE

1.0       INTRODUCTION    

Food borne illness caused by consumption of foods contaminated with pathogenic bacteria or their toxins has been of great public health concern. In recent times, consumers are even more concerned of the processed foods they eat not only because of the high risk of contamination but also because of the added synthetic preservatives which may be hazardous to health. Food additives such as monosodium glutamate, aspartame, saccharin, sodium cyclamate, sulfites, nitrates, nitrites and antibiotics have all been reported to cause clinical conditions manifesting variously as headache, nausea, weakness, mental retardation, seizures, cancer and anorexia (Rangan and Barceloux, 2009; Wroblewska, 2009). The increasing demand for food with longer shelf life, food with little or no chemical preservatives coupled with the concern about toxic effects of some preservatives has resulted in increased pressure to find alternatives for better healthcare.  Therefore, there is a considerable interest to stop the disease outbreaks caused by pathogenic and/or spoilage food microorganisms among food processors, food safety researchers and regulatory agencies (Marij et al., 2009).

An antimicrobial is any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host. Antimicrobial agents, are a large variety of chemical compounds and physical agents that are used to destroy microorganisms or to prevent their development (Lai et al., 2004). Antimicrobial agents of plant origin have been documented and spices are among those perceived to have great potentials for use as antimicrobial agents (Arora and Kaur, 1999; Okeke et al., 2001).  Spices are defined by Corn (1999) as dried seeds, fruits, roots, barks, leaves or vegetables used in nutritionally insignificant quantities as food additives for the purpose of flavour, colour or as preservative that kill harmful bacteria or suppress their growth.

 Spices, which include plant materials of medicinal importance, have been used for the treatment of human ailments way back in the history of man. In Nigeria, some spices are used for the preparation of special types of soup. These include soup for newly delivered mothers to accelerate blood flow leading to the elimination of blood clots from her womb and blood system. Some have been recommended for fast relief of ailments such as cholera, diarrhoea, dysentery and wound sepsis (Inyang, 2003, Olumsimbo et al., 2011).

It is now recognized that spices and herbs may fulfil more than one function in foods to which they are added. These include imparting flavour, prolonging the storage life of foods by their bacterostatic or bacterocidal activity, in addition to being nutrients. These appeal to consumers who tend to question the safety of synthetic food additives (Eruteya and Odunfa, 2009). The medicinal and preservative values of spices have been attributed to the presence of bioactive antimicrobial compounds (Lai and Roy, 2004).

Piper guineense (Igbo: Uziza) is a flowering vine in the family Piperaceae, cultivated for its fruit which is usually dried and used as spice for seasoning. In the dried form the fruit is often referred to as peppercorn or simply pepper. Pepper gets its spicy heat mostly from the piperine compound which has been reported to exhibit antimicrobial properties detectable both in the outer fruit and in the seed (Oladosun et al., 2012).

The fruits of the guinea pepper (Piper guineense, Uziza in Igbo) is a common spices and condiments included in a variety of indigenous Nigerian recipes particularly among the Igbos (Okeke et al., 2001). In a recent survey, respondents in the region indicated that the spice act as a stimulants and laxatives, used to smoothen the skin and cure fever, cough and stomach disorders. They are also used as abortificients to treat amenoria and cleanse the womb after childbirth (Okeke, 1998).

1.1 AIMS AND OBJECTIVES

The aim of this research is to determine the antimicrobial effect of piper guineense (uziza against some food contaminating organisms and to determine the quantity of the spices needed to inhibit the growth of the organisms.

The Objectives are;

To determine the antimicrobial activity of the spices against a variety of test organisms

To determine the minimal concentrations of the spices needed to inhibit the growth of the organisms.

To determine the phytochemical compounds present in the leave and seed of the piper guineense (uziza).

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