department of nuclear agriculture and radiation processing, school of nuclear and allied science

154 Pages·2017·7.62 MB·English

University of Ghana http://ugspace.ug.edu.gh

UNIVERSITY OF GHANA

DEPARTMENT OF NUCLEAR AGRICULTURE
AND RADIATION PROCESSING, SCHOOL OF
NUCLEAR AND ALLIED SCIENCE

DEVELOPMENT OF STARTER CULTURE
FOR FERMENTATION OF MILLET INTO FURA
AND PRESERVATION OF FURA BY
GAMMA RADIATION

BY
COSMOS AMANKONA
(10103969)

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA,
LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR
THE AWARD OF MPHIL RADIATION PROCESSING DEGREE
(FOOD SCIENCE AND POST HARVEST TECHNOLOGY)

JUNE, 2016
University of Ghana http://ugspace.ug.edu.gh

DECLARATION
This thesis is the result of research conducted by Cosmos Amankona in the Department of
Nuclear Agriculture and Radiation Processing of the School of Nuclear and Allied Sciences
(SNAS), University of Ghana, under the supervision of Dr. Wisdom Kofi Amoa-Awua and
Prof. Mrs. Victoria Appiah.
Except for the references of other peoples‘ work which have been duly cited, this theses has
not been presented either in whole or in part for another degree elsewhere.

Signed……………………………… …………………
Cosmos Amankona Date
(Student)
Signed…………………… ……………………………..
Dr. Wisdom Kofi Amoa-Awua Date
(Supervisor)
Signed…………………… ……………………………..
Prof. Mrs. Victoria Appiah Date
(Co-Supervisor)

i
University of Ghana http://ugspace.ug.edu.gh

DEDICATION
This thesis is dedicated to my family and friends, especially my wife for always being there
for me in difficult times.

ii
University of Ghana http://ugspace.ug.edu.gh

ACKNOWLEDGEMENT
Thanks to the Almighty God for giving me the needed strength and wisdom to come out
successfully with this work.
I am highly grateful to DANIDA for sponsoring this work under the GREEN GROWTH
project. I am also grateful to my experienced supervisors, Dr. Wisdom Kofi Amoa- Awua
and Prof. Mrs. Victoria Appiah for their constant guidance, tolerance and understanding
throughout the work.
I am also grateful to Messrs. Theophillus Annan and Alexander K.H. Appiah as well as Mrs.
Amy Atter and Dr. Mrs. Owusu, for their Assistance and advice throughout the work, not
forgetting to thank Mr. Emmanuel Tetteh for always making all laboratory apparatus
available for me.
I am also grateful to all the lecturers of the Department of Nuclear Agriculture and Radiation
Processing of the School of Nuclear and Allied Sciences (SNAS), University of Ghana, for
the knowledge impartation and making my study a success.
Lastly, I would like to express my heartfelt gratitude to all members of my family, my wife
Georgina; my Mum, Anastasia; my sisters Helina, Patience and Henrietha; my dear
children, Bright, Prince, Benedicta and Bertina; not forgetting my friends Rebecca and Joyce,
for their immense encouragement and financial support during the study period.

iii
University of Ghana http://ugspace.ug.edu.gh

TABLE OF CONTENTS
DECLARATION .................................................................................................................... i
DEDICATION ....................................................................................................................... ii
ACKNOWLEDGEMENT ................................................................................................... iii
TABLE OF CONTENTS ...................................................................................................... iv
LIST OF TABLES ................................................................................................................ ix
LIST OF FIGURES ................................................................................................................ x
ABSTRACT .......................................................................................................................... xi
CHAPTER ONE ...................................................................................................................... 1
INTRODUCTION ..................................................................................................................... 1
1.1 RATIONALE OF THE STUDY ...................................................................................... 3
1.2 MAIN OBJECTIVE ......................................................................................................... 4
1.2.1 SPECIFIC OBJECTIVES: ........................................................................................ 4
CHAPTER TWO ..................................................................................................................... 5
LITERATURE REVIEW .......................................................................................................... 5
2.1 THE PEARL MILLET GRAIN ....................................................................................... 5
2.2 FERMENTATION ........................................................................................................... 5
2.2.1 Historical Perspective of Fermentation ..................................................................... 7
2.2.2 Classification of Fermented Foods ............................................................................ 8
2.3 STARTER CULTURES ................................................................................................ 10
2.3.1 Bacteria .................................................................................................................... 10
2.3.2 Yeasts....................................................................................................................... 11
2.3.3 Moulds ..................................................................................................................... 12
2.4 FUNCTIONS OF STARTER CULTURES ................................................................... 13
2.5 FACTORS TO CONSIDER IN SELECTING LACTIC ACID BACTERIA STARTER
CULTURES FOR CEREAL FERMENTATION ................................................................ 14
2.5.1 Fast Acidification .................................................................................................... 14
2.5.2 Good Antimicrobial properties ................................................................................ 15
2.5.4 Good Probiotic Effects ............................................................................................ 16
2.5.5 Nutritional Quality of the Fermented Food ............................................................. 16
2.5.6 Starch hydrolysis ..................................................................................................... 17
2.5.7 Exopolysaccharide Formation ................................................................................. 18
2.6 STARTER CULTURES IN AFRICAN CEREAL FERMENTATION ........................ 18
iv
University of Ghana http://ugspace.ug.edu.gh

2.6.1 Kisra......................................................................................................................... 19
2.6.2 Ogi ........................................................................................................................... 20
2.6.3 Uji ............................................................................................................................ 20
2.6.4 Mawe ....................................................................................................................... 22
2.6.5 Mahewu ................................................................................................................... 22
2.7 LACTIC ACID BACTERIA AND THEIR USES IN FOOD ....................................... 23
2.8 CLASSIFICATION OF LACTIC ACID BACTERIA .................................................. 23
2.9 CHARACTERIZATION AND IDENTIFICATION OF MICROORGANISMS IN
FERMENTED FOODS ........................................................................................................ 24
2.9.1 Phenotypic methods ................................................................................................. 24
2.9.2 Genotypic methods .................................................................................................. 26
2.10 ANTIMICROBIAL COMPOUNDS PRODUCED BY LACTIC ACID BACTERIA 27
2.10.1 Organic Acids and Low pH ................................................................................... 28
2.10.2 Hydrogen Peroxide ................................................................................................ 29
2.10.3 Carbon dioxide ...................................................................................................... 30
2.10.4 Diacetyl .................................................................................................................. 30
2.10.5 Bacteriocins ........................................................................................................... 31
2.11 FOOD IRRADIATION ................................................................................................ 33
2. 12 SOURCES OF IONISING RADIATION ................................................................... 34
2.12.1 Gamma rays ........................................................................................................... 34
2.12.2 Electron-beam machines ........................................................................................ 34
2.12.3 X-rays .................................................................................................................... 35
2.13 APPLICATION OF FOOD IRRADIATION .............................................................. 36
2.13.1 Reduction of pathogenic microorganisms ............................................................. 36
2.13.2 Decontamination .................................................................................................... 37
2.13.3 Extension of shelf-life ........................................................................................... 38
2.13.4 Disinfestation ......................................................................................................... 38
CHAPTER THREE ............................................................................................................... 41
MATERIALS AND METHODS ............................................................................................. 41
3.1 Study area and design ..................................................................................................... 41
3.2 Sample Collection and Preparation ................................................................................ 41
3.3 Chemical Analysis.......................................................................................................... 41
3.3.1 Determination of pH ................................................................................................ 41
3.3.2 Determination of Titratable Acidity ........................................................................ 42
v
University of Ghana http://ugspace.ug.edu.gh

3.4 MICROBIOLOGICAL ANALYSIS ............................................................................. 42
3.4.0 Enumeration of microorganisms ............................................................................. 42
3.4.1 Homogenization and Serial Dilution ....................................................................... 42
3.4.2 Enumeration of Aerobic Mesophiles ....................................................................... 42
3.4.3 Enumeration and Confirmation of Total Coliforms ................................................ 42
3.4.4 Enumeration of Lactic Acid Bacteria ...................................................................... 43
3.4.5 Enumeration of Yeasts ............................................................................................. 43
3.4.6 Isolation of Lactic Acid Bacteria ............................................................................. 43
3.4.7 Isolation of Yeasts ................................................................................................... 43
3.5 CHARACTERISATION OF LAB ISOLATES ............................................................. 43
3.5.1 Characterization of Lactic Acid Bacteria Isolates by Gram Reaction ..................... 43
3.5.2 Characterisation of Lactic Acid Bacteria Isolates by Catalase Reaction ................. 44
3.5.3 Oxidase Test ............................................................................................................ 44
3.5.4 Microscopic Examination ........................................................................................ 44
3.5.5 Growth at Different Temperatures .......................................................................... 44
3.5.6 Salt Tolerance Test .................................................................................................. 45
3.5.7 Growth at Different pH ............................................................................................ 45
3.5.8 Identification of Lactic Acid Bacteria ..................................................................... 45
3.6.1 Macroscopic and Microscopic Examination of Yeast ............................................. 45
3.6.2 Identification of Yeast Isolates ................................................................................ 45
3.7 Antimicrobial Studies ................................................................................................. 45
3.8 TECHNOLOGICAL PROPERTIES OF IDENTIFIED LACTIC ACID BACTERIA . 46
3.8.1 Rate of Acidification of Millet Dough by LAB ...................................................... 46
3.8.2 Production of Exopolysaccharides (EPS) by LAB Isolates..................................... 47
3.8.3 Tests for Amylase Secretion by LAB Isolates ......................................................... 47
3.8.4 Test for Protease Secretion by LAB Isolates ........................................................... 48
3.9 DEVELOPMENT OF STARTER CULTURE .............................................................. 48
3.9.1 Irradiated Millet Flour ............................................................................................. 48
3.9.2 Starter Cultures ........................................................................................................ 48
3.9.3 Inoculation Trials ..................................................................................................... 48
3.9.3.1 Fermentation with Single Starter Culture ............................................................. 49
3.9.3.2 Fermentation with Combined Starter Culture ...................................................... 49
3.10 Survival of Enteric Pathogens in Fermenting Dough ............................................... 49
vi
University of Ghana http://ugspace.ug.edu.gh

3.11 SHELF LIFE STUDIES ............................................................................................... 50
3.11.1 Dose Optimization ................................................................................................. 50
3.11.2 Storage ................................................................................................................... 50
CHAPTER FOUR .................................................................................................................. 52
RESULTS ................................................................................................................................ 52
4.1 Field Study ..................................................................................................................... 52
4.2 Acidification of steep water and dough during spontaneous fermentation .................... 52
4.3 Changes in Microbial Population during Steeping and Dough Fermentation ............... 54
4.3.1 Population of Lactic Acid Bacteria (LAB) .............................................................. 54
4.3.2 Population of Yeasts ................................................................................................ 55
4.3.2. Population of aerobic mesophiles ........................................................................... 56
4.3.4 Population of total coliforms ................................................................................... 57
4.4 Phenotypic characterization of Lactic Acid Bacteria ..................................................... 58
4.5 Characterisation and Identification of Yeasts ................................................................ 61
4.6 Technological properties of Lactic acid Bacteria Isolates ............................................. 61
4.6.1 Rate of Acidification by Lactic Acid Bacteria Isolates ........................................... 61
4.6.2 Amylase Secretion exopolysaccharide production and protease secretion by Lactic
Acid Bacteria Isolates ....................................................................................................... 63
4.6.3 Antimicrobial Interaction between Lactic Acid Bacteria isolates ........................... 65
4.6.4 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates ........ 65
4.6.5 Antimicrobial Activity of Lactic Acid Bacteria against Some Common Enteric
Pathogens .......................................................................................................................... 66
4.7 Starter culture trials ........................................................................................................ 67
4.7.1 Changes in Microbial Population ............................................................................ 67
4.7.2 Microbial Counts during Dough Fermentation with combined Starter Cultures .... 69
4.7.3 Acidification of Fermenting Dough in Fermentation Trials with Starter Cultures . 70
4.7.4 Acidification of Fermenting Dough in Fermentation Trials with combined Starter
cultures.............................................................................................................................. 71
4.7.5 Survival of Enteric Pathogens ................................................................................. 72
4.8 STORAGE OF FURA SAMPLES ................................................................................. 74
4.8.1 Dose optimization .................................................................................................... 74
4.8.2 Shelf Life Studies .................................................................................................... 76
CHAPTER FIVE ................................................................................................................... 78
DISCUSSION .......................................................................................................................... 78
vii
University of Ghana http://ugspace.ug.edu.gh

5.1 Processing of Fura ............................................................................................................. 78
5.2 Acidification during spontaneous fermentation ................................................................. 78
5.3 Lactic acid bacteria involved in Fura fermentation ........................................................... 79
5.4 Yeasts involved in fura Fermentation ........................................................................ 81
5.5 Antimicrobial activity of Lactic Acid Bacteria against Common Enteric Pathogens .... 82
5.6 Microbial Interactions during Fura Fermentation ......................................................... 84
5.7 Technological Properties ................................................................................................ 85
5.8 Starter culture selection .................................................................................................. 86
5.9 Shelf life studies ............................................................................................................. 88
CHAPTER SIX ...................................................................................................................... 89
CONCLUSIONS AND RECOMMENDATIONS .................................................................. 89
6.1 Conclusions .................................................................................................................... 89
6.2 Recommendation(S) ....................................................................................................... 89
CHAPTER SEVEN ................................................................................................................ 90
REFERENCES ........................................................................................................................ 90
APPENDIX ............................................................................................................................ 119

viii
University of Ghana http://ugspace.ug.edu.gh

LIST OF TABLES
Table 4.1 Population of Lactic Acid Bacteria .......................................................................... 54
Table 4.2 Population of Yeasts ................................................................................................ 56
Table 4.3 Population of aerobic mesophiles ............................................................................ 57
Table 4.4 Population of total coliforms ................................................................................... 58
Table 4.5 Phenotypic characteristics of lactic acid bacteria isolated from steeping water and
fermenting dough ..................................................................................................................... 60
Table 4.6 Amylase Secretion, exopolysaccharide (EPS) production and protease secretion by
Lactic Acid Bacteria Isolates ................................................................................................... 64
Table 4.7 Antimicrobial Interaction between Lactic Acid Bacteria isolates ........................... 65
Table 4.8 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates ......... 66
Table 4.9 Antimicrobial activity of lactic acid bacteria against pathogen indicator- strains ... 67
Table 4.10 mean microbial counts (log CFU/g) for fermentations with single starter cultures
.................................................................................................................................................. 68
Table 4.11 Mean microbial counts (log CFU/g) during dough fermentation with combined
starter cultures .......................................................................................................................... 70
Table 4.12 Count (log CFU/g) for survival of enteric pathogens inoculated into spontaneous
and mixed culture fermentation of millet dough ...................................................................... 73
Table 4.13.The microbial counts (CFU/g) for dose optimization for storage of Fura samples
.................................................................................................................................................. 75
Table 4.14 Population of Aerobic Mesophiles and Yeast and Moulds before irradiation of
Fura samples ............................................................................................................................ 77

ix