DEPARTMENT OF DEPARTMENT OF MICROBIOLO ...

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Ugboaku, Edith J.

FACULTY OF BIOLOGICAL SCIENCES

DEPARTMENT OF DEPARTMENT OF DEPARTMENT OF DEPARTMENT OF MICROBIOLOMICROBIOLOMICROBIOLOMICROBIOLOGYGYGYGY

Antibacterial Activity of Piper guineense, Xylopia aethiopica

and Allium cepa against Bacteria Isolated from Spoilt Soup

Preparations

MARTIN, HANNAH CHINENYE

PG/M.Sc/10/57261

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Preparations

BY

MARTIN, HANNAH CHINENYE

PG/M.Sc/10/57261

Department of Microbiology

UNIVERSITY OF NIGERIA, NSUKKA

DECEMBER, 2014

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TITLE PAGE



Preparations

BY

MARTIN HANNAH CHINENYE

PG/M.Sc/10/57261

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER

OF SCIENCE (M.Sc.) DEGREE IN MEDICAL MICROBIOLOGY

SUPERVISOR: PROF C.U. IROEGBU.

DECEMBER, 2014

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CERTIFICATION

Miss Martin Hannah Chinenye, a post graduate student in the department of Microbiology,

majoring in medical microbiology has satisfactorily completed the requirements for course work

and research for the degree of masters in science (M.Sc) in Microbiology. The work embodied in

this project is original and has not been submitted in part or full for either diploma or degree of

this university or any other university.

______________________ _______________________

Prof A. N. Moneke Prof. C. U. Iroegbu

Head, Supervisor

Department of Microbiology, Department of Microbiology,

University of Nigeria, Nsukka. University of Nigeria, Nsukka.

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DEDICATION

This work is dedicated to God almighty whose love and grace saw me through this program.

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ACKNOWLEDGEMENT

My heartfelt gratitude goes to my supervisor Prof. C. U. Iroegbu for his fatherly

disposition, attention, guidance and patience in the course of this research project.

My thanks also goes to Mr. A. A. Ngene and Dr. A. C. Ike for their support during this

research and also Prof K. F. Chah who provided me with some test organisms used in this

research

I lack both words and space to appreciate all my friends in this department and my

friends outside the department for their help in several ways, especially Chinazor Araonu, Obudu

Uche, Joy, Patricia Kalu, Akudo Osuji, Iyke Ibe. God bless all of you.

Finally I wish to thank my parents Mr and Mrs Martin Ibekwe, and my lovely husband

Mr. Chrys Duru for their great love and care.

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TABLE OF CONTENTS

Title page - - - - - - - - - - i

Certification - - - - - - - - - - ii

Dedication - - - - - - - - - - iii

Acknowledgement - - - - - - - - - iv

Table of contents - - - - - - - - - v

List of tables - - - - - - - - - - vi

List of appendices - - - - - - - - - vii

Abstract - - - - - - - - - viii

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW- - - 1

1.2: LITERATURE REVIEW- - - - - - - - 4

1.2.1 General Characteristics of Piper guineense - - - - - 7

1.2.2 General Characteristics of Xylopia aethiopica - - - - - 8

1.2.3 General Characteristics of Allium cepa - - - - - - 9

1.2.4 Review of Some Medicinal Spices- - - - - - 11

1.2.5 Some Spices reported to Possess Antibacterial Properties - - - 13

1.2.6 Some Major Groups of Antimicrobial Phytochemicals from Plants- - - 15

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1.2.7 Description of Test Organisms- - - - - - - - 19

CHAPTER TWO: MATERIALS AND METHOD- - - - - - 24

2.1 Collection and Identification of Plant Materials - - - - - 24

2.2 Isolation and identification of Microorganisms - - - - - 24

2.3 Test Microorganism- - - - - - - - 24

2.4 Sample Preparation and Extraction Procedures - - - - - 25

2.5 Media Preparation - - - - - - - - - 25

2.6 Preparation of Crude Plant Extracts - - - - - - 26

2.7 Determination of Antimicrobial Activity of Extracts- - - - - 26

2.8 Determination of Minimum Inhibitory Concentration (MIC) and

Minimum Bactericidal Concentration (MBC) of Crude Extracts- - - 27

2.9 Phytochemical Screening of the Plant Extract- - - - - - 27

2.9.1 Test for Alkaloids - - - - - - - - 28

2.9.2 Test for Flavonoids- - - -- - - - - - 28

2.9.3 Test for Glycosides- - - - - - - - - 28

2.9.4 Test for Saponins - - - - - - - - - 29

2.9.5 Test for Tannins.- - - - - - - - - 29

2.9.5 Test for Fats and Oil - - - - - - - - 29

2.10 Screening of ground Spices for Inhibitory Activity against Test Organisms- 29

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CHAPTER THREE RESULT- - - - - - - - 31

3.1 Isolation and Characterisation of Test Organisms- - - - 31

3.2 Yield from Aqueous and Ethanol Extractions- - - - - - 31

3.3 Chemical Constituents in Ethanol, Cold and Hot water of the Extracts- - - 34

3.4 Antimicrobial Activity of the Extracts- - - - - - - 34

3.5 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC) of the Extracts- - - - - - - 43

3.6 Screening of Spices for Inhibitory Activity against Test Organisms - - 43

CHAPTER FOUR: DISCUSSION - - - - - - - 51

REFRENCES 55

APPENDICES 60

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List of Tables

Table Title Page

1 Characterization of theTest Organisms- - - - - 32

2 Yields from the Ethanol and Aqueous Extractions - - - 33

3 Chemical Constituents in Ethanol, Hot and Cold Water Extracts of

Piper guineense- - - - - - - - 36


Xylopia aethiopica- - - - - - - - 37


Allium cepa- - - - - - - - - 38

6 Inhibition of Microrganisms by Hot Water Extract of Piper guineense 39

7 Inhibition of Microrganisms by Hot Water Extract of Xylopia aethiopica 40

8 Inhibition of Microrganisms by Cold Water Extract of Allium cepa 41

9 Inhibition of Microrganisms by Cold Water Extract of Xylopia aethiopica 42

10 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

of Ethanol Extracts of X.aethiopicaI, P.guineense and A.cepa 44


of Cold Water Extracts of X.aethiopicaI, P.guineense and A.cepa 45


of Hot Water Extracts of X.aethiopicaI, P.guineense and A.cepa - 46

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13 Effect of Spice Combination (X. aethiopica: P. guineense ) on Test Organisms 47

14 Effect of Spice Combination ( A. cepa: X. aethiopica) on Test Organisms 48

15 Effect of Spice Combination (P. guineense : A.cepa) on Test Organisms 49

16 Effect of Spice Combination (X. aethiopica: P. guineense: A.cepa ) on

Test Organisms - - - - - - - 50

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Appendices

Appendix Title Page

1 Univariate Analysis of Variance- - - 60

2 Post Hoc Tests- - - - - 72

3 Laboratory Media and Reagents - - - 95

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ABSTRACT

The antibacterial activity of ethanol, hot water and cold water extracts of Allium cepa, Xylopia

aethiopica and Piper guineense were determined against bacteria isolated from spoilt egusi soup

and veterinary unit of the university. Antimicrobial testing was by both broth –dilution and agar

diffusion methods. The spices tested, Allium cepa, Xylopia aethiopica and Piper guineense were

extracted with cold, hot water and with ethanol. The ethanol extract of Piper guineense gave the

highest yield of extract 4.8g (32%). The highest IZD (14.7±0.3mm) was achieved with hot water

extract at concentration of 400mg/ml against Escherichia coli. The hot water extract also had

activity against Bacillus sp (IZD= 12.7±0.6mm) isolated from spoilt egusi-soup. The Bacillus

strain isolated from spoilt pepper-soup was not susceptible to the hot water extract and indeed

any other extract even at the highest concentration. The most susceptible test organism was

E.coli with IZD ranges of 12.0± 0.1mm (obtained from cold water extract of X. aethiopica at

400mg/ml) to 14.7±0.2mm (from hot water extract of Piper guineense at 400mg/ml). The least

susceptible were Enterobacter sp and Proteus sp. which were only susceptible to cold water

extract of A. cepa (IZD= 10.0± 0.2mm and 12.0±0.2mm both at 400mg/ml, respectively).

Ethanol extracts showed no activity against any test organisms. Result show that the spices have

potentials for use as food preservatives while still acting as food condiments.

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CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1. 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 (Marija et al., 2009). 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

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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, diarrhea, dysentery and wound sepsis (Inyang, 2003,

Olumsimbo et al., 2011).

It is now recognized that spices and herbs may fulfill 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).

Xylopia aethiopica (Igbo: Uda) is an evergreen, aromatic tree of the Annonaceae family

that can grow up to 20m high. It is a plant used both as a spice and as a herb. It has been reported

in folklore that X. aethiopica is very potent in curing several ailments including cough,

rheumatism and nerve pains as well as in elimination of blood clots when used to prepare

peppersoup for newly delivered mothers (Ekpo et al., 2012).

Allium cepa (common onion) is a biennial garden plant, it is usually thought of as a

vegetable, and it also has a long medicinal use history. Principally, the fleshy bulb that grows

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below the ground is used as medicine and as food; but other parts of the plant have also been

used in traditional medicine for the treatment of various ailments (Azu et al., 2007).

The fruits of the guinea pepper (Piper guineense, Uziza in Igbo) and seeds of the African

pepper (Xylopia aethiopica, Uda in Igbo) are common spices and condiments included in a

variety of indigenous Nigerian recipes particularly among the Igbos of southern Nigeria (Okeke

et al., 2001). In a recent survey, respondents in the region indicated that the two spices act as

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).

Studies in the past decades confirm that the growth of both Gram-negative and Gram-

positive food borne bacteria, yeasts and mold can be inhibited by spices (Eruteya and Odunfa,

2009). Monodora myristica, Piper guineense and Xylopia aethiopica were screened for fungi-

toxic activity of their essential oils against mycelial growth of 3 food contaminants, Aspergillus

fumigatus, Aspergillus nidulans and Mucor hiemalis. The essential oils from all the spices were

fungi-toxic to varying degrees (Nwaiwu and Imo, 1999). Johnson and Vaugh (1969) reported the

inhibitory activity of reconstituted onion and garlic preparations against Salmonella typhimurium

and Escherichia coli. According to Shelef (1983) garlic inhibited Salmonella typhymurium,

Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, mycotoxigenic

Aspergillus and Candida albicans.

1.1.1. Statement of Problem

Although a number of spices have been reported in research or folklore to exhibit

antimicrobial activity against different types of microorganisms, this varies widely depending on

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the type of spices or herbs, test medium and microorganism. Besides, some of these claims still

need to be authenticated through scientific testing. Food preparations are known to be spoiled by

microbial contaminants that cause them to ferment and sour; some of these contaminants are

potent pathogens causing food poisoning or food infection when they multiply to high doses.

Thus many local foods need preservation without synthetic preservatives and need to be free

from pathogens. Thus, this study was undertaken to evaluate the antimicrobial and, by

implication, the potential preservative effects of these natural spices used in making soup on

some food spoilage isolates.

1.1.2. Aim

The aim of this research is to determine the antimicrobial effect of three spices used in

preparing pepper-soup on some food contaminating organisms and to determine the quantity of

the spices needed to inhibit the growth of the organisms.

1.1.3. Objectives

- To isolate and characterize microorganisms associated with spoilage of egusi- and

pepper-soups.

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

organisms including those isolated from spoilt egusi soup and peppersoup

- To determine the minimal concentrations of the spices needed to inhibit the

growth of the organisms.

1.2. Literature Review

Plants and their products have been used by humans in diverse ways, and the most

common uses are as food, spices and medicines. The uses of spice are not limited to flavouring

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agents. They possess potent medicinal properties such as antimicrobial activity, antioxidants,

anticancer, anorexia, bronchitis and rheumatic complaints and as a post-operative antiemetic. The

medicinal property under review is the antimicrobial activity. The antimicrobial property can be

merely inhibitory or cytostatic or total killing or cytocidal. The main advantage of using the

herbal antimicrobial drug is that there are little or no side effects. Such side effects as depletion of

the normal intestinal flora, bone marrow depression, dysentery, local inflammation, damage to the

liver and kidney are largely overcome by using herbal preparations either as drug or as spices

(Saha Rajekhar et al., 2012).

Spoilage is a metabolic process that causes food to be undesirable or unacceptable for

human consumption due to changes in sensory and nutritional characteristics (Doyle, 2007).

Prevention of pathogenic and spoilage microorganisms in food is usually achieved by using

chemical preservatives but they have been associated with many carcinogenic and teratogenic side

effects as well as residual toxicity. There is, also, growing concern about microbial resistance

towards conventional preservatives; consumers tend to be suspicious of chemical additives,

hence, the exploration and exploitation of naturally occurring antimicrobial herbal preparations

are receiving increasing attention in food preservation (George et al., 2010). There has been an

increasing consumer demand for foods free of or low in, added synthetic preservatives because

synthetic preservatives could be toxic to humans (Bedin, et al., 1999). Concomitantly, consumers

have also demanded for wholesome and safe food with long shelf lives. These requirements are

often contradictory and have put pressure on the food industry for progressive removal of

chemical preservatives and adoption of natural alternatives to control food borne pathogens and

spoilage microorganisms (Brull and Coote, 1999). Many plant derived products such as spices,

fruit preparations, vegetable preparations or extracts have been used for centuries for the

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preservation and extension of the shelf life of foods (Chattopadhyay and Bhattacharyya, 2007).

Numerous naturally occurring antimicrobials are present plant tissues and many studies have

evaluated the antimicrobial activities of several plant extracts, including Sesamum radiatum

(Shittu et al., 2007), Allium cepa (Agatemor, 2009), Olives, Chardonnay grapes, black

raspberries and orange essential oils (George et al.,2010).

Spices are the most common plant materials with potential antimicrobial properties that

are used in foods; and they have been used traditionally for thousands of years by many cultures

for preserving foods and as food additives to enhance aroma and flavour (Souza et al., 2005).

Spices may be indigenous or exotic, aromatic or with strong taste, but used in all cultures to

enhance the taste of foods. Spices may come in form of rhizomes, bulbs, barks, flower buds,

stigmas, fruit, seeds and leaves. They are categorized into tiny wild fruits, nuts, herbs, spices and

leafy vegetables. Some of them are not only used for food, but also in folklore medicine for

treatment of minor ailments. Spice ingredients produced from roots, barks, probably evolved as

part of the defense mechanisms of leaves, bulbs, stems flowers and seeds of certain plants

against microbial invasion. Each spice has a unique aroma and flavor, which is derived from

chemical constituents of the plant, generally designated “secondary metabolites” and so called

because they are secondary to the plant’s basic metabolism. Most spices contain dozens of

secondary metabolites or compounds. These are the plant’s recipes for survival-legacies of their

co-evolutionary races against biotic enemies. Some of the secondary metabolites that are active

against microorganisms fall into groups of compounds generically known as alkaloids,

flavonoids, glycosides etc. Spices include leaves (bay, mint, rosemary, coriander, laurel,

oregano), flowers (clove), bulbs (garlic, onion), fruits (cumin, red chilli, black pepper), stems

(coriander, cinnamon), rhizomes (ginger) and other plant parts (Shelef, 1983).

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1.2.1 General Characteristics of Piper guineense

Piper guineense also known as African black Pepper, Ashanti Pepper, Benin Pepper,

False Cubeb, Guinea Cubeb, Uziza Pepper is very similar to Piper nigrum which is the true

pepper of commerce from which black and white peppers are processed (Isawumi, 1984). It has

more than 700 species widely distributed throughout the tropical and subtropical regions of the

world. It is known with different vernacular names in Nigeria: Igbo (Uziza), Yoruba (Iyere) and

Ibibios of Akwa Ibom (Odusa). P. guineense has culinary, medicinal, cosmetic and insecticidal

uses (Dalziel, 1955).

The plants that provide Ashanti pepper are climbing vines that can grow up to 20m in

length. These are native to tropical regions of central and Western African and are Semi-

cultivated in countries such as Nigeria where the leaves are used as a flavoring in soups recipes.

It is used in West African cuisine where it imparts "heat" (piquantness) and a spicy, pungent

aroma to classic West African "soups" (stews).

Ethnomedicinal uses of Piper guineense

Piper guineense has culinary, medicinal, cosmetic and insecticidal uses (Dalziel, 1955).

P. guineense insecticidal activity against Zonocerus variegatus is attributable to the piperine-

amide constituent of the plant. The leaves are considered aperitive, carminative and eupeptic.

They are also used for the treatment of cough, bronchitis, intestinal diseases and rheumatism

(Essiett and Ibanga, 2012). In Chinese folk medicine; black pepper is used to treat epilepsy.

Piperine, the active component of black pepper blocks convulsions induced by ‘kainte’ but not

by glutamate. It is also used in Chinese medicine for the treatment of rheumatism, toothache and

stomach ache. It is crushed and eaten by pregnant women in Caspian Littoral of Iran where

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esophageal cancer rate is high. Black pepper has been prepared in the form of pills as a remedy

for cholera and syphilis. It has also been used in tooth powder for toothache and for sore throat

and hoarseness. It could be chewed to reduce throat inflammation. Other applications of black

pepper include treatment of boils, hair loss and skin diseases. It alleviates itching and paralysis.

A mixture of black pepper and honey serves as a remedy for night blindness. Black pepper is

also useful in hepatitis as well as urinary and reproductive disorders.

1.2.2 General Characteristics of Xylopia aethiopica

Xylopia aethiopica is known by different names in different languages – English (negro

peppr), Yoruba (eru), Igbo (Uda), Hausa (kimbara) and Kugbo (alilaar). It is widely cultivated

in West Africa, Central and Southern Africa. Xylopia aethiopica is an angiosperm of the family

Annonaceae. It grows into a tall tree of about 20m high and 75cm stem girth. The fruits are

rather small and look like twisted bean-pods in clusters of up to 40 green or red monocarps when

fresh but turn dark brown when dry (Burkill, 1985). It has been reported, that there are between

100 and 150 species of Xylopia distributed throughout the tropical regions of the world,

particularly Africa, among which are, X. aethiopica, X. brasiliensis, X. frutenscens, X.

grandiflora, X. aromatic. It is used as a spice and possesses great nutritional and medicinal

values in folklore (Oluwatosin et al, 2010). The various extracts from Xylopia spp. have been

shown to possess antiseptic and analgesic properties, and insecticidal activity against adult

mosquitoes, several leaf-eating insects and houseflies. Various parts of the plant have been

traditionally employed in different therapeutic preparations (Konning et al., 2004).

Phytochemichal evaluation shows that X. aethiopica is rich in alkaloids, tannins, flavonoids,

steroids, oligosaccharides and has tolerable levels of cyanogenic glycosides (Ijeh et al., 2004).

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Ethnomedicinal uses of Xylopia aethiopica

X. aethiopica has an attractive aroma and has been applied in ethnomedicine in the

treatment of cough, bronchitis and dysentery (Iwu, 1986). In Ivory Coas,t the fruit extract is used

as tonic to encourage female fertility, for ease of childbirth and as a woman remedy after child

birth for relief of pains in the ribs, chest, lumbargo, neuralgia and in treatment of boils and skin

eruptions (Acquaya et al., 2002); for increasing menstrual flow and was accordingly deemed to

have abortifacient properties (Burkill, 1985; Nwafor and Gwotmut, 2006)). Some of its

investigated uses include termite anti-feedant activity (Murray, 1995) and antiseptic properties

(Iwu, 1995). Xylopia aethiopica has a wide variety of applications; the very odorous roots of the

plant are employed in West Africa in tinctures, administered orally to expel worms and other

parasitic animals from the intestines, or in teeth-rinsing and mouth-wash extracts against

toothaches. Crushed powdered fruits can also be mixed with shea butter fat and coconut oil and

used as creams, cosmetic products, and perfumes (Burkill 1985), and the dried fruits are also

used as spices in the preparation of two special local soups named “obe ata” (Yoruba) and “isi-

ewu” (Igbo) taken widely in the southwest and southeastern parts of Nigeria.

1.2.3 General Characteristics of Allium cepa

Allium cepa (Onion) which belongs to the family Alliaceae, is also known as ‘garden

onion’or ‘bulb’onion. It is one of the oldest cultivated vegetables in history. Onion is a biennial

plant, growing from a subterranean bulb. It can grow up to 70 cm in height. It has an erect stem

and an umbel of soft, white to pink flowers on its top. Its underground bulb carries small,

shallow roots. Above ground, the onion shows only a single vertical shoot; the bulb grows

underground, and is used for energy storage, leading to the possibility of confusion with a tuber

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which it is not. The leaves are bluish-green and hollow, the bulbs are large, fleshy and firm.

Three main varieties of onion are available viz: red, white and purple skinned. Onions are easily

propagated, transported and stored. Onions are effective against common cold, heart disease,

diabetes, osteoporosis, coughs and sore throat. They also act as bacteristatic. Certain chemical

compounds believed to have anti-inflammatory, anti-cholesterol, anticancer and antioxidant

properties including quercetin are present in onions. They are high in flavonoids which is

concentrated on the outer layer of the flesh. Onions are also rich in Calcium, iron, phosphorus,

vitamin C, riboflavin, niacin, thiamine, carotene and polphenols than other allium vegetables.

Ethnomedicinal uses of Allium cepa

Onion has a great variety of medicinal uses. It is considered to have antihelmintic,

antioxidant, antiseptic, carminative, diuretic, expectorant, febrifuge and vulnerary properties.

Onion is said to help in cases ranging from the common cold to heart disease and diabetes. In

traditional medicine, Onion had been used for colds, coughs, flu and bronchitis. During winter

times, onion juice sweetened with honey can be used for prevention of common cold. It is also

said that chewing of fresh Onion can kill germs in mouth and soothe toothache. Recent studies

are showing its beneficial effect in the treatment of high blood pressure and high blood

cholesterol (Azu et al., 2007). It can be a good medicine for prevention of cardiovascular

disease, and even certain head and neck tumors. Some studies suggest that high consumption of

Onion, along with Garlic lowers the possibility of appearance of stomach cancers by 40% (Indu

et al., 2006). It can also be used for prevention of osteoporosis and in treatment of blisters, boils

and topical scars.

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1.2.4. A Review of other Medicinal Spices

Medicinal plants are of great importance to the health of individuals and communities.

The medicinal value of plants lies in some chemical substances that produce a definite

physiological action in the human body. Many of these indigenous medicinal plants are used as

spices and food plant. They are sometimes added to foods meant for pregnant and nursing

mothers for medicinal purposes (Edeoga et al., 2005).

Ginger, the rhizome of Zingiber officinale, is one of the most widely used species of the

ginger family Zingiberaceae and is a common condiment for various foods and beverages.

Ginger is a creeping perennial on a thick tuberous rhizome which spreads underground. The

odour and taste are characteristically aromatic and pungent. The plant is indigenous to southern

Asia and is cultivated in a number of countries including India. The medicinal part of the herb is

the dried roots. It is now recongnised as a drug of choice for nausea and vomiting. It has been

found useful in pregnancy-related morning sickness. In rheumatoid arthritis and Osteoarthritis, it

is used as natural pain reliever and an inflammatory agent. It is also used in curing ulcer and

preventing heart attack and stroke (Samir and Amrit, 2003). Raw ginger is chewed to stimulate

the flow of saliva and to relax congested nostrils. Ginger tea is prescribed for cough, colds and

influenza (Gill, 1992). The juice of the rhizome served with honey is a very efficacious remedy

for cough and asthma (Okanla et al., 1990). It is recommended for ailments of the digestive

system, rheumatism and piles.

Allium sativum, commonly known as garlic is a specie in the onion family

Amaryllidaceae. It is known that Allium sativum possesses antimicrobial, antiprotozoal,

antimutagenic, antiplatelet and antihyperlipidemic properties. Allium sativum has been used in

world cuisines as well as in herbal medicine for thousands of years. It is used to prevent heart

12

diseases (including arteriosclerosis and high blood pressure) and cancer including stomach and

colon cancer. Allium sativum has been found to reduce platelet aggregation and hyperlipidemia

(Kojuri et al., 2007).

Studies have shown other relevant spices such as Monodora myristica seed used as

condiment in West Africa, and a decoction of the seed used to treat guinea worm infection. The

seeds are used as a remedy for constipation when mixed with palm oil. Roasted and powdered

seeds of the plant are very effective in curing stomach ache. The seeds are rubbed on the

forehead to cure headache (Gill, 1992). Another spice, cinnamon or Cinnamomum zeylanicum,

found in the inner bark of Cinnamomun trees, is commonly used in cooking for its aroma, flavor,

and taste. Historically, cinnamon has been used by the Egyptians for embalming, presumably

based on its antimicrobial properties. Eugenol and cinnamaldehyde are the two major chemical

components in cinnamon that are responsible for its health benefits. Eugenol, a phenol

compound, inhibits mold and adds flavor and aroma to bakery items. It also contains antiviral

properties in vitro. Additionally, eugenol and cinnamaldehyde inhibit Helicobacter pylori growth

at a low pH, showing their efficacy in eliminating the bacteria present in the human stomach (Ali

et al., 2005). The electronegative cinnamaldehyde also inhibits amino acid decarboxylase.

Cinnamaldehyde interferes with electron transfers and reacts with nitrogen-containing

compounds, resulting in impeded growth of microorganisms.

Clove (Syzygium aromaticum) has been proven to be active against many types of

bacteria. Cloves are the dried immature flower buds of a tropical tree of the Myrtaceae family.

These trees are native to Indonesia but also cultivated in other tropical regions. Cloves have been

used for centuries as natural medicine against illnesses such as diarrhea, ringworm and nausea

and have been shown to be effective in reducing toothache. They also have a strong inhibitory

13

effect against microbes and are able to kill species of bacteria and fungi such as Staphylococcus

aureus, Listeria monocytogenes, and Aspergillus. This property is attributed to the presence of

eugenol, the phenol compound also found in cinnamon. Eugenol makes up the majority of clove

bud oil. Other spices such as Curcuma longa (Turmeric), Piper nigrum (Black pepper) etc have

been found to have varied medicinal values (Shelef, 1983).

1.2.5 Some spices Reported to Possess Antibacterial Properties.

The antimicrobial properties of substances are desirable tools in control of infections and

food spoilage or food preservation. Antimicrobial activity depends on the type of spice or herb,

type of food and microorganism, as well as on the chemical composition and constituents of

extracts and essential oils. The antimicrobial activity of some plant extracts on some organisms

associated with fish spoilage was studied by George et al. (2010). They observed that

marceration in hot water was the best extraction method followed by ethanol and cold water

extraction methods. Among the plant materials they evaluated, Citrus paradise had the best

antimicrobial activity followed by Piper guineense and Carica papaya.

Aqueous and ethanol extracts of Occimum gratissum and P. guineense leaves were

screened for antibacterial activity against Escherichia coli and Staphylococcus aureus by Nwinyi

et al. (2009). Both extracts were found to exhibit selective inhibition against the isolates. Ethanol

extracts showed more inhibitory effect compared to the aqueous extracts.

Antimicrobial activity of some extracts of Allium sativum (garlic), Myristica fragrans

(nutmeg), Zingiber officinale (ginger), Allium cepa (onion) and Piper nigrum (pepper) was

evaluated against 20 different strains of Escherichia coli, 8 serotypes of Salmonella, Listeria

mononcytogenes and Aeromonas hydrophila by Indu et al. (2006). The result showed that garlic,

14

ginger and nutmeg showed some inhibitory activity on different test organisms while extracts of

Onion and Pepper did not show any activity against the test organisms.

Olusimbo et al. (2011) studied the aqueous and ethanol extracts of four spices (Monodora

myristica, Piper.guineense, Xylopia aethiopica and Tetrapleura tetraptera) on some pathogens.

The aqueous extracts had antimicrobial activity on all test organisms used while the ethanol

extracts were less active. The antimicrobial activity of essential oils of Xylopia aethiopica was

evaluated on some Gram-positive and Gram-negative pathogens by Fleischer et al. (2008), the

fresh and dried fruits, leaf, stem bark and root bark essential oils showed varied activity on the

test organisms except on Escherichia coli.

The effect of Eugenia aromatic (clove), Allium sativum (garlic) and Piper guineense

(brown pepper), three spices commonly used in the preparation of suya against Bacillus spp,

Enterobacter spp Aspergillus niger and Rhizopus stolonifer were studied by Eruteya and Odunfa

(2009). The sensitivity of the organism revealed that Clove is outstanding compared to the much

studied garlic and that Gram positive bacteria showed higher susceptibility to spices than Gram

negative organisms. R. stolonifer showed higher sensitivity to brown pepper than it did to garlic.

The growth of A. niger was not completely inhibited by brown pepper or a combination of both.

The percentage composition of these three spices affected their inhibitory effects on

microorganisms in suya condiment.

Azu et al. (2007) investigated the antimicrobial activity of raw and aqueous extract of

Allium cepa and Zingiber officinale against Staphlococcus aureus and Pseudomonas aeruginosa

that are common cause of nosocomial infection and urinary tract infection using cup–plate

diffusion technique. The result showed that ethanol extract of ginger gave the widest zone of

inhibition against the two test organisms at the concentration of 0.8gml-1

. However,

15

Pseudomonas aeruginosa was more sensitive to the extract of onion bulbs compared to

Staphylococcus aureus. It was also observed that the solvent of extraction and its varying

concentrations affected the sensitivity of the two organisms to the plant materials. The minimum

inhibitory concentration (MIC) of ginger extracts on the test organisms ranged from 0.1gml-1

-

0.2gml-1

, showing that ginger was more effective and produced marked inhibitory effect on the

two test organisms compared to the onion extracts. This investigation indicates that, though both

plants had antibacterial activity on the two test organisms, ginger had more inhibitory effect thus

confirming their use in folk medicine.

Marija and Nevena (2009) reviewed the antimicrobial activity of essential oils of widely

used spices and herbs, such as garlic, mustard, cinnamon, cumin, clove, bay, thyme, basil,

oregano, pepper, ginger, sage, rosemary etc., against most common bacteria and fungi that

contaminate food (Listeria spp., Staphylococcus spp., Salmonella spp., Escherichia spp.,

Pseudomonas spp., Aspergillus spp., Cladosporium spp. and many others). The result shows that

cinnamon, cloves and mustard have very strong antimicrobial potential, cumin, oregano, sage,

thyme and rosemary show medium inhibitory effect, and spices such as pepper and ginger have

weak inhibitory effect.

1.2.6 Some Major Groups of Antimicrobial Phytochemicals from Plants.

Phytochemicals are non nutritive compounds found in plant and which may have

protective or disease preventive properties. These phytochemicals are mostly secondary

metabolites of which over 10,000 have been isolated. In many cases, these substances serve as

plant defense mechanism against predation by microorganism, insects and herbivores. However,

it has been demonstrated that these chemical substances can also protect human against diseases.

They include the following;

16

Alkaloids

They are natural plant compounds with a basic character and usually contain one or more

nitrogen atom in a heterocyclic ring. They are usually colourless, crystalline, non volatile solids

which are insoluble in water but soluble in ethanol, ether, chloroform and other organic solvents.

Only very few liquids are soluble in water. Most alkaloids have a bitter taste and are optically

active. Most alkaloids are physiologically active while some are extremely poisonous. The first

medically useful example of an alkaloid was morphine isolated in 1805 fom the opium Papaver

somniferum. Many alkaloids are commonly found to have antimicrobial properties. The

mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and har

ane is attributed to their ability to intercalate with DNA.

Flavonoids

Flavonoids are a class of water soluble plant pigments. They are a group of polyphenolic

compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain.

Since the flavonoids are known to be synthesized by plants in response to microbial infection, it

should not be surprising that they have been found in vitro to be effective antimicrobial

substances against a wide array of microorganisms. Their activity is probably due to their ability

to complex with extracellular and soluble proteins and to complex with bacterial cell wall (Ajali,

2004). More lipophilic flavonoids may also disrupt microbial membranes. Human studies

suggest that flavonoids may reduce the risk of cardiovascular disease and stroke (Knek et al.,

1997).

Saponins

Saponins are glycosides with distinctive foaming characteristics. They are natural

detergents found in certain plants. They are found in many plants especially certain desert plants.

17

They got their name from the soapwort plant (Saponaria) the root of which was used historically

as a soap. Saponins have detergent or surfactant properties because they contain both water

soluble and fat soluble component. Saponins are amphipathic compounds, possessing both

hydrophilic and lipophilic portions. They are, therefore, surface active and can be used as

emulsifiers. Molecular weight is of the order 180-2000 Daltons. At concentrations below 200-

500 ppm saponins exist as monomers; above 200-500ppm, they aggregate as micelles with a

molecular weight of approximately 100,000 Dalton. Some saponins are sweet while others are

bitter.

The antifungal and antibacterial properties of saponins are important in cosmetic

application in addition to their emollient effects. Saponins have both current and potential

applications in animal and human nutrition, in pig and poultry raising facilities and in dog and

cat foods. Saponins have ammonia binding activity when added to the diet,can bind to ammonia

and certain other odoriferous components in the excreta and prevent them from being released

into the air. It is however interesting that human do not suffer severe poisoning from saponins.

Tannins

Tannins is a general descriptive name for a group of polymeric phenolic substances

capable of tanning leather or precipitating gelatin from solution, a property known as

astringency. Their molecular weight range from 500 to 3000 kD and are found in almost every

plant part: bark, wood, leaves, fruits and roots. Tannins are divided into two groups,

hydrolysable and condensed tannins. Hydrolysable tannins are based on Gallic acid, usually as

multiple esters with D-glucose; while the more numerous condensed tannins often called

proanthocyanidins are derived from flavonoid monomers. Tannins may be formed by

18

condensation of flavan derivatives which have been transported to woody tissues of plants.

Alternatively; tannins may be formed by polymerization of quinone units.

This group of compounds has received a great deal of attention in recent years, since it

was suggested that the consumption of tannin-containing beverages, especially green teas and

red wines, can cure or prevent a variety of illness (Herbert, 1989). Many human physiological

activities, such as stimulation of phagocytic cells, host mediated tumor activity, and a wide range

of anti-infective activities have been assigned to tannins. One of their molecular actions is to

complex with proteins through so-called nonspecific forces such as hydrogen bonding and

hydrophobic effects, as well as by covalent bond formation. Thus, their mode of antimicrobial

action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope

transport proteins etc.

Phenolics and Polyphenols

Some of the simplest bioactive phytochemicals consists of a single phenolic ring.

Cinnamic and caffeic acids are common representatives of a wide group of phenylpropane-

derived compounds which are in the highest oxidation state. The common herbs, Tarragon and

Thyme, both contain caffeic acid, which is effective against viruses (Wild, 1994), bacteria

(Bratner and Grein, 1994) and fungi (Duke, 1985).

Catochol and pyrogallol both are hydroxylated phenols shown to be toxic to

microorganisms. Catochol has two –OH groups, and pyrogallol has three. The site(s) and number

of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to

microorganisms, with evidence that increased hydroxylation results in increased toxicity

(Geissman, 1963). In addition, some authors have found that more highly oxidized phenols are

inhibitors. The mechanisms thought to be responsible for phenolic toxicity to microorganisms

19

include enzyme inhibition by the oxidized compounds, possibly through more non specific

interactions with the proteins (Mason and Wesserman, 1987). Phenolic compounds possessing a

C3 side chain at lower level of oxidation and containing no oxygen are classified as essential oils

and often cited as antimicrobial as well. Eugenol is a well characterized representative found in

clove oil. Eugenol is considered bacteriostatic against both fungi and bacteria (Duke, 1985).

1.2.7 Description of Test Organisms

Food borne pathogens are widely distributed in the environment and may be a significant

cause of mortality and morbidity in the population (Indu et al., 2006). Escherichia coli is a

significant food borne hazard in many countries around the world. Infection often causes

haemorrhagic diarrhoea, and occasionally to kidney failure and death. Salmonella is another

bacteria that is the cause of food borne illness mainly from foods of animal origin throughout the

world. Staphylococcus aureus and Bacillus cereus cause foodborne illness due to their ability to

form heat stable toxins (WHO, 2007).

Salmonella spp

Salmonella species are gram negative, aerobic, rod-shaped, zoonotic bacteria that can

infect humans, birds, reptiles, and other animals. Salmonella spp. are a group of bacteria which

reside in the intestinal tract of human beings and warm blooded animals and are capable of

causing disease. They are members of the Enterobacteriaceae group. The genus Salmonella

contains 2 species: Salmonella enterica and Salmonella bongori. Salmonella enterica is an

important agent of food borne illness.

Salmonella spp. are not particularly heat resistant and most serotypes are killed by normal

cooking conditions, i.e. cooking to a core temperature of 75ºC instantaneously or an equivalent

20

time temperature combination, e.g. 70ºC for 2 minutes. However, a few highly heat resistant

serotypes have been reported, e.g. S. senftenberg 775W and S. irumu. Heat resistance is

influenced by water activity (aw), nature of the solutes and pH of the suspending medium.

Greater heat resistance is observed for cells in sucrose compared with NaCl at the same aw

values. The incidence of various Salmonella species seems to vary with geographic location and

the types of food consumed. Imported birds and animals may serve to introduce different

Salmonella species to the local area that can cause new and devastating outbreaks. They are the

causative agents of typhoid fever, enteric fever, gastroenteritis and septicemia.

Escherichia coli

Escherichia coli is a facultative, anaerobic, motile, gram negative rods that ferment

sugars to produce acid and gas. It belongs to the family Enterobacteriaceae which are bacteria

that normally live in the intestines of animals; including humans.There are approximately 100

strains of E. coli most of which are beneficial as normal flora. Although E. coli inhabit the

intestinal tract as beneficial microorganisms, there are also strains of E. coli that are known to

produce toxins. E.coli strains that contain enterotoxins and other virulence factors including

invasiveness and colonization factors cause diarrheal disease. Four such strains have been

identified. The National Center for Infectious Diseases in the United States , Centers for Disease

Control (CDC), particularly warns of the dangers posed by the rare strain E. coli O157:H7, a

pathogenic strain isolated from manure from cattle, sheep, pigs, deer and poultry. This strain can

cause severe diarrhea and kidney damage. Young children, the elderly, and those with weakened

immune systems are the most vulnerable. It is this particular strain that has been highly

publicized. E.coli is also a major cause of urinary tract infections and noscomial infections

including septicemia and meningitis.

21

Staphylococcus spp

They are gram positive cocci and occur most commonly as irregular cluster of spherical cells.

They are mesophilic non spore formers; however they are generally highly resistant to drying,

especially when sequestered in organic matter such as blood, pus and tissue fluids. They are

capable of surviving outside the body for extended period of time, even up to several months.

The genus Staphylococcus comprises both of pathogenic and non-pathogenic organisms. Most

Staphylococci are indigenous to skin surfaces and mucus membranes of the upper respiratory

tract. Breaks in the skin and mucus lining may serve as portals of entry to the underlying tissue.

With the possibility of infection by virulent strains, the three major species include S. aureus, S.

saprophyticus and S. epidermidis. Strains of the last two species are generally avirulent,

however, under special circumstances where a suitable portal of entry is provided. S. epidermidis

may be the aetiological agent for skin lesion and endocarditis and S. saprophyticus has been

implicated in some urinary tract infections. S.aureus is mainly associated with the skin and

mucous membrane of warm blooded vertebrates but is often isolated from food products, dust

and water. Some species are opportunistic pathogens of human and animals or produce

extracellular toxins.

Enterobacter spp

Enterobacter spp are in the family Enterobacteriaceae. Enterobacter spp are facultatively

anaerobic gram negative bacilli, motile by means of peritrichous flagella and have class 1

fimbriae. They produce acid upon glucose fermentation are methyl red negative and Voges-

Proskauer positive, with an optimal growth temperature of 30oC, about 80% are encapsulated

(Hart, 2006). They are widely distributed in nature occurring in fresh water, soil sewage plants,

vegetable and animals and human feces. Several strains of this bacterial organism are pathogenic

22

and cause opportunistic infection in immunocompromised hosts and in those who are on

mechanical ventilation. The urinary and respiratory tracts are the most common site of infection.

The genus Enterobacter is a member of the coliform group of bacteria. It does not belong

to the fecal coliform group of bacteria as does E.coli because it is incapable of growth at 44.5oC

in the presence of bile salts. Two clinically important species from this genus are E. aerogenes

and E.cloacae (Cabral, 2010).

Proteus spp

Proteus is a genus of gram negative proteobacteria, which includes pathogens responsible

for many urinary tract infections. Proteus exhibit characteristic swarming and they are part of the

normal flora of the gastrointestinal tract. It occurs in intestine of humans and a wide variety of

animal; also occur in manure, soil and polluted water. Three of the Proteus species, P. vulgaris,

P. mirabilis and P. penneri, are pathogenic to humans causing chronic urinary tract infections,

bacteremia, pneumonia and focal lesions. These species only become pathogenic if present

outside the gastro intestinal tract. Proteus also hydrolyzes urea, which alters the pH of urine and

may lead to the formation of kidney stones. Some Proteus species are motile, and all are oxidase

negative, urease positive, aerobic, rod shaped bacilli that do not ferment lactose.

Bacillus spp

Bacillus is a genus of gram positive rod shaped bacteria and a member of the phylum

Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive

for the enzyme catalase. Bacillus includes both free living and pathogenic species. Under

stressful conditions, the cell produces oval endospores that can dormant for extended periods

(Madigan, 2005). They are found in a wide range of habitats, a few species are pathogenic to

vertebrates and invertebrates.

23

Two Bacillus species are considered medically significant: B. anthracis which causes

anthrax and B. cereus which causes a food borne illness similar to that of Staphylococcus. The

type species is B. subtilis an important model organism. It is also a food spoiler, causing ropiness

in bread and related food. Some environmental and commercial strains B. coagulans may play a

role in food spoilage of highly acidic tomato based products (Ryan and Ray, 2004).

24

CHAPTER TWO

MATERIALS AND METHOD

2.1 Collection and Identification of Plant Material

The plant material of the spices Piper guineeense (guinea pepper), Xylopia aethiopica

(African pepper) and Allium cepa (common onion) were purchased locally from Ogige market in

Nsukka. The plant seeds were identified by Mr Ugwuozor of Plant Science and Biotechnology

Department, University of Nigeria, Nsukka.

2.2 Isolation and identification of Microrganisms

Egusi soup which was observed to have spoilt after 24 h storage without reheating and

pepper-soup perceived to spoil after 48 h were obtained from a restaurant and cultured to isolate

contaminant and presumed spoilage organisms using standard bacteriological techniques. A

loopful of each spoilt soup sample was inoculated on sterile nutrient agar plates and incubated at

37oC for 24h. Discrete colonies obtained on the plate were isolated and purified by streaking and

re-isolation three successive times in nutrient agar plates. The pure cultures were subsequently

characterized and tentatively identified on the basis of their cultural, morphological and

biochemical properties with reference to Bergey’s Manual of Determinative Bacteriology, 8th

edition (Bergey and Breed, 1957)

2.3 Test Microorganism

The test organisms were a strain each of Escherichia coli, Enterobacter sp. and

Proteus sp. isolated from egusi soupand two strains of Bacillus spp. – one isolated from egusi-

soup and the other from pepper soup. Other test bacterial strains, namely, Staphylococcus sciuri,

25

Staphylococcus. aureus and Salmonella guineum, were obtained from Professor Char of the

Department of Veterinary Microbiology, University of Nigeria, Nsukka.

2.4 Sample Preparation and Extraction Procedures

The seeds of Piper guineense and X. aethiopica were spread and dried in the sun for 3

to 4 days and then pulverised with a mechanical grinder. The bulbs of Allium cepa were washed

and then air dried at room temperature for one week and pulverised into fine powder using an

electric milling machines.

A 15 g sample of each powdered plant tissue was soaked for 24 h in 100 ml of

absolute ethanol. After 24 h, the extracts were filtered using a clean muslin cloth and then

filtered with Whatman No.1 filter paper. The filtrates were evaporated under forced air current

and the extract obtained. The same procedure was repeated for hot water and cold water in

extraction of the various powdered plant materials. All extracts were stored dry in sterile

containers and refrigerated until used for phytochemical analyses and antimicrobial testing.

2.5 Media Preparation

All media used were prepared according to the manufacturer’s directives. Nutrient

Agar (Fluka) was prepared by suspending 23grams of the medium in one liter of distilled water

and sterilized by autoclaving at 121oC for 15 mins and checked for sterility at 37

oC for 24h. The

Muller- Hinton Agar (Biotec) was prepared by suspending 38 grams of the medium in one liter

of distilled water. It was mixed well and boiled for about one minute and sterilized in an

autoclave at 121oC for 15 mins and checked for sterility at 37

oC for 24h. The Nutrient Agar was

used for plating out the organisms as well as storing them in slants while the Muller- Hinton

Agar was used for sensitivity test.

26

2.6 Preparation of Crude Plant Extracts

A 2 g amount of extract was weighed out, using a Mettler balance, and then dissolved in

5 ml of dimethyl sulphoxide (ethanol extract) or 5 ml of water (water extracts). Subsequently,

each solution was serially diluted two-fold to obtain 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125

mg/ml concentrations.

2.7 Determination of Antimicrobial Activity of Extracts

The antimicrobial activity was evaluated using the agar well diffusion method as

described by Okeke et al. (2001). The dried extracts were reconstituted as described above. Prior

to use, the stock cultures of the test organisms were sub-cultured on nutrient broth and incubated

at 37oC for 12 h. The concentration of the 12 h culture was adjusted to 0.5 McFarland Standard (

i.e. about 105 cfu/ml). A 0.1 ml volume of the standard suspension (10

5 cfu/ml) of each test

bacterial strain was spread evenly on Muller Hinton agar plates using sterile glass rod spreader

and the plates were allowed to dry at room temperature. Subsequently, 6 mm-diameter wells

were bored on the agar and100 µl of each reconstituted plant extract was pipetted into triplicate

wells. After holding the plates at room temperature for 1 hour to allow diffusion of extract into

the agar, they were incubated at 37oC for 24 h and the inhibition zone diameter (IZD) was

measured to the nearest mm. Antimicrobial activities were expressed as the IZD (mm) produced

by the plant extracts.

27

2.8 Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal Concentration (MBC) of Crude Extracts

The MIC and MBC of the extracts for each susceptible test organism were determined

by a modification of the broth macro tube dilution method described by Okeke et al. (2001) and

Okoli et al. (2002). Two-fold serial dilutions of the reconstituted extract 400 mg/ml were made

in nutrient broth to achieve a concentration range of approximately 3.125- 400 mg/ml. A 0.1 ml

suspension of the test organism was inoculated into 1 ml of each concentration of the extract in

duplicates. A tube containing nutrient broth only was seeded with the test organism as described

above to serve as control. All culture tubes were incubated at 37oC for 24 h. Growth was scored

visually by the turbidity of the culture. The least concentration showing no growth was taken to

be the MIC.

To determine the MBC, 0.1 ml inoculum was taken from each of the last three

consecutive tubes in which there was no growth and sub cultured on Muller-Hinton Agar plates.

After incubation at 37oC for 24 h, the plates were observed for bacterial growth. The least

concentration showing no growth was taken as the MBC.

2.9 Phytochemical Screening of the Plant Extract

The phytochemical screening was carried out using the methods described by

Farnsworth (1996), Harbone (1998) and Sofowara (1993).

28

2.9.1 Test for Alkaloids

A 0.2 g weight of each plant extracts was boiled with 5 ml of 2% hydrochloric acid in

a water bath for 10 min. The mixture was filtered and 1 portion of each extract treated with 2

drops of the following reagents.

1) Drangendroff’s Reagent: A red precipitate indicates the presence of alkaloids

2) Mayer’s Reagent: A red precipitate indicates the presence of alkaloids

3) Picric Acid (1%). A yellow precipitate indicates the presence of alkaloids.

2.9.2 Test for Flavonoids

A 0.2 g weight of the extracts was heated with 10 ml of ethyl acetate in a boiling water

bath for 3 minutes. The mixture was cooled and filtered. The filtrate was used for the following

test.

1. Ammonium test. Approximately 4 ml of the filtrate was shaken with 1 ml of dilute

ammonia (1%). The layers were allowed to separate and a yellow colour in the ammonia layer

indicates the presence of flavonoid.

2. Aluminum Chloride test. About 4 ml of the filtrate was shaken with 1 ml of dilute 1%

aluminum chloride solution and observed for yellow coloration in the aluminum chloride layer.

2.9.3 Test for Glycosides

This was performed by weighing 2 g of the extract and adding to 30 ml of distilled

water. The mixture was heated for 5 min in a boiling water bath, allowed to cool and filtered.

Fehlings solution A and B were added to the filtrate until it turned alkaline when tested with red

litmus paper. The alkaline mixture was heated in a boiling water bath for 2 minutes. A brick red

precipitate indicates the presence of glycosides.

29

2.9.4 Test for Saponins

To conduct this test, 0.1 g of the extract was boiled with 5 ml of distilled water in a boiling water

bath for 2 min. The mixture was filtered while still hot and allowed to cool. The filtrate was used

for the following tests.

Frothing test: Exactly 1ml of the filtrate was diluted with 4ml of distilled water. The mixture

was vigorously shaken and then observed on standing for a stable froth.

Emulsion Test: To conduct this test, 2 drops of olive oil was added to 1ml of the filtrate. The

mixture was shaken and observed for the formation of emulsion.

2.10.5 Test for Tannins.

To perform this test, 1 g of the extract was boiled with 5 ml of 45% ethanol (45ml of

ethanol in 100ml of distilled water) for 5 min, the solution was filtered and the filtrate treated

with ferric chloride solution. Using a pipette, 2 drops of ferric chloride was added to 1 ml of the

filtrate. A greenish black precipitate indicates the presence of tannins.

2.10.6 Test for Fats and Oil.

About 0.2 g of the extract was pressed between filter paper and the paper observed. A

control was also prepared by placing 2 drops of olive oil in filter paper. Translucency of the filter

paper indicates the presence of fats and oil.

2.10 Screening of ground Spices for Inhibitory Activity against Test Organisms

This was carried out using the method described by Eruteya and Odunfa (2009).

Different concentrations(0.5%, 1.5%, and 3.0%) of grounded powder of Piper guineense,

Xylopia aethiopica and Alium cepa were added to nutrient agar before autoclaving at 1210C for

30

15 min. Using pour plate method 0.1 ml of 18-24 h of the different test organisms was added to

the sterilized nutrient agar containing the different spice concentrations respectively. Plates

without spice but with organisms served as control while plates with spice but no organism

served as standard. The plates were incubated at 370Cfor 24 h. After 24 h the plates were

checked for the concentration that inhibited the growth of the organisms. The inhibitory activity

was recorded by reduction in colony count on agar.

31

CHAPTER THREE

RESULT

3.1 Isolation and Characterization of Test Organisms

A total of seven organisms were used in this study; four, namely, E. coli, Proteus sp.,

Enterobacter sp.and Bacillus strain isolated from spoilt egusi; and three – Staph sciuri, Staph

aureus and Salmonella guineum obtained from Veterinary Microbiology Department. The

Bacillus strain isolated from pepper soup was not susceptible to the spices preparations at the

preliminary screening and, therefore, was not used further. The three strains obtained from

Veterinary Microbiology Department were re-characterised to confirm their identity. All test

organisms were tentatively identified or had their identity confirmed using standard

bacteriological techniques as shown in Table 1.

3.2 Yield from Aqueous and Ethanol Extractions

The spices were extracted with cold water, hot water and ethanol, respectively. The

highest yield was achieved with ethanol P. guineense 4.8g (32%), followed by cold water P.

guineense, 3.7g (24.7%). The least were cold water A. cepa and hot water X. aethiopica. Table 2.

32

Table 1: Characterization of the Test Organisms

Test strain/

(Code)

Cell morphology

and Gram reaction

Biochemical Characteristics

Tentative

organism Catalase Lactose Mannose Glucose Sucrose Citrate Motility Sporulation

ES-1 Gram – rod + - + + + + + - Enterobacter

sp

ES-2 Gram – rod + - + + + - + - Escherichia

coli

ES-3 Gram – rod - - -

-

+ + - + - Proteus sp

ES-4 Gram + rod - + - - - + + Bacillus sp

VS-1 Gram + Cocci + - + `ND + ND ND ND Staphylococc

us sciuri

VS-2 Gram + cocci + + + ND + ND ND ND Staphylococc

us aureus

VS-3 Gram - rod + ND ND + ND + + ND Salmonella

guineum

Key: Key: ES - Egusi Isolate; VS – Vet Isolate; ND – Not Determined

33

Table 2: Yield from the Ethanol and Aqueous Extractions

Extract Weight of

spice(g)

Weight of

Extract(g)

Percentage

Yield

Ethanol X. aethiopica 15 1.9 12.7%

Cold-Water X. aethiopica 15 0.6 4%

Hot-Water X. aethiopica 15 1.1 7.3%

Ethanol P. guineense 15 4.8 32%

Hot-Water P. guineense 15 3.7 10.7%

Cold-Water P. guineense 15 1.6 24.7%

Ethanol A. cepa 15 0.8 5.3%

Hot-Water A. cepa 15 0.7 4.7%

Cold-Water A. cepa 15 0.6 4%

34

3.3 Chemical Constituents in Ethanol, Cold and Hot water of the Extracts.

The secondary metabolites tested were alkaloids, flavonoids, glycosides, Saponins,

Tannins, Fats and oil. Alkaloids were detected in low or moderate amounts from extracts of all

the spices except ethanol extract of P. guineense and A. cepa and Cold water extracts of X.

aethiopica and A. cepa. Flavonoid was not detected in any of the extracts. Glycosides were

detected in high amounts in cold water extracts of P. guineense, in moderate amount in hot

water P. guineense and in low amount in ethanol extract of A. cepa and not at all in the other

extracts. Saponins were detected in moderate amount in ethanol P. guineense, low amount in

cold and hot water extracts of P. guineense and ethanol extract of X. aethiopica but not in

others.

Cold water extracts of P. guineense and X. aethiopica yielded tannins in high amount

while hot water extracts of P. guineense and ethanol extracts of A. cepa yielded moderate

amount. Cold water extracts of X. aethiopica and A. cepa had no detectable tannin. With the

exception of hot water extract of A. cepa and ethanol extract of A. cepa in which fats and oil

occurred in high amounts, it was moderately present in the other extracts. (Table 3-5).

3.4 Anti-bacterial Activity of the Extracts

Tables 6-9 show the inhibition zone diameter (IZD) measurement obtained for

antibacterial activity of the extracts. The highest inhibition zone diameter was achieved with

hot water extract of P. guineense on E.coli 14±0.2mm at a concentration of 400mg/ml

followed by the activity of P. guineense against Bacillus spp 12.7±0.2mm at a concentration of

400mg/ml (Table 6).

35

Table 7 shows the activity of X. aethiopica on E.coli, Bacillus spp, Staph sciuri and

Salmonella guineum. The extract exhibited activity at 400 and 200mg/ml with E. coli showing

the highest IZD 13.1±0.1mm at 400mg/ml. Proteus, Staph aureus and Enterobacter spp

showed no susceptibility at any concentration. The hot water extracts of all the three spices

showed no activity against Proteus and Enterobacter spp. Similarly, the hot water extract of A.

cepa did not show activity against any of the test organisms.

A 400mg/ml concentration of cold extract of A. cepa showed activity of 10±0.2mm

against Enterobacter spp and 12±0.1mm against other test organisms except Bacillus spp

which showed no susceptibility to any of the cold water extracts (Table 8).

As shown in table 9, cold water extract of X. aethiopica was not active against most test

organisms even at 400mg/ml concentration except E.coli, which showed susceptibility to 400,

200 and 100mg/ml, and slightly against Staph aureus IZD 7.8±0.1mm. Finally, cold water

extract of P. guineense and ethanol extract of all the spices showed no activity on any of the

test organisms.

36

Table 3: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Piper

guineense

Metabolites Ethanol Hot water Cold water

Alkaloids _ ++ ++

Flavonoids _ _ _

Glycosides _ ++ +++

Saponins ++ + +

Tannins + ++ +++

Fats and Oil ++ ++ ++

KEY

_ Absent

+ Low

++ Moderate

+++ High

37

Table 4: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Xylopia

aethiopica


Alkaloids + + _

Flavonoids _ _ _

Glycosides _ _ _

Saponins + _ _

Tannins ND ++ +++

Fats and oil ++ ++ ++

KEY

_ Absent

+ Low

++ Moderate

+++ High

ND Not determined

38

Table 5: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Allium

cepa


Alkaloids _ ++ _

Flavonoids _ _ _

Glycosides + _ ND

Saponins _ _ ND

Tannins ++ + ND

Fats and oil +++ +++ ++

KEY

- Absent

+ Low

++ Moderate

+++ High

ND Not determined

39

Table 6: Inhibition of Microorganisms by Hot Water Extract of Piper guineense

Test organisms

Inhibition Zone Diameter (IZD mm) achieved at each extract

concentration

400 200 100 50 25 12.5 6.25 3.125

Escherichia coli 14.7±0.3 9.9±0.2 7.9±0.1 0.0 0.0 0.0 0.0 0.0

Salmonella guineum 8.2±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bacillus spp

Staphylococcus aureus

12.7±0.6

8.0±0.1

10.8±0.3

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Staphylococcus sciuri 9.1±0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Enterobacter spp

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0

KEY

0.0 = No Activity

40

Table 7: Inhibition of Microorganisms by Hot water Extract of Xylopia aethiopica


concentration

Test Organisms 400 200 100 50 25 12.5 6.25 3.125

Escherichia coli 13.1±0.2 11±0.2 10.1±0.1 0.0 0.0 0.0 0.0 0.0

Salmonella guineum 9.9±0.1 7.7±0.3 0.0 0.0 0.0 0.0 0.0 0.0

Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bacillus spp 9.1±0.1 7.3±0.3 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus aureus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus sciuri 10.1±0.1 7.4±0.1 0.0 0.0 0.0 0.0 0.0 0.0

Enterobacter spp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

KEY

0.0 = No Activity

41

Table 8: Inhibition of Microorganisms by Cold Water Extract of Allium cepa


concentration

Test organism 400 200 100 50 25 12.5 6.25 3.125

Escherichia coli 12.0±0.2 10.2±0.2 9.8±0.2 0.0 0.0 0.0 0.0 0.0

Salmonella guineum 12.0±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteus 12±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bacillus spp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus aureus 12±0.2 10±0.2 10±0.1 0.0 0.0 0.0 0.0 0.0

Staphylococcus sciuri 12±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Enterobacter spp 10±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

KEY

0.0 = No Activity

42

Table 9: Inhibition of Microorganism by Cold Water Extract of Xylopia aethiopica


concentration

Test Organisms 400 200 100 50 25 12.5 6.25 3.125

Escherichia coli 12±0.1 10±0.1 7.2±0.1 0.0 0.0 0.0 0.0 0.0

Salmonella guineum 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Bacillus spp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus aureus 7.8±0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Staphylococcus sciuri 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Enterobacter spp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

KEY

0.0 = No Activity

43

3.5 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration

(MBC) of the Extracts

The result revealed that the MIC and MBC of hot water extracts were generally lower

than those of cold water and ethanol extracts. The least activity was recorded for the ethanol

extracts of all the spices (Table 10).

Cold water extract of P. guineense showed no bacterial activity while cold water extract

of X. aethiopica and A. cepa had MBC values ranging from 50mg/ml to 100mg/ml against all

test organisms except Proteus (Table 11). The highest activity was recorded for hot water P.

guineense with MIC and MBC values of 3.125mg/ml and 25 mg/ml, respectively, for both

Salmonella guineum and Staph. sciuri followed by hot water extracts of A. cepa with MIC and

MBC values of 3.125mg/ml and 50mg/ml, respectively, for both E. coli and Staph. sciuri.

None of the extract showed bactericidal activity against Bacillus spp (Table 12).

3.6 Screening of Spices for Inhibitory Activity against Test Organisms

Tables 13-16 show the effects of combinations (ratio of 1:1 or 1:1:1) of ground spices used

against each test organisms.The combination of X. aethiopica and P. guineense had

inhibitory effect on S.guineum, Staph aureus and Enterobacter spp at concentrations of 3%

(Table 13). The combination of A. cepa : X. aethiopica at concentration of 3% had inhibitory

effect on all test organisms except Staph aureus and E.coli (Table 14). The combination of P.

guineense and A .cepa and X. aethiopica, P. guineense and A.cepa had an inhibitory effect at

concentration of 3.0% on all the test organisms (Table15 & 16).

44

Table 10: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

of Ethanol Extract of X. aethiopica, P. guineense and A.cepa

Test organism Piper guineense

(mg/ml)

Xylopia aethiopica

(mg/ml)

Alium cepa

(mg/ml)

MIC MBC MIC MBC MIC MBC

Escherichia coli 50 _ 100 _ 100 _

Salmonella guineum 25 _ 50 _ 50 50

Proteus 50 _ 100 _ 50 _

Bacillus spp 100 _ 100 _ 50 _

Staphylococcus sciuri 50 _ 50 _ _ _

Enterobacter spp 50 _ 25 _ _ _

Staphylococcus aureus 100 _ 50 _ _ _

KEY

- = No Activity

45


of Cold Water Extract of X. aethiopica, P. guineense and A. cepa


(mg/ml)

Xylopia aethiopica

(mg/ml)

Allium cepa

(mg/ml)

MIC MBC MIC MBC MIC MBC

Escherichia coli 100 _ 100 400 50 50

Salmonella guineum 100 _ 100 _ 25 50

Proteus _ _ 100 _ _ _

Bacillus spp 100 _ 100 _ 50 100

Staphylococcus sciuri 50 _ 50 100 100 100

Enterobacter spp _ _ 100 _ 100 50

Staphylococcus aureus _ _ 50 400 50 100

KEY

- = No Activity

46


of Hot Water Extract of X. aethiopica, P. guineense and A. cepa


(mg/ml)

Xylopia aethiopica

(mg/ml)

Allium cepa

(mg/ml)

MIC MBC MIC MBC MIC

MBC

Escherichia coli 25 100 3.125 100 3.125 50

Salmonella guineum 3.125 25 50 200 12.5 50

Proteus 25 25 3.125 _ 12.5 100

Bacillus spp 3.125 _ 3.125 _ 12.5 _

Staphylococcus sciuri 3.125 25 3.125 400 3.125 50

Enterobacter spp 25 100 50 _ 6.25 100

Staphylococcus aureus 3.125 50 3.125 _ 3.125 100

KEY

- = No Activity

47

Table 13: Effect of Spice Combination (X. aethiopica: P. guineense ) on Test Organisms

Test organisms No Spice 0.5% 1.5% 3.0%

Escherichia coli +++ +++ +++ +

Salmonella guineum +++ +++ ++ _

Proteus +++ ++ ++ +

Bacillus spp +++ +++ ++ +

Staphylococcus aureus +++ +++ +++ ++

Staphylococcus sciuri +++ +++ +++ _

Enterobacter spp +++ +++ ++ _

KEY

+++ = Abundant growth

++ = Growth (numerous separate colonies)

+ = limited growth

- = No growth

48

Table 14: Effect of Spice Combination (P. guineense: A. cepa) on Test Organisms


Escherichia coli +++ ++ ++ +

Salmonella guineum +++ +++ _ _

Proteus +++ +++ ++ _

Bacillus spp +++ +++ ++ _

Staphylococcus aureus +++ +++ +++ ++

Staphylococcus sciuri +++ +++ + _

Enterobacter spp +++ +++ ++ _

KEY



+ = limited growth

- = No growth

49

Table 15: Effect of Spice Combination (Alium cepa: X.aethiopica) on Test Organisms

Test Organisms No Spice 0.5% 1.5% 3.0%

Escherichia coli +++ ++ + _

Salmonella guineum +++ +++ _ _

Proteus +++ +++ + _

Bacillus spp +++ +++ +++ _

Staphylococcus aureus +++ +++ +++ _

Staphylococcus sciuri +++ + _ _

Enterobacter spp +++ +++ + _

KEY



+ = limited growth

- = No growth

50

Table 16: Effect Spice Combination (X. aethiopica : P. guineense : A. cepa ) on Test

Organisms


Escherichia coli +++ ++ + _

Salmonella guineum +++ ++ _ _

Proteus +++ +++ ++ _

Bacillus spp +++ +++ +++ _

Staphylococcus aureus +++ ++ + _

Staphylococcus sciuri +++ ++ ++ _

Enterobacter spp +++ + + _

KEY



+ = limited growth

- = No growth

51

CHAPTER FOUR

DISCUSSION

Additives preserve foods by inhibiting microbial growth or inhibiting enzyme activity

in case of fruits and forestalling spoilage. It is on this premise that spices are being tested for

antibacterial properties; and presumably those with antibacterial activity could be further

studied for use as natural preservatives for recipes where they are used for spicing or as

condiments. This trend is being promoted because of the safety concerns surrounding the use

of chemical additives for food preservation. In this study, the preservative effect of three

Nigerian spices Piper guineense, Xylopia aethiopica and Allium cepa used in the preparation of

pepper-soup was evaluated.

The phytochemical screening of the plant crude extracts revealed the presence of

Alkaloids, Glycosides, Saponnins, Tannins, Fats and oil in varying proportions and the absence

of Flavonoids. These compounds have variously been reported to possess antimicrobial activity

(Okeke et al., 2001, Mahajan et al., 2008). The absence of flavonoids was reported in all the

extracts and flavonoids have been found in–vitro to be effective antimicrobial substance

against a wide array of microorganisms (Azu et al., 2007; Ijeh et al, 2004; Ekpo et al; 2012).

The results show that there are differences in phytochemical constituents extractable by the

different solvents (cold water, hot water and ethanol) used. These differences seem to be

reflected in the difference in spectrum and degree of antibacterial activity of extracts on the test

organisms. Almost all the metabolites detected have been suspected to contribute to

antimicrobial activity of extracts in other reports (Okeke et al., 2001, Mahajan et al., 2008).

For example, tannins have been found to form irreversible complexes with proline–rich

proteins resulting in the inhibition of the cell protein synthesis; besides, herbs that contain

52

tannin are astringent in nature and are used for treating intestinal disorders such as diarrhea and

dysentery but this use has not been specifically found to be due to their antimicrobial activities

(Shimada, 2006 and Dharmanda, 2003). There was no experiment in this work designed to

trace the specific active compounds in the extracts.

From this investigation, the aqueous extract of the spices have more potential as an

antimicrobial agent than its ethanolic extracts. This result is in agreement with Ijeh et al.

(2005), who reported the higher susceptibility of test organisms to aqueous extract of O.

grattisimum and X. aethiopica, Olusimbo et al. (2011) who reported the antimicrobial potential

of the aqueous extract of Piper guineense and Azu et al. (2007) who reported the antibacterial

properties of the water soluble extracts of onion. The high level of activity observed in the

aqueous extracts against the bacterial pathogens showed that the active components were

soluble in water. This property is very desirable as these spices are used as condiments in food

preparation (Olusimbo et al., 2011). This also supports the extensive inclusion of these spices

in folklore medicinal preparations in various cultures in Africa. For example it is believed that

Piper guineense stimulates the production of hydrochloric acid in the stomach and promotes

the health of the digestive tract (Olusimbo et al., 2011).

It was observed that the hot water extracts of Piper guineense and Xylopia aethiopica

showed activity against all the test organisms except Proteus spp and Enterobacter spp. (Table

6 & 7). This may mean that heating of these spices during cooking would not inactivate the

ingredients that are active against the suscepetible bacterial strains. In other words the spices in

the cooked food samples may already be active against some contaminants, thereby providing

a measure of food preservation. It was also observed that hot water extract of Allium cepa did

not show any activity. This may be explained by the fact that the antimicrobial substance in the

53

onion extracts, which are mainly phenolic compounds are heat labile and might have been

denatured during the extraction process (Azu et al., 2007).

The cold water extract of Allium cepa exhibited activity at (400mg/ml) on Salmonella

guineum, Proteus, Staphylococcus scuiri, Enterobacter spp, Escherichia coli and Staph. aureus

and inactivity on Bacillus spp. This would imply that for A. cepa to be applied in food

preservation, its cold water extract should not be subjected to heating or cooking. This leads to

the speculation that chewing of raw onions may reduce presence or number/type of pathogens

in the oral cavity, thus helping to maintain oral hygiene. The non-susceptibility of Bacillus spp

may be explained by the physiology of the strain, since the organism has the ability to form

resting spores under adverse environmental conditions, it may also form resting spores when

exposed to theeffect of the extracts. Specific investigation needs to be carried out on the

differential reactions of the vegetative cells and spores of Bacillus sp to exposure to different

concentrations of cold extracts of A. cepa. Also, the cold water extract of Xylopia aethiopica

had activity only on E. coli at 400mg/ml while cold water Piper guineense was not active

against the test organism, suggesting that cold water as a solvent could dissolve the active

constituents of Allium cepa better compared to X. aethiopica and P. guineense extract (Azu et

al, 2007). It is not known to what extent the activity of cold water extract of X. aethiopica

could affect the intestinal E. coli in vivo and whether this could partly explain the diarrhea that

results in some individuals after consumption of much pepper.

It was also observed that there were differences in susceptibility of the test

microorganisms used in the research to each extract. Escherichia coli was the most inhibited

organism by P. guineense, A. cepa and X. aethiopica with a mean inhibition zone diameter

(IZD) of 3.99mm while the least inhibited was Enterobacter spp with a mean inhibition zone

54

diameter of 0.314mm by cold water A. cepa. Escherichia. coli was best inhibited by hot water

extract of Piper guineense with an average IZD of 14.7±0.3mm. Varied susceptibility of each

test organisms to the extracts usually reflect the differences in physiology of individual

bacterial species as stated by Garret et al., (2000) or differences in the quantity and quality of

the active ingredients (Cowan, 1999; Adetiyi et al., 2004), extraction methods employed, the

dosage of extract applied and the diffusion properties of these extracts in the agar. (Ekwenye

and Elegalam, 2005).

A 50:50 combination of the extracts of A.cepa and X.aethiopica had the same activity

(IZD) as the ground spice material applied directly in 1:1 proportions also. The later was not

estimated by IZD measurement but by reduction in colony count in agar. All materials used in

this assay were heat- treated. In the first place the activity shown by both after heat treatment

can only come from heat-stable components present in both the extract and in the unextracted

spice preparation. Both materials having relatively equivalent activity may indicate that it is the

same components that show activity in them. A residual activity after heat treatment is

desirable since almost all recipes in which the spices are applied are cooked at high heat. This

means that the heat stable antimicrobial constituent confers a measure of preservation on the

food for a period.

CONCLUSION

This research shows that the spices have a measure of antimicrobial actvivity and also

potentials for use as preservative. More studies need to be carried out to determine how these

spices can be used as preservatives- in what form, what quantity and how long.

55

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60

APPENDIX 1

UNIANOVA Clearance BY Organisms Conc

/METHOD=SSTYPE(3)

/INTERCEPT=INCLUDE

/EMMEANS=TABLES(Organisms)

/EMMEANS=TABLES(Conc)

/EMMEANS=TABLES(Organisms*Conc)

/PRINT=DESCRIPTIVE

/CRITERIA=ALPHA(.05)

/DESIGN=Organisms Conc Organisms*Conc.

UNIANOVA Clearance BY Organisms Conc Groups

/METHOD=SSTYPE(3)

/INTERCEPT=INCLUDE

/POSTHOC=Organisms Conc Groups(LSD)

/EMMEANS=TABLES(Organisms)

/EMMEANS=TABLES(Conc)

/EMMEANS=TABLES(Groups)

/EMMEANS=TABLES(Organisms*Conc)

/EMMEANS=TABLES(Organisms*Groups)

/EMMEANS=TABLES(Organisms*Conc*Groups)

/PRINT=DESCRIPTIVE

/CRITERIA=ALPHA(.05)

/DESIGN=Organisms Conc Groups Organisms*Conc Organisms*Groups Conc*Groups Organisms*Conc*Groups.

Descriptive Statistics

Dependent Variable:Clearance

Organisms Conc Groups Mean Std. Deviation N

E.coli 400 mg/ml Hot water Piper guineense 14.733 .2517 3

Hot water Xylopia aethiopica 13.100 .1732 3

Cold water Allium cepa 12.000 .2000 3

Cold water Xylopia aethiopica 11.967 .1528 3

Total 12.950 1.1882 12

200 mg/ml Hot water Piper guineense 9.867 .2309 3




Total 10.267 .4868 12


61




Total 8.767 1.3020 12

50 mg/ml Hot water Piper guineense .000 .0000 3

Hot water Xylopia aethiopica .000 .0000 3

Cold water Allium cepa .000 .0000 3

Cold water Xylopia aethiopica .000 .0000 3

Total .000 .0000 12





Total .000 .0000 12

12.5 mg/ml Hot water Piper guineense .000 .0000 3




Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12

Total Hot water Piper guineense 4.058 5.6488 24

62

Hot water Xylopia aethiopica 4.279 5.6974 24

Cold water Allium cepa 4.004 5.3147 24

Cold water Xylopia aethiopica 3.650 4.9647 24

Total 3.998 5.3330 96

Salmonella spp 400 mg/ml Hot water Piper guineense 8.167 .2887 3




Total 7.508 4.7473 12





Total 1.967 3.5592 12





Total .000 .0000 12





Total .000 .0000 12

25 mg/ml Hot water Piper guineens .000 .0000 3




Total .000 .0000 12




63


Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total 1.184 3.2044 96

Proteus 400 mg/ml Hot water Piper guineense .000 .0000 3




Total 3.025 5.4731 12





Total .000 .0000 12





Total .000 .0000 12

64





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12

Total Hot water Piper guineense .000 .0000 24




Total .378 2.1166 96

Bacillus spp 400 mg/ml Hot water Piper guineense 12.667 .5774 3


65



Total 5.433 5.8340 12





Total 4.533 4.9089 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





66

Total .000 .0000 12





Total .000 .0000 12





Total 1.246 3.3893 96

Staphylococcus aureus 400 mg/ml Hot water Piper guineense 8.000 .0000 3




Total 6.942 4.5242 12





Total 2.500 4.5235 12





Total 2.508 4.5378 12





Total .000 .0000 12


67




Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12




Cold water Xylopia aethiopica .979 2.6464 24

Total 1.494 3.5412 96

Staphylococcus sciuri 400 mg/ml Hot water Piper guineense 9.067 .1155 3




Total 7.792 4.8235 12




68


Total 1.858 3.3622 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12

69





Total 1.206 3.2615 96

Enterobacter spp 400 mg/ml Hot water Piper guineense .000 .0000 3




Total 2.508 4.5390 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12



70



Total .000 .0000 12





Total .000 .0000 12





Total .000 .0000 12

Total Hot water Piper guineense .000 .0000 24




Total .314 1.7553 96

Total 400 mg/ml Hot water Piper guineense 7.519 5.4082 21




Total 6.594 5.5513 84

200 mg/ml Hot water Piper guineense 2.952 4.7920 21




Total 3.018 4.4799 84

100 mg/ml Hot water Piper guineense 1.124 2.8210 21




71

Total 1.611 3.5141 84





Total .000 .0000 84





Total .000 .0000 84





Total .000 .0000 84





Total .000 .0000 84





Total .000 .0000 84




Cold water Xylopia aethiopica .661 2.4437 168

Total 1.403 3.5720 672

72

Post Hoc Tests

Groups

Multiple Comparisons

Clearance

LSD

(I) Groups (J) Groups

Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound

Upper

Bound

Hot water Piper guineense Hot water Xylopia aethiopica -.085* .0087 .000 -.102 -.068

Cold water Allium cepa -.517* .0087 .000 -.534 -.500

Cold water Xylopia aethiopica .788* .0087 .000 .771 .805

Hot water Xylopia aethiopica Hot water Piper guineense .085* .0087 .000 .068 .102

Cold water Allium cepa -.432* .0087 .000 -.449 -.414

Cold water Xylopia aethiopica .873* .0087 .000 .856 .890

Cold water Allium cepa Hot water Piper guineense .517* .0087 .000 .500 .534

Hot water Xylopia aethiopica .432* .0087 .000 .414 .449

Cold water Xylopia aethiopica 1.305* .0087 .000 1.288 1.322

Cold water Xylopia aethiopica Hot water Piper guineense -.788* .0087 .000 -.805 -.771

Hot water Xylopia aethiopica -.873* .0087 .000 -.890 -.856

Cold water Allium cepa -1.305* .0087 .000 -1.322 -1.288

Based on observed means.

The error term is Mean Square(Error) = .006.

*. The mean difference is significant at the .05 level.

Conc


Clearance

LSD

(I) Conc (J) Conc

Mean Difference



Lower Bound Upper Bound

73

400 mg/ml 200 mg/ml 3.576* .0123 .000 3.552 3.600

100 mg/ml 4.983* .0123 .000 4.959 5.007

50 mg/ml 6.594* .0123 .000 6.570 6.618

25 mg/ml 6.594* .0123 .000 6.570 6.618

12.5 mg/ml 6.594* .0123 .000 6.570 6.618

6.25 mg/ml 6.594* .0123 .000 6.570 6.618

3.125 mg/ml 6.594* .0123 .000 6.570 6.618

200 mg/ml 400 mg/ml -3.576* .0123 .000 -3.600 -3.552

100 mg/ml 1.407* .0123 .000 1.383 1.431

50 mg/ml 3.018* .0123 .000 2.994 3.042

25 mg/ml 3.018* .0123 .000 2.994 3.042

12.5 mg/ml 3.018* .0123 .000 2.994 3.042

6.25 mg/ml 3.018* .0123 .000 2.994 3.042

3.125 mg/ml 3.018* .0123 .000 2.994 3.042

100 mg/ml 400 mg/ml -4.983* .0123 .000 -5.007 -4.959

200 mg/ml -1.407* .0123 .000 -1.431 -1.383

50 mg/ml 1.611* .0123 .000 1.587 1.635

25 mg/ml 1.611* .0123 .000 1.587 1.635

12.5 mg/ml 1.611* .0123 .000 1.587 1.635

6.25 mg/ml 1.611* .0123 .000 1.587 1.635

3.125 mg/ml 1.611* .0123 .000 1.587 1.635

50 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570

200 mg/ml -3.018* .0123 .000 -3.042 -2.994

100 mg/ml -1.611* .0123 .000 -1.635 -1.587

25 mg/ml .000 .0123 1.000 -.024 .024

12.5 mg/ml .000 .0123 1.000 -.024 .024

6.25 mg/ml .000 .0123 1.000 -.024 .024

3.125 mg/ml .000 .0123 1.000 -.024 .024

25 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570

200 mg/ml -3.018* .0123 .000 -3.042 -2.994

100 mg/ml -1.611* .0123 .000 -1.635 -1.587

50 mg/ml .000 .0123 1.000 -.024 .024

74

12.5 mg/ml .000 .0123 1.000 -.024 .024

6.25 mg/ml .000 .0123 1.000 -.024 .024

3.125 mg/ml .000 .0123 1.000 -.024 .024

12.5 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570

200 mg/ml -3.018* .0123 .000 -3.042 -2.994

100 mg/ml -1.611* .0123 .000 -1.635 -1.587

50 mg/ml .000 .0123 1.000 -.024 .024

25 mg/ml .000 .0123 1.000 -.024 .024

6.25 mg/ml .000 .0123 1.000 -.024 .024

3.125 mg/ml .000 .0123 1.000 -.024 .024

6.25 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570

200 mg/ml -3.018* .0123 .000 -3.042 -2.994

100 mg/ml -1.611* .0123 .000 -1.635 -1.587

50 mg/ml .000 .0123 1.000 -.024 .024

25 mg/ml .000 .0123 1.000 -.024 .024

12.5 mg/ml .000 .0123 1.000 -.024 .024

3.125 mg/ml .000 .0123 1.000 -.024 .024

3.125 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570

200 mg/ml -3.018* .0123 .000 -3.042 -2.994

100 mg/ml -1.611* .0123 .000 -1.635 -1.587

50 mg/ml .000 .0123 1.000 -.024 .024

25 mg/ml .000 .0123 1.000 -.024 .024

12.5 mg/ml .000 .0123 1.000 -.024 .024

6.25 mg/ml .000 .0123 1.000 -.024 .024




75

Homogeneous Subsets

Organisms


Clearance

LSD

(I) Organisms (J) Organisms

Mean Difference




E.coli Salmonella spp 2.814* .0115 .000 2.791 2.836

Proteus 3.620* .0115 .000 3.597 3.642

Bacillus spp 2.752* .0115 .000 2.729 2.775

Staphylococcus aureus 2.504* .0115 .000 2.482 2.527

Staphylococcus sciuri 2.792* .0115 .000 2.769 2.814

Enterobacter spp 3.684* .0115 .000 3.662 3.707

Salmonella spp E.coli -2.814* .0115 .000 -2.836 -2.791

Proteus .806* .0115 .000 .784 .829

Bacillus spp -.061* .0115 .000 -.084 -.039

Staphylococcus aureus -.309* .0115 .000 -.332 -.287

Staphylococcus sciuri -.022 .0115 .058 -.044 .001

Enterobacter spp .871* .0115 .000 .848 .893

Proteus E.coli -3.620* .0115 .000 -3.642 -3.597

Salmonella spp -.806* .0115 .000 -.829 -.784

Bacillus spp -.868* .0115 .000 -.890 -.845

Staphylococcus aureus -1.116* .0115 .000 -1.138 -1.093

Staphylococcus sciuri -.828* .0115 .000 -.851 -.806


Bacillus spp E.coli -2.752* .0115 .000 -2.775 -2.729

Salmonella spp .061* .0115 .000 .039 .084

Proteus .868* .0115 .000 .845 .890


Staphylococcus sciuri .040* .0115 .001 .017 .062

76


Staphylococcus aureus E.coli -2.504* .0115 .000 -2.527 -2.482

Salmonella spp .309* .0115 .000 .287 .332

Proteus 1.116* .0115 .000 1.093 1.138

Bacillus spp .248* .0115 .000 .225 .271

Staphylococcus sciuri .288* .0115 .000 .265 .310

Enterobacter spp 1.180* .0115 .000 1.158 1.203

Staphylococcus sciuri E.coli -2.792* .0115 .000 -2.814 -2.769

Salmonella spp .022 .0115 .058 .000 .044

Proteus .828* .0115 .000 .806 .851

Bacillus spp -.040* .0115 .001 -.062 -.017



Enterobacter spp E.coli -3.684* .0115 .000 -3.707 -3.662

Salmonella spp -.871* .0115 .000 -.893 -.848

Proteus -.065* .0115 .000 -.087 -.042

Bacillus spp -.932* .0115 .000 -.955 -.910

Staphylococcus aureus -1.180* .0115 .000 -1.203 -1.158

Staphylococcus sciuri -.893* .0115 .000 -.915 -.870




Homogeneous Subsets

Estimated Marginal Means

1. Organisms


Organisms Mean Std. Error



E.coli 3.998 .008 3.982 4.014

77

Salmonella spp 1.184 .008 1.168 1.200

Proteus .378 .008 .362 .394

Bacillus spp 1.246 .008 1.230 1.262

Staphylococcus aureus 1.494 .008 1.478 1.510

Staphylococcus sciuri 1.206 .008 1.190 1.222

Enterobacter spp .314 .008 .298 .330

2. Conc


Conc Mean Std. Error



400 mg/ml 6.594 .009 6.577 6.611

200 mg/ml 3.018 .009 3.001 3.035

100 mg/ml 1.611 .009 1.594 1.628

50 mg/ml 7.344E-17 .009 -.017 .017

25 mg/ml 7.344E-17 .009 -.017 .017

12.5 mg/ml 7.344E-17 .009 -.017 .017

6.25 mg/ml 7.344E-17 .009 -.017 .017

3.125 mg/ml 4.837E-16 .009 -.017 .017

3. Groups


Groups Mean Std. Error



Hot water Piper guineense 1.449 .006 1.437 1.461

Hot water Xylopia aethiopica 1.535 .006 1.522 1.547

Cold water Allium cepa 1.966 .006 1.954 1.978

Cold water Xylopia aethiopica .661 .006 .649 .673

78

4. Organisms * Conc


Organisms Conc Mean Std. Error



E.coli 400 mg/ml 12.950 .023 12.905 12.995

200 mg/ml 10.267 .023 10.221 10.312

100 mg/ml 8.767 .023 8.721 8.812

50 mg/ml 2.288E-16 .023 -.045 .045

25 mg/ml 2.288E-16 .023 -.045 .045

12.5 mg/ml 2.288E-16 .023 -.045 .045

6.25 mg/ml 3.891E-16 .023 -.045 .045

3.125 mg/ml -7.118E-15 .023 -.045 .045

Salmonella spp 400 mg/ml 7.508 .023 7.463 7.554

200 mg/ml 1.967 .023 1.921 2.012

100 mg/ml -1.099E-15 .023 -.045 .045

50 mg/ml -4.341E-16 .023 -.045 .045

25 mg/ml -4.341E-16 .023 -.045 .045

12.5 mg/ml -4.712E-16 .023 -.045 .045

6.25 mg/ml -4.036E-16 .023 -.045 .045

3.125 mg/ml -2.641E-15 .023 -.045 .045

Proteus 400 mg/ml 3.025 .023 2.980 3.070

200 mg/ml -1.339E-16 .023 -.045 .045

100 mg/ml 5.240E-16 .023 -.045 .045

50 mg/ml 2.003E-16 .023 -.045 .045

25 mg/ml 2.743E-16 .023 -.045 .045

12.5 mg/ml 1.492E-16 .023 -.045 .045

6.25 mg/ml -2.168E-17 .023 -.045 .045

3.125 mg/ml -6.482E-16 .023 -.045 .045

Bacillus spp 400 mg/ml 5.433 .023 5.388 5.479

200 mg/ml 4.533 .023 4.488 4.579

100 mg/ml 3.090E-17 .023 -.045 .045

79

50 mg/ml -3.613E-17 .023 -.045 .045

25 mg/ml -7.784E-17 .023 -.045 .045

12.5 mg/ml -1.373E-16 .023 -.045 .045

6.25 mg/ml -4.328E-16 .023 -.045 .045

3.125 mg/ml 8.762E-16 .023 -.045 .045

Staphylococcus aureus 400 mg/ml 6.942 .023 6.896 6.987

200 mg/ml 2.500 .023 2.455 2.545

100 mg/ml 2.508 .023 2.463 2.554

50 mg/ml -1.910E-16 .023 -.045 .045

25 mg/ml -2.017E-16 .023 -.045 .045

12.5 mg/ml -3.184E-16 .023 -.045 .045

6.25 mg/ml -8.340E-16 .023 -.045 .045

3.125 mg/ml 7.693E-16 .023 -.045 .045

Staphylococcus sciuri 400 mg/ml 7.792 .023 7.746 7.837

200 mg/ml 1.858 .023 1.813 1.904

100 mg/ml 9.828E-16 .023 -.045 .045

50 mg/ml 6.636E-16 .023 -.045 .045

25 mg/ml 5.711E-16 .023 -.045 .045

12.5 mg/ml 3.349E-16 .023 -.045 .045

6.25 mg/ml 7.713E-17 .023 -.045 .045

3.125 mg/ml 4.728E-16 .023 -.045 .045

Enterobacter spp 400 mg/ml 2.508 .023 2.463 2.554

200 mg/ml -1.593E-15 .023 -.045 .045

100 mg/ml -2.192E-15 .023 -.045 .045

50 mg/ml 8.256E-17 .023 -.045 .045

25 mg/ml 1.535E-16 .023 -.045 .045

12.5 mg/ml 7.280E-16 .023 -.045 .045

6.25 mg/ml 1.740E-15 .023 -.045 .045

3.125 mg/ml 1.167E-14 .023 -.045 .045

5. Organisms * Groups

80


Organisms Groups Mean Std. Error



E.coli Hot water Piper guineense 4.058 .016 4.026 4.090



Cold water Xylopia aethiopica 3.650 .016 3.618 3.682

Salmonella spp Hot water Piper guineense 1.021 .016 .989 1.053



Cold water Xylopia aethiopica -8.341E-16 .016 -.032 .032

Proteus Hot water Piper guineense 5.464E-16 .016 -.032 .032

Hot water Xylopia aethiopica 5.763E-16 .016 -.032 .032



Bacillus spp Hot water Piper guineense 2.933 .016 2.901 2.965


Cold water Allium cepa -9.836E-16 .016 -.032 .032

Cold water Xylopia aethiopica 5.312E-16 .016 -.032 .032

Staphylococcus aureus Hot water Piper guineense 1.000 .016 .968 1.032

Hot water Xylopia aethiopica -1.527E-15 .016 -.032 .032


Cold water Xylopia aethiopica .979 .016 .947 1.011

Staphylococcus sciuri Hot water Piper guineense 1.133 .016 1.101 1.165




Enterobacter spp Hot water Piper guineense -1.739E-15 .016 -.032 .032




81

6. Organisms * Conc * Groups


Organisms Conc Groups Mean Std. Error



E.coli 400 mg/ml Hot water Piper guineense 14.733 .046 14.643 14.824




200 mg/ml Hot water Piper guineense 9.867 .046 9.776 9.957








50 mg/ml Hot water Piper guineense 3.216E-16 .046 -.090 .090








12.5 mg/ml Hot water Piper guineense 2.472E-16 .046 -.090 .090






82

Cold water Allium cepa 2.441E-16 .046 -.090 .090


3.125 mg/ml Hot water Piper guineense -2.800E-15 .046 -.090 .090




Salmonella spp 400 mg/ml Hot water Piper guineense 8.167 .046 8.076 8.257




200 mg/ml Hot water Piper guineense -1.173E-15 .046 -.090 .090






















83







Proteus 400 mg/ml Hot water Piper guineense -2.293E-15 .046 -.090 .090


























84







Bacillus spp 400 mg/ml Hot water Piper guineense 12.667 .046 12.576 12.757


























85







Staphylococcus aureus 400 mg/ml Hot water Piper guineense 8.000 .046 7.910 8.090


























86







Staphylococcus sciuri 400 mg/ml Hot water Piper guineense 9.067 .046 8.976 9.157


























87







Enterobacter spp 400 mg/ml Hot water Piper guineense -1.988E-14 .046 -.090 .090


























88







Descriptive Statistics


Organisms Conc Mean Std. Deviation N

E.coli 400 mg/ml 14.733 .2517 3

200 mg/ml 9.867 .2309 3

100 mg/ml 7.867 .1155 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total 4.058 5.6488 24

Salmonella spp 400 mg/ml 8.167 .2887 3

200 mg/ml .000 .0000 3

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total 1.021 2.7603 24

Proteus 400 mg/ml .000 .0000 3

200 mg/ml .000 .0000 3

89

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total .000 .0000 24

Bacillus spp 400 mg/ml 12.667 .5774 3

200 mg/ml 10.800 .3464 3

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total 2.933 5.2156 24

Staphylococcus aureus 400 mg/ml 8.000 .0000 3

200 mg/ml .000 .0000 3

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total 1.000 2.7027 24

Staphylococcus sciuri 400 mg/ml 9.067 .1155 3

200 mg/ml .000 .0000 3

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

90

3.125 mg/ml .000 .0000 3

Total 1.133 3.0632 24

Enterobacter spp 400 mg/ml .000 .0000 3

200 mg/ml .000 .0000 3

100 mg/ml .000 .0000 3

50 mg/ml .000 .0000 3

25 mg/ml .000 .0000 3

12.5 mg/ml .000 .0000 3

6.25 mg/ml .000 .0000 3

3.125 mg/ml .000 .0000 3

Total .000 .0000 24

Total 400 mg/ml 7.519 5.4082 21

200 mg/ml 2.952 4.7920 21

100 mg/ml 1.124 2.8210 21

50 mg/ml .000 .0000 21

25 mg/ml .000 .0000 21

12.5 mg/ml .000 .0000 21

6.25 mg/ml .000 .0000 21

3.125 mg/ml .000 .0000 21

Total 1.449 3.6684 168


Source

Type III Sum of

Squares df Mean Square F Sig.

Corrected Model 2245.940a 55 40.835 3.363E3 .000

91

Intercept 352.930 1 352.930 2.906E4 .000

Organisms 328.695 6 54.783 4.512E3 .000

Conc 1043.897 7 149.128 1.228E4 .000

Organisms * Conc 873.348 42 20.794 1.712E3 .000

Error 1.360 112 .012

Total 2600.230 168

Corrected Total 2247.300 167

a. R Squared = .999 (Adjusted R Squared = .999)

Estimated Marginal Means

1. Organisms


Organisms Mean Std. Error



E.coli 4.058 .022 4.014 4.103

Salmonella spp 1.021 .022 .976 1.065

Proteus 1.654E-16 .022 -.045 .045

Bacillus spp 2.933 .022 2.889 2.978

Staphylococcus aureus 1.000 .022 .955 1.045

Staphylococcus sciuri 1.133 .022 1.089 1.178

Enterobacter spp -4.777E-16 .022 -.045 .045

2. Conc


Conc Mean Std. Error



400 mg/ml 7.519 .024 7.471 7.567

200 mg/ml 2.952 .024 2.905 3.000

100 mg/ml 1.124 .024 1.076 1.171

50 mg/ml -9.640E-17 .024 -.048 .048

92

25 mg/ml -9.640E-17 .024 -.048 .048

12.5 mg/ml -9.640E-17 .024 -.048 .048

6.25 mg/ml -9.640E-17 .024 -.048 .048

3.125 mg/ml 6.498E-16 .024 -.048 .048

3. Organisms * Conc


Organisms Conc Mean Std. Error



E.coli 400 mg/ml 14.733 .064 14.607 14.859

200 mg/ml 9.867 .064 9.741 9.993

100 mg/ml 7.867 .064 7.741 7.993

50 mg/ml 1.082E-15 .064 -.126 .126

25 mg/ml 1.082E-15 .064 -.126 .126

12.5 mg/ml 1.378E-15 .064 -.126 .126

6.25 mg/ml 1.516E-15 .064 -.126 .126

3.125 mg/ml -1.688E-15 .064 -.126 .126

Salmonella spp 400 mg/ml 8.167 .064 8.041 8.293

200 mg/ml -1.168E-15 .064 -.126 .126

100 mg/ml -1.124E-16 .064 -.126 .126

50 mg/ml -5.481E-16 .064 -.126 .126

25 mg/ml -4.741E-16 .064 -.126 .126

12.5 mg/ml -5.605E-16 .064 -.126 .126

6.25 mg/ml -4.458E-16 .064 -.126 .126

3.125 mg/ml 1.367E-16 .064 -.126 .126

Proteus 400 mg/ml 1.808E-15 .064 -.126 .126

200 mg/ml -2.610E-16 .064 -.126 .126

100 mg/ml 1.806E-16 .064 -.126 .126

50 mg/ml 1.148E-16 .064 -.126 .126

25 mg/ml 2.481E-16 .064 -.126 .126

12.5 mg/ml 3.646E-16 .064 -.126 .126

93

6.25 mg/ml 1.804E-16 .064 -.126 .126

3.125 mg/ml -1.312E-15 .064 -.126 .126

Bacillus spp 400 mg/ml 12.667 .064 12.541 12.793

200 mg/ml 10.800 .064 10.674 10.926

100 mg/ml -1.284E-15 .064 -.126 .126

50 mg/ml -9.801E-16 .064 -.126 .126

25 mg/ml -1.032E-15 .064 -.126 .126

12.5 mg/ml -3.042E-16 .064 -.126 .126

6.25 mg/ml -9.927E-16 .064 -.126 .126

3.125 mg/ml 4.614E-16 .064 -.126 .126

Staphylococcus aureus 400 mg/ml 8.000 .064 7.874 8.126

200 mg/ml -7.353E-16 .064 -.126 .126

100 mg/ml -2.870E-16 .064 -.126 .126

50 mg/ml -5.007E-16 .064 -.126 .126

25 mg/ml -4.415E-16 .064 -.126 .126

12.5 mg/ml -9.236E-16 .064 -.126 .126

6.25 mg/ml -1.254E-15 .064 -.126 .126

3.125 mg/ml 9.624E-16 .064 -.126 .126

Staphylococcus sciuri 400 mg/ml 9.067 .064 8.941 9.193

200 mg/ml -1.022E-15 .064 -.126 .126

100 mg/ml -2.222E-16 .064 -.126 .126

50 mg/ml -4.730E-16 .064 -.126 .126

25 mg/ml -4.322E-16 .064 -.126 .126

12.5 mg/ml -7.885E-16 .064 -.126 .126

6.25 mg/ml -1.233E-15 .064 -.126 .126

3.125 mg/ml 2.935E-16 .064 -.126 .126

Enterobacter spp 400 mg/ml -7.867E-15 .064 -.126 .126

200 mg/ml -3.620E-15 .064 -.126 .126

100 mg/ml -7.477E-16 .064 -.126 .126

50 mg/ml 6.299E-16 .064 -.126 .126

25 mg/ml 3.745E-16 .064 -.126 .126

12.5 mg/ml 1.589E-16 .064 -.126 .126

94

6.25 mg/ml 1.555E-15 .064 -.126 .126

3.125 mg/ml 5.695E-15 .064 -.126 .126

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