ANTIMICROBIAL ACTIVITY OF HONEY AND ETHANOLIC EXTRACT

OF GINGER AGAINST FOOD PATHOGENIC BACTERIA

MUSA IBRAHIM HAULE

A SPECIAL PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF

SCIENCE IN FOOD SCIENCE AND TECHNOLOGY OF SOKOINE

UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA.

2017

i
 ABSTRACT

The antimicrobial activity of ethanolic extract of ginger, honey and the mixture of

honey and ethanolic extract of ginger was investigated against two common food

borne bacteria Staphylococcus spp. and Salmonella spp. using disk diffusion test.

Staphylococcus spp. was susceptible to the extracts with the average inhibition zone

diameter 30.12 3.62 mm, while Salmonella spp. was resistant, with no zone of

inhibition. However; honey and the combination of honey with ethanolic extract of

ginger were active against Staphylococcus spp. with average susceptible zone of

inhibition diameter 28.25 10.68 mm and 28.31 4.32 mm, respectively and inactive

against Salmonella spp. with zone of inhibition mm diameter. The

separate use of ethanolic extract of ginger had no antimicrobial effects on the studied

bacteria. Statistical analysis showed that there was significant difference in the

inhibitory activity (P 0.05) in both combined and separate extracts. The combined

extracts showed the highest antimicrobial activity as compared to honey and the

ethanolic extract of ginger. The study on the physicochemical effects on bacteria

susceptibility showed that; test bacteria was susceptible on low acidic pH

(4.18 0.84 5.80 1.06) and high osmotic concentration of extracts

(37.04 5.69 56.18 13.83) %Brix. The increase in dilution decreases osmotic

concentration of the extracts, hence the decrease in their antimicrobial activity. But

for a unit increase in acidity, bacteria susceptibility was decreased. The results

indicated the potential for use of honey or combination of honey and ginger as food

preservatives.

i
 DECLARATION

I, MUSA IBRAHIM HAULE, do hereby declare to the Senate of Sokoine University

of Agriculture, that this report is my own original work done within the period of

registration and that it has neither been submitted nor being concurrently submitted

in any other institution.

. ..

Musa Ibrahim Haule Date

The above declaration is confirmed

..

Prof. Jovin.K. Mugula Date

(Supervisor)

ii
 COPYRIGHT

No part of this report may be reproduced, stored in any retrieval system, or

transmitted in any form or by any means without prior written permission of the

author or Sokoine University of Agriculture (SUA) in that behalf.

iii
 ACKNOWLEDGEMENT

I would like to thank my supervisor Prof. Jovin K Mugula, for continuous guidance

throughout the study. Also I would like to thank Mrs Gaudensia Mchotika, and Mr

Stewart Mwanyika of the Department of Food Technology, Nutrition and Consumer

Sciences, College of Agriculture (SUA) and Mr P. Mkuchu of the Department of

Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine (SUA)

for their technical assistances.

Special thanks to my beloved parents Mwl. Ibrahim J. Haule, and Mrs Halima I.

Haule and my brother Dr Daniel I. Haule, for their support to conduct this study at

Sokoine University of Agriculture (SUA), my sisters Ms Christabella I. Haule and

Ms Eunice I. Haule and finally special thanks to my beloved Pastors; Mrs Kaaneny

Mashoo, Mr Ipyana Mwanginda and Mr Mwambuchi for the encouragement and

both spiritual and financial support. The successful of this report is product of their

support.

iv
 DEDICATION

I dedicate this work to my beloved father; Mwl. Ibrahim J. Haule and mother, Mrs

Halima I. Haule for their hard work in raising me, and all their support in all fields of

my life including all my academic achievements.

v
 TABLE OF CONTENTS

ABSTRACT ................................................................................................................. i

DECLARATION ........................................................................................................ ii

COPYRIGHT ............................................................................................................ iii

ACKNOWLEDGEMENT ........................................................................................ iv

DEDICATION ............................................................................................................ v

TABLE OF CONTENTS .......................................................................................... vi

LIST OF TABLES .................................................................................................... ix

LIST OF FIGURES ................................................................................................... x

LIST OF APPENDICES ......................................................................................... xii

LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS ......................... xiii

CHAPTER ONE ........................................................................................................ 1

1.0 INTRODUCTION ................................................................................................ 1

1.1 Food quality and safety .......................................................................................... 1

1.2 Justification ............................................................................................................ 1

1.3 Objectives ............................................................................................................... 2

1.3.1 General objective ............................................................................................. 2

1.3.2 Specific objectives ........................................................................................... 2

CHAPTER TWO ....................................................................................................... 3

2.0 LITERATURE REVIEW.................................................................................... 3

2.1 Salmonella and staphylococci food poisoning ....................................................... 3

vi
2.2 Bacterial growth limiting factors ........................................................................... 3

2.3 Foods associated with salmonella and staphylococci poisoning ........................... 4

2.4 Food borne pathogenic bacteria contamination routes........................................... 4

2.4.1 Water ............................................................................................................... 5

2.4.2 Processing equipment ...................................................................................... 5

2.5 Herbs and spices in food preservation ................................................................... 5

2.6 Antimicrobial activity of pure honey ..................................................................... 6

2.7 Antimicrobial activity of ginger ............................................................................. 6

2.8 Antimicrobial resistance bacteria ........................................................................... 7

2.9 Controlling microorganisms in food processing ................................................... 7

CHAPTER THREE ................................................................................................... 8

3.0 MATERIALS AND METHODS ........................................................................ 8

3.1 Sample collection and bacteria isolation. ............................................................... 8

3.1.1 Sample identification ....................................................................................... 8

3.1.2 Preparation of Extracts .................................................................................... 8

3.1.3 Ethanolic extraction and purification .............................................................. 9

3.1.4 Preparation of pure honey ............................................................................. 10

3.1.5 Antimicrobial formulation ............................................................................. 10

3.2 Antibacterial susceptibility testing ....................................................................... 11

3.2.1 Media preparation .......................................................................................... 11

3.2.2 Disk diffusion method ................................................................................... 11

3.2.3 Measuring inhibition zone ............................................................................. 12

3.3 Determination of osmotic concentrations of antimicrobials ............................ 13

3.3.1 Determination of pH ...................................................................................... 14

vii
3.4 Study design ......................................................................................................... 14

3.5 Data analysis ........................................................................................................ 14

CHAPTER FOUR .................................................................................................... 14

4.0 RESULTS AND DISCUSSION ........................................................................ 15

4.1 Results .................................................................................................................. 15

4.1.1 Physico-chemical properties of extracts ........................................................ 15

4.1.1.1 Osmotic concentration ............................................................................ 15

4.1.1.2 Acidity .................................................................................................... 15

4.1.2 Effects of extracts on bacterial susceptibility ................................................ 16

4.1.2.1 Effects of osmotic concentration on bacterial susceptibility .................. 16

4.1.2.2 Effects of acidity on bacterial susceptibility ........................................... 17

4.2 Discussion ............................................................................................................ 19

4.3 Bacterial susceptibility ......................................................................................... 19

CHAPTER FIVE ...................................................................................................... 21

5.0 CONCLUSIONS AND RECOMMENDATIONS ........................................... 21

5.1 Conclusions .......................................................................................................... 21

5.2 Recommendations ................................................................................................ 21

REFERENCES ......................................................................................................... 22

APPENDIX ............................................................................................................... 27

viii
 LIST OF TABLES

Table 1: Physicochemical characteristics of extracts ................................................. 15

Table 2: Inhibition zone diameter (mm) .................................................................... 20

Table 3: Bacterial susceptibility ................................................................................. 20

ix
 LIST OF FIGURES

Figure 1: Scatter diagram showing the effects of osmotic concentration on

bacterial susceptibility ................................................................................ 17

Figure 2: Scatter diagram showing the effects of acidity on bacterial

susceptibility ............................................................................................. ..18

x
 LIST OF PLATES

Plate 1: Purification by Filtration of ethanolic extracts using Whatman

Filter paper grade no. 1 ................................................................................ 9

Plate 2: Honey at different concentrations ................................................................. 10

Plate 3: Ditching bores on the settled Agar media ..................................................... 12

Plate 4: Measuring Zone of Inhibition diameter (mm) .............................................. 13

Plate 5: Activity of Honey: Staphylococcus spp. (left), Salmonella spp.

(right) ......................................................................................................... 19

Plate 6: Activity of ginger: Staphylococcus spp. (right), Salmonella spp.

(left) ........................................................................................................... 20

xi
 LIST OF APPENDICES

Appendix 1: Test significance differences on Antimicrobial and bacterial

Susceptibility ....................................................................................... 27

Appendix 2: Test of significance difference: Antimicrobial and Inhibition

zone diameter (mm) ............................................................................. 27

xii
 LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS

%: Percentage

: Volume by volume

: Weight by volume

ml: Micro-millilitre

AMR: Antimicrobial resistance

ANOVA: Analysis of variances

AST: Antimicrobial Susceptibility Testing

Aw: Water activity

BSAC: British Society for Antimicrobial Chemotherapy

: Degree centigrade

Dr: Doctor

GIT: Gastro-intestinal tract

H: Hour

MDR: Multi-drug resistance

MHA: Mueller Hinton Agar

Mm: Millimetre

N: Sample size

P: Probability

pH: Hydrogen ions concentration

Prof: Professor

RTE; Ready to eat foods

S. d: Standard deviation

xiii
S: Second

Spp.: Species

SUA: Sokoine University of Agriculture

TSS: Total Soluble Solids

: Standard deviation

xiv
 CHAPTER ONE

1.0 INTRODUCTION

Spices have been used for centuries by many cultures to enhance the flavour and

aroma in foods (Macwan et al., 2016). Early culture recognized the value of using

spices and herbs in preserving foods and for their medicinal values (Ahmed et al.,

2013). Research by Babatunde and Adewumi, (2015) indicated that the growth of

both Gram-positive and Gram-negative food pathogenic bacteria can be inhibited by

ginger and other spices extracts. Honey produced by stingless bee (Apis mellifera)

showed antimicrobial activity against both Gram-positive and Gram-negative

bacteria.

1.1 Food quality and safety

Food quality is defined as the degree of excellence of food as determined by its taste,

appearance, and nutritional quality, as well as in bacteriological or keeping quality

(Singham, Birwal and Yadav, 2015). These attributes influences the value of a

product to the consumer. In the urban areas food borne disease resulted from

unhygienic handling of foods by street food vendors (Mukantwali et al., 2013). This

has been reported as a major public health concern and thus jeopardizing the health

status of thousands of urban dwellers.

1.2 Justification

The use of organic chemical preservatives (plant derived chemicals) in food

processing replaces the use of inorganic chemicals preservatives (sodium chloride,

benzoic acid, sodium acetate, acetic acid, citric acid and sodium benzoate). The aim

1
of this study was activity of the combination of ginger extract and honey on

microbial growth.

1.3 Objectives

1.3.1 General objective

The general objective of this study was to evaluate combined antimicrobial activity

of honey and ethanolic extract of ginger against common food-borne pathogenic

bacteria

1.3.2 Specific objectives

The specific objectives of this study were:

i. To evaluate the antimicrobial combined efficacy of honey and ginger on

foodborne Salmonella spp. and Staphylococcus spp.

ii. To study the physicochemical effects (acidity and osmotic concentration) of

honey, ethanolic extract of ginger and the combination of honey and the

ethanolic extract of ginger against Salmonella spp. and Staphylococcus spp.

2
 CHAPTER TWO

2.0 LITERATURE REVIEW

Many forms of bacteria poisoning can be prevented through either cooking foods

thoroughly and eating them quickly or refrigerating it effectively. However many

toxins are not destroyed by heat treatment. Food borne illness can also be minimized

by hand washing, preventing cross contamination, proper storage and maintaining

cooking temperatures (Danyluk et al., 2012).

2.1 Salmonella and staphylococci food poisoning

Salmonellosis is the commonly food borne disease resulted from ingestion of viable

cells. Salmonella are rod shaped Gram-negative bacteria, which are mostly causing

typhoid and non-typhoid diseases (gastro intestinal) (Malik et al., 2015). They are

normally found in intestinal tract of animals (Kunwar, Harpreet and Vipra, 2013).

Poultry and poultry products are known to be mainly primary vehicles of salmonella

through the value chain such as cooking and storage procedures/community kitchen

practices (Makelele et al., 2015). Staphylococcus spp. is Gram-positive spherical-

shaped non-spore forming bacteria occurring in soil, water and air. It is a facultative

anaerobe so can grow under both aerobic and anaerobic conditions. They are also

found in the nasal passage, oral cavity and gastro intestinal tract (GIT) of human.

They grow better aerobically than anaerobically (Jay, 2000).

2.2 Bacterial growth limiting factors

Number of environmental factors such as temperature, osmotic concentration, water

activity (Aw), hydrogen ions concentration (pH), and presence of oxygen and

3
composition of the food determines the growth and survival of organism. Several

chemical preservatives, including sorbets and benzoates, inhibit bacterial growth.

The effectiveness of these preservatives increases as the pH is reduced (Ahmed et al.,

2013). Acids inhibit microbial growth by lowering the pH, affecting the proton

gradient across biological membrane, acidifying the cytoplasm and interfering with

the chemical transport across cell membrane. Acid environment not only limits

microbial growth but also enhances destruction of microorganisms synergistically

with other antimicrobial systems.

2.3 Foods associated with salmonella and staphylococci poisoning

Ready to eat foods (RTE) such as meat and meat products, salads containing eggs,

improperly treated vegetable salads, chicken and bakery products are commonly

associated with staphylococcus and salmonella food poisoning. Tonsils and skin of

pigs, chickens and turkeys often harbour Staphylococcus spp., and are also potential

sources of contamination (Johler et al., 2015).

2.4 Food borne pathogenic bacteria contamination routes

Generally foods are contaminated with salmonella spp., E. coli, Clostridium spp.,

Bacillus spp., and Staphylococcus spp. An unusual high stomach pH level (lower

acidity) greatly reduces the number of bacteria required to cause symptoms

(Makelele et al., 2015). The contamination of foods can occur anywhere in the

growing, harvesting, cleaning and transportation chain (Mukantwali et al., 2013;

Malik et al., 2015).

4
2.4.1 Water

Water used for diluting pesticides, irrigation, and washing represents a possible

source of contamination (Mathur, Joshi and Harwani, 2014).Foods are raw

agricultural commodities and can also be exposed to contamination from animals,

birds, insects, and from domestic and agricultural wastes.

2.4.2 Processing equipment

The contact surfaces of equipment used in harvesting, storage, and packing of the

agricultural produce may also be contaminated with rodent or animal manure

(Kunwar, Harpreet and Vipra, 2013). Other possible sources of contamination

include poor handling practices of foods and the conditions under which it is stored

and transported (Reddi et al., 2015)

2.5 Herbs and spices in food preservation

Ginger crude oil extracts are better than other extract. The use of natural

preservatives is another way to retain colour and restore flavour. Herbs and spices

were mostly used for food flavouring. In ancient times, it has been used as medicine

against variety of diseases (Ahmed et al., 2013). A number of spices showed

antimicrobial activity against different types of microorganisms. According to

Witkowska et al., (2013), the activity depends on the type of spice or herb, type of

food and microorganism, as well as on the chemical composition and content of

extracts and essential oils.

5
2.6 Antimicrobial activity of pure honey

Honey was found to inhibit bacteria due to its biochemical composition and

characteristics such as; high osmotic concentration, hydrogen peroxide generation,

flavonoids and proteineous compounds (Akinnibosun and Iterdjere, 2013; Purbafrani

et al., 2014). These result to lowering pH while increasing the osmotic pressure

which shrink and disrupt the bacteria cell wall. Honey has been reported to be

effective in a number of human pathologies (Fikselov et al., 2014). A large number

of in vitro and limited clinical studies have confirmed the broad-spectrum

antimicrobial (antibacterial, antifungal, antiviral properties of honey. The activity is

determined by the acidity (low pH), osmotic effect and high sugar concentration

(Elijah et al., 2015). Depending on the osmolarity effects which inhibit bacteria, the

sugar concentration of honey ties up water molecules so that bacteria would have

insufficient water (Aw0.93) to support their growth. The viscosity of pure honey

creates a physical barrel that inhibits the contamination by infectious agents present

in the air (Alvarez-suarez et al., 2014).

2.7 Antimicrobial activity of ginger

The main active phytochemical present in ginger are gingerols, shogoals and

paradols, chemical structure particularly to the presence of hydrophilic functional

group such as hydroxyl group, phenolic components and lipophilicity of some

essential oils component (Sulieman et al., 2015). These compounds are actively

against bacteria which inhibits multiplication of colon bacteria. According to studies

by Witkowska et al., (2013), the activity against bacteria depends on its

concentration.

6
2.8 Antimicrobial resistance bacteria

Microbes evolve to become more or fully resistant to antimicrobials which

previously could treat it the condition is known as Antimicrobial resistance (AMR).

Microbes resistant to multiple antimicrobials are called multidrug resistant (MDR). It

has been reported by Hamid, (2011) that the resistance arises through one of three

ways: Natural resistance in certain types of bacteria; Genetic mutation; or by one

species acquiring resistance from another. The resistance can appear spontaneously

due to random mutations; or more commonly following gradual build-up over time,

and because of misuse of antimicrobials.

2.9 Controlling microorganisms in food processing

Microorganism must be kept under control so that food can be safely prepared in

restaurants and homes. Processes and techniques used to control microbes varies

greatly and can be grouped as inanimate objects and living tissues (Jay, 2000).

7
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Sample collection and bacteria isolation.

The study used one sample of ginger which was selected randomly at the market and

one sample of honey. Ginger rhizomes were purchased from Morogoro market. Pure

honey was purchased from the College of Forestry, Wildlife and Tourism at SUA.

The test bacteria cultures were isolated in the College of Veterinary and Medical

Science, Department of Veterinary Microbiology and Parasitology, Research

laboratory at SUA.

3.1.1 Sample identification

The identification of honey quality was performed by water test method as described

in (Mybeeline, 2015). A small sample of honey was poured into a 100 ml beaker

half-filled of water; honey dropped down to the bottom of the cup without mixing up

with water except when was stirred. The controls of preserved culture of common

food pathogenic microorganism follows the similar procedures as mentioned in the

Microbiological guidelines.

3.1.2 Preparation of Extracts

Crude extract were prepared according to the procedure described by Plotto, (2002).

Fresh ginger rhizomes were washed with clean and safe water to remove debris and

rinsed thoroughly by double distilled sterile water. Size reduction was performed to

the fresh ginger rhizomes using a sterile knife before blended using electric blender.

Samples were with sterilized distilled water at the concentration of 100.0 %, 90.0 %,

8
80.0 % and 70.0 % . The crude extract was filtered using whatman grade no.1

filter paper. Only 50.0 ml of each extract solutions were taken as the test sample and

packaged into the sterile bottles A1, A2, A3 and A4 respectively. A crude extracts

were stored in the refrigerator as aqueous stock for further use. All procedures were

done in a sterile condition.

3.1.3 Ethanolic extraction and purification

The extraction and purification of extracts was carried out according to the method

used by Babatunde and Adewumi, (2015). The ethanolic extraction was done by

dissolving 100 ml of 70.0 % (v/v) ethanol into 50.0 ml of each of ginger crude

extracts and packaged in a sterilized conical flask of 100.0 ml by sealing with a foil

paper. The crude extracts were kept on ambient temperature for proper dissolving of

solvent into crude extracts. After 72 h the extract in the conical flask were placed in

the rotary evaporator to remove ethanol; extracts were filtered and stored into six

different sterilized bottle jars G1, G2, G3 and G4.

Plate 1: Purification by Filtration of ethanolic extracts using Whatman Filter

paper grade no. 1

9
3.1.4 Preparation of pure honey

Pure honey was filtered with a sterile white/muslin cloth to remove debris; filtered

pure honey were replicated into four by diluting with sterilized distilled water at

100.0 %, 90.0 %, 80.0 % and 70.0 % ( honey to make 10.0 ml of each

concentrations. All samples were stored in the different sterilized bottles H1, H2, H3

and H4 respectively for further use.

Plate 2: Honey at different concentrations

3.1.5 Antimicrobial formulation

The formulation of the extracts was adopted from Akinnibosun and Iterdjere, (2013)

and slightly modification by Babatunde and Adewumi, (2015). Sample of each

concentration of honey were measured using a sterile measuring cylinder 5.0 ml each

and put into separate sterilized test tubes. Then 5.0 ml each concentration of

ethanolic extracts of ginger were pipetted into each test tubes containing honey. The

contents were mixed by the use of Rotary Mixer. The conical flasks were covered

with foil paper, shaken and left to stand for 72 h. After 72 h, the suspensions were

10
filtered and the filtrates were concentrated using Rotatory Evaporating Machine at

40. The total soluble solids (TSS) was measured and expressed as %Brix and pH

were recorded at each test samples prior to the Antimicrobial Susceptibility Testing

(AST) to the test pathogenic bacteria (Salmonella spp. and Staphylococcus spp.). All

samples were stored in a refrigerator for further use

3.2 Antibacterial susceptibility testing

This test was carried out to classify bacteria as Susceptible (S), Intermediate (I) or

Resistant (R) as described by Mindy et al., (2003).

3.2.1 Media preparation

Mueller-Hinton agar was prepared according to the manufacturer recommendations.

Therefore 19.0 g of the MHA was measured and shaken with 500 ml of sterile

distilled water. The media was then autoclaved (121 /15 min) and cooled to 45.

To a well sterilized petri plates (90 mm diameter) the media was then poured to half

it. The media was left to cool until solidify in the plates.

3.2.2 Disk diffusion method

Antimicrobial susceptibility test was performed by a standardized method for disk

diffusion (Kirby-Bauer Method) method as described in the BSAC Methods for

Antimicrobial Susceptibility Testing by Wootton, (2013). Each strain of bacteria was

tested into different sets of plates, which were simultaneously processed for each

strain. Twelve plates were used for each bacteria strain at different concentration of

extracts. With the use of a sterile glass Pasteur pipettes, four equidistant bores were

punched into the petri-plates.

11
Plate 3: Ditching bores on the settled Agar media

Each Petri plate was swabbed on with strain inoculum streaked thoroughly all over

the surface of the agar. All bore were filled up with 2 ml of antimicrobial using a

precise eppendorf. All plates were incubated at 37 C and inhibition zones were

measured after 24 h.

3.2.3 Measuring inhibition zone

Measuring the zone of inhibition was performed as described by Ortez and Rankin,

(2005) in the BSAC Manual of Antimicrobial Susceptibility Testing. After

incubation, the zones of inhibition of the growth of the microorganisms around the

disks were measured. The area where the bacteria have not grown enough to be

visible indicated that the extracts inhibited the bacteria from growing or killed the

bacteria. The effectiveness of antimicrobial was shown by the presence of growth

inhibition zones surrounding an antimicrobial impregnated disk. It was measured by

keeping the lid of the plate in place, using a ruler, the diameter of the disk plus the

surrounding clear area was measured and expressed in millimetres (mm).

12
Plate 4: Measuring Zone of Inhibition diameter (mm)

Since zone of inhibition approximates the minimum antibiotic concentration

sufficient to prevent growth of the test isolate, this zone of inhibition was compared

against a reference standard which contains measurement ranges and their equivalent

qualitative categories of Susceptible (S), Intermediate (I) or Resistant (R).

Physico-chemical analyses

This test was done to measure the amount of Total Soluble Solids (TSS) and the

acidity that present in the extracts.

3.3 Determination of osmotic concentrations of antimicrobials

The standard digital Brix hydrometer was used to determine osmotic concentration of

the samples as described in the Manual of Food Analysis (Moisture and Total

Solids Analysis) by Bradley, (2010). The total soluble solids that present in each

antimicrobial samples was measured and expressed as %Brix.

13
3.3.1 Determination of pH

The standard digital pH-meter was used to measure pH of the samples. Firstly; pH

was standardized by buffering at 7.0 and 4.0; distilled water was used for rinsing the

electrodes as described by Metrohm, (2009). Exactly 5 ml of ginger ethanolic

extracts were poured in the clean beaker and agitated by vigorously shaking for 45 s

and then pH for each concentration was determined. Similar procedure was

performed for the determination of the pH of honey and the combination of honey

and ethanolic extracts of ginger.

3.4 Study design

The sample of honey was diluted with sterile distilled water into different

concentration; 100 %, 90 %, 80 % and 70 % . Similarly the replicates were

performed to ginger ethanolic extract. While the combined extracts were made by

adding 5 ml of each sample of honey into each sample of ethanolic extracts of

ginger; resulting into 16 replicates (n=16) of the combined extracts and 4 individual

extracts (n=4) and of honey and 4 individual extracts of ethanolic extract of ginger

(n=4); total of 24 samples (n=24) of extracts to be tested for antibacterial activity.

3.5 Data analysis

Data analysis were performed in combination with MS-Excel software for Windows,

data were subjected to R-Commander version 3.3-2 for the analysis of variance

(ANOVA) to determine the significance differences (P0.05) and the mean \values

were expressed as mean and standard deviation ( ).

CHAPTER FOUR

14
4.0 RESULTS AND DISCUSSION

4.1 Results

4.1.1 Physico-chemical properties of extracts

4.1.1.1 Osmotic concentration

Among the studied samples, it has found that honey contained more osmotic

concentration (56.18 13.83 %Brix) as compared to ethanolic extract of ginger

(14.88 0.25 %Brix) and the combined extracts of ginger and honey (37.04 5.69

%Brix).

4.1.1.2 Acidity

The physicochemical test has shown that the combined extracts of honey and the

ethanolic extract of ginger were more acidic (4.18 0.84) as compared to honey itself

and the ethanolic extract of ginger. The ethanolic extracts of ginger were at neutral

pH (7.11 0.22) while honey itself was slightly acidic with average pH of 5.80 1.06.

Table 1: Physicochemical characteristics of extracts

Extract pH %Brix

Honey (n=4) 5.80 1.06ab 56.18 13.83a

Ginger (n=4) 7.11 0.22c 14.83 0.25c

Combined (Ginger and Honey) (n=16) 4.18 0.84a 37.04 5.69ab

Value are expressed as mean S.d

Mean values in each column with different superscripts are significantly different

at .

15
4.1.2 Effects of extracts on bacterial susceptibility

Susceptibility of Staphylococcus spp., to honey and combination of honey and

ethanolic extract of ginger in this study were primarily described as the effects of the

physico-chemical properties of the extracts (i.e. acidity expressed as pH and osmotic

concentrations expressed as %Brix).

4.1.2.1 Effects of osmotic concentration on bacterial susceptibility

For each unit increase in osmotic concentration of the extracts, the bacterial

susceptibility was increased by the factor 0.67. When the osmotic concentration of

the extracts was reduced, bacteria susceptibility was also reduced by 0.71 mm. This

showed that bacteria susceptibility was highly dependent of the osmotic

concentration of the extracts. There was intermediate association between bacteria

susceptibility and the osmotic concentration by . Therefore the increase in

dilution decreases osmotic concentration of the extracts, hence the decrease in the

activity.

16
 60

Inhibition zone diameter (mm) 50 y = 0.6651x - 0.7115

R = 0.6037
40

30

20

10

0
0 20 40 60 80
%Brix

Inhibition zone diameter (mm)

Linear (Inhibition zone diameter (mm))

Figure 1: Scatter diagram showing the effects of osmotic concentration on

bacterial susceptibility

4.1.2.2 Effects of acidity on bacterial susceptibility

This study has found that for a unit increase in acidity, bacteria susceptibility

decreased by a factor 5.63. Neglecting the effect of acidity bacteria susceptibility was

by 51.38 mm. Therefore it implies that there were other antimicrobial properties

associated within honey rather than its acidic property. The result showed that there

was perfect negative association by between bacteria susceptibility and

acidity of the extracts for Staphylococcus spp. It was found that pH values where the

test bacteria exhibit highest susceptible zone of inhibition was acidic (4.45 1.14).

The results showed that the bacterial susceptibility decreased as pH increased

(i.e.4.45 1.14 5.07 0.94 6.56 1.23; susceptible, intermediate and resistant

respectively).

17
 40

35
Inhibition zone diameter (mm)

30

25

20

15

y = -5.6296x + 51.384
10 R = 0.4327

0
0 2 4 6 8
pH

Inhibition zone diameter (mm)

Linear (Inhibition zone diameter (mm))

Figure 2: Scatter diagram showing the effects of acidity on bacterial

susceptibility

18
4.2 Discussion

4.3 Bacterial susceptibility

According to Ogodo and Ekeleme (2013), ethanolic extract of ginger rhizome

showed activity by zone of inhibition diameter 10.09 mm and 13.05 mm for both

salmonella spp. and staphylococcus spp. respectively. Where similar study have been

performed by Rajsekhar, Chandaker, and Upmanyu (2012), ethanolic extract of

ginger showed zone of inhibition diameter of 30.00 mm. Honey produced by

stingless bees (Apis mellifera) showed antimicrobial activity against both Gram-

positive and Gram-negative bacteria (Onyeneto, Nwachukwu and Nwogwugwu,

2015). Several researchers have shown that honey exerts antimicrobial activities

against various bacteria when used at its higher concentrations (Akinnibosun and

Iterdjere, 2013). But; a study by Alvarez-suarez et al., (2014) using Manuka honey

showed activity against both Gram-negative and Gram-positive bacteria at pH 3.5-

4.5. This has proved the deviation from this study against observation from other

scholars where in this study, honey and the mixture of honey and the ethanolic

extract of ginger showed activity against Gram-negative bacteria only as shown in

plate 4 and 5 below.

Plate 5: Activity of Honey: Staphylococcus spp. (left), Salmonella spp. (right)

19
Plate 6: Activity of ginger: Staphylococcus spp. (right), Salmonella spp. (left)

Table 2: Inhibition zone diameter (mm)

Extract Inhibition zone diameter (mm)

Honey (n=4) 28.25 10.69a

Ginger (n=4) 0.00 0.00b

Combined (Ginger and Honey) (n=16) 28.31 4.22a

Value are expressed as mean S.d

Mean values in each column with different superscripts are significantly different

at .

Table 3: Bacterial susceptibility

Antimicrobial Staphylococcus spp. Salmonella spp.

Honey (n=4) 2.50 1.00a 0.00 1.00a

Ginger (n=4) 1.00 0.00b 0.00 0.00a

Combined (Ginger and Honey) (n=16) 2.90 0.08a 0.00 0.00a

Value are expressed as mean S.d

Mean values in each column with different superscripts are significantly different

at . KEY: 1: Resistant, 2; Intermediate and 3: Susceptible

20
 CHAPTER FIVE

5.0 CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

This study shows that pure honey purchased from the College of Forestry, Wildlife

and Tourism at SUA Morogoro Tanzania, is susceptible to Staphylococcus spp.

which showed greater average antimicrobial activity of 28.25 10.69 mm zone of

inhibition; while the ethanolic extracts of ginger rhizomes from Mawenzi market in

Morogoro, had no antimicrobial activity against both tested bacteria isolates

(Salmonella spp. and Staphylococcus spp.). The mixture of honey and ethanolic

extract of ginger showed greater antimicrobial activity of 28.31 4.32 mm against

Staphylococcus spp. Since foods preserved with natural additives have become

popular due to greater consumer awareness and concern regarding synthetic chemical

additives therefore; honey and the mixture of honey and ethanolic extract of ginger

can be used as an alternative source in preserving foods and beaverages (i.e. meat,

snacks, fruit juices and salads) and preventing Staphylococcal food poisoning.

5.2 Recommendations

According to this study the following further studies are recommended:-

a. Evaluating the shelf-life and quality attributes of foods preserved with

honey, the mixture of honey and ethanolic extract of ginger.

b. Evaluating the activity of honey and the mixture of honey and ethanolic

extract of ginger from different locations in Tanzania against other common

Gram-negative and Gram-positive food pathogenic bacteria.

c. Examining the antimicrobial resistance factors associated in ginger grown

from different locations in Tanzania against tested bacteria isolate.

21
 REFERENCES

Ahmed, N., Singh, J., Kour, H. and Gupta, P. (2013) Naturally Occurring

Preservatives in Food and their Role in Food Preservation, International

Journal of Pharmaceutical and Biological Archieves. India, 4(1), pp. 22

30.

Akinnibosun, F. and Iterdjere, E. (2013) Evaluation of the antibacterial properties

and synergistic effect of Garcinia kola Heckel ( Family: Guttiferae ) seed

extract and honey on some bacteria, African Journal of Microbiology

Research. Nigeria, 7(3), pp. 174180.

Alvarez-suarez, J., Gasparrini, M., Forbes-hern, T., Mazzoni, L. and Giampieri, F.

(2014) The Composition and Biological Activity of Honey: A Focus on

Manuka Honey. Italy. Available at: [http://awww.mdpi.com/journal/foods]

(Accessed: 3 October 2016).

Babatunde, O. and Adewumi, A. (2015) Effects of ethanolic extract of garlic ,

roselle and ginger on quality attributes of chicken patties, African journal

of Biotechnology. Nigeria, 14(8), pp. 688694. doi: 10.5897/

AJB2014.14120.

Bradley, R. (2010) Compositional analysis of foods: Moisture and Total Solids

Analysis, in Nielsen, S. (ed.) Food Analysis, pp. 9498.

Danyluk, M., Schneider, K., Harris, L. and Worobo, R. (2012) Outbreaks of

Foodborne Disease Associated with Fruit and Vegetable Juices , 1416.

USA.

Elijah, M., Imohiosen, O., Lamidi, B. and Umar, M. (2015) Biochemical screening

of pure honey and its antibacterial activity on some bacterial isolates

22
 compared with a common antibiotics, International Journal of

Pharmaceutical Science Invention. Nigeria, 4(5), pp. 1520.

Fikselov, M., Kaniov, M., Hleba, L., Mellen, M., Vukovi, N. and Dugan, M.

(2014) Antimicrobial and Antioxidant Activity of Natural Honeys of

Different Origin, Animal Science and Biotechnologies. Slovakia.

Hamid, M. (2011) Resistance pattern of coagulase positive Staphylococcus aureus

clinical isolates from Asir region , Kingdom of Saudi Arabia, Journal of

Microbiology and Antimicrobials. Available at:

[http://www.academicjournals.org/JMA] (Accessed: 4 August 2016).

Jay, M. (2000) Staphylococcal Gastro-enteritis, in Sesma, J. (ed.) Modern Food

Microbiology. 6th edn. Gaithersburg,Maryland: Aspen Publishers,Inc., p.

625.

Johler, S., Giannini, P., Jermini, M., Baumgartner, A. and Stephan, R. (2015) Further

Evidence for Staphylococcal Food Poisoning Outbreaks Caused by egc -

Encoded Enterotoxins, Toxins. Switzerland. Available at:

[http://www.mdpi.com/journal/toxins] (Accessed: 4 August 2016).

Kunwar, C., Harpreet, M. and Vipra, M. (2013) Outbreak investigation: Salmonella

food poisoning, Medical Journal Armed force India. India, 9(5), pp. 388

391.

Macwan, S., Dabhi, K., Aparnathi, K. and Prajapati, J. (2016) Essential Oils of Herbs

and Spices: Their Antimicrobial Activity and Application in Preservation

of Food, International Journal of Current Microbiology and Applied

Sciences. India.

Makelele, L., Kazadi, Z., Oleko, R., Foma, R., Kabwang, R., Ngbolua, K. and

Gdeon, B. (2015) Microbiological quality of food sold by street vendors in

23
 Kisangani , Democratic Republic of Congo, African Journal of Food

Science. Congo.

Malik, K., Anwar, N., Khan, I., Mushtaq, M. and Noor, S. (2015) Isolation and

Identification of Salmonella Species from Dahi Bhalay , Fruit Chaat and

Fruit Juices Collected From Different Localities of Lahore, Bulletin of

Environment, Pharmacology and Life Sciences. India.

Mathur, A., Joshi, A. and Harwani, D. (2014) Microbial Contamination of Raw

Fruits and Vegetables, Internet Journal of Food Safety. India.

Metrohm (2009) Quality Control in the Food and Beverage Industry, Food Analysis.

Switzerland. Available at: [http://www.food.metrohm.com/] (Accessed: 7

March 2017).

Mindy, P., Cheryl, B., Elliott, J., Facklam, R., Tanja, P. and Wells, J. (2003) Manual

for the Laboratory Identification and Antimicrobial Susceptibility Testing

of Bacterial Pathogens of Public Health Importance in the Developing

World, World Health Organization (WHO): Department of Communicable

Disease Surveillance and Response. USA.

Mossong, J., Decruyenaere, F., Moris, G., Ragimbeau, C., Olinger, M., Johler, S.,

Perrin, M., Hau, P. and Weicherding, P. (2015) Investigation of a

staphylococcal food poisoning outbreak combining case control ,

traditional typing and whole genome sequencing methods , Luxembourg ,

June 2014. Luxembourg. doi:

Mukantwali, C., Laswai, H., Tiisekwa, B., Wiehler, S. and Brat, P. (2013) Could

good hygienic practices reduce the microbial population on pineapple

fruits?, Academia Journal of Agricultural Research. Tanzania: Academia

publishing.

24
Mybeeline (2015) The Water Test to Spot Fake Honey: How to check the purity of

honey at home?, Conducting tests for purity of honey at home. Available at:

[https://www.mybeeline.co/en/p/how-to-check-the-purity-of-honey-at-

home] (Accessed: 22 July 2016).

Ogodo, A. and Ekeleme, U. (2013) In-vitro antibacterial activity of garlic cloves and

ginger rhizomes on food-borne pathogens, International Journal of Basic

and Applied Sciences. Nigeria: Science Publishing Corporation. Available

at: [http://www.sciencepubco.com/index.php/IJBAS] (Accessed: 29

September 2016).

Onyeneto, T., Nwachukwu, I. and Nwogwugwu, N. (2015) The Effect of pH and

Chemical Preservatives on the Growth of Bacterial Isolates from

Commercial Samples of Fruit Juices Sold in South Eastern Nigeria,

International Journal of Research Studies in Biosciences (IJRSB). Nigeria,

3(10), pp. 141145.

Ortez, J. and Rankin, I. (2005) Test methods, in Coyle, M. (ed.) Mannual of

Antimicrobial Susceptibility Testing. 2nd edn. USA: American Society for

Microbiology, p. 236.

Plotto, A. (2002) Ginger: Post-Production Management for Improved Market

Access, Post-harvest Compendium. USA.

Purbafrani, A., Amirhosein, S., Bayyenat, S., Moghaddam, H. and Saeidi, M. (2014)

The Benefits of Honey in Holy Quran, International Journal of

Pediatrics. Iran, 2(9), pp. 6773.

Rajsekhar, S., Chandaker, A., Upmanyu, N. and Kuldeep, B. (2012) Spice as

antimicrobial agents: a review, International Research Journal of

pharmacy. India, 3(2), pp. 49.

25
Reddi, S., Kumar, R., Balakrishna, N. and Rao, V. (2015) Microbiological quality of

street vended fruit juices in Hyderabad , India and their association between

food safety knowledge and practices of fruit juice vendors, International

Journal of Current Microbiology and Applied Sciences. India, 4(1), pp.

970982.

Singham, P., Birwal, P. and Yadav, B. (2015) Importance of Objective and

Subjective Measurement of Food Quality and their Inter-relationship,

Journal of Food Processing and Technology. Available at:

[https://www.omicsonline.org/open-access/importance-of-objective-and-

subjective-measurement-of-food-quality-and-their-interrelationship-2157-

7110-1000488.php?aid=59908] (Accessed: 13 July 2017).

Sulieman, A., Eltayeb, F., Sulieman, S. and Osman, N. (2015) Activity of Zingiber

officinale ( Ginger ) Oil against Bacteria Isolated from Children Throat,

International Journal of Microbiology. India. Available at:

[http://www.microbiozjournals.com] (Accessed: 3 May 2017).

Witkowska, A., Hickey, D., Alonso-gomez, M. and Wilkinson, M. (2013)

Evaluation of Antimicrobial Activities of Commercial Herb and Spice

Extracts Against Selected Food-Borne Bacteria, Journal of Food

Research. Ireland: Canadian Center of Science and Education, 2(4), pp. 37

54.

Wootton, M. (2013) Disc Diffusion Method for Antimicrobial Susceptibility Testing,

BSAC Methods for Antimicrobial Susceptibility Testing. Ireland: British

Society for Antimicrobial Chemotherapy (BSAC).

26
 APPENDIX

Appendix 1: Test significance differences on Antimicrobial and bacterial

Susceptibility

Response Susceptibility

Df Sum Sq. Mean Sq. F value Pr(>F)

Antimicrobial 2 5.625 2.81250 3.3863 0.04266 *

Residuals 45 37.375 0.83056

Signif. Codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Appendix 2: Test of significance difference: Antimicrobial and Inhibition zone

diameter (mm)

Response Inhibition zone diameter (mm)

Df Sum Sq. Mean Sq. F value Pr(>F)

Antimicrobial 2 1334.8 667.41 3.4797 0.03934 *

Residuals 45 8631.1 191.80

Signif. Codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

27