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 i 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) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION This thesis is dedicated to my family and friends, especially my wife for always being there for me in difficult times. University of Ghana http://ugspace.ug.edu.gh iii 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. University of Ghana http://ugspace.ug.edu.gh iv 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 University of Ghana http://ugspace.ug.edu.gh v 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 University of Ghana http://ugspace.ug.edu.gh vi 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 University of Ghana http://ugspace.ug.edu.gh vii 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 University of Ghana http://ugspace.ug.edu.gh viii 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 University of Ghana http://ugspace.ug.edu.gh ix 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 University of Ghana http://ugspace.ug.edu.gh x LIST OF FIGURES Fig. 4.1. (a-d) Acidification during fermentation of millet into Fura………………………...53 Fig.4.2. Changes in pH during dough fermentation by Lactic Acid Bacteria isolates ............ 62 Fig.4.3. Titratable acidity during acidification of fermenting dough by lactic acid bacteria .. 63 Fig.4.4. (a-b) pH and Titratable Acidity of Fermenting Dough in Fermentation Trials with Starter Cultures ........................................................................................................................ 71 Fig. 4.5 (a-b) pH and Titratable Acidity of Fermenting Dough in Fermentation Trials with combined Starter Cultures........................................................................................................ 72 Fig. 4.6a Population of Aerobic Mesophiles during the storage of Fura samples ................... 77 Fig. 4.6b Population of Yeasts and Moulds during the storage of Fura samples .................... 77 University of Ghana http://ugspace.ug.edu.gh xi ABSTRACT Lactic Acid Bacteria (LAB) are the most widespread of organisms responsible for food fermentation and have been applied as commercial starter cultures in many food industries. A study was conducted to develop a starter culture for the fermentation of millet into Fura and to extend the shelf life of Fura by gamma radiation. The isolation, characterization and identification of the LAB and yeasts responsible for Fura fermentation was carried out using physiological methods. A brief survey was carried out in Dome and Nima in Accra to observe and confirm the processing operations documented in literature and also obtain samples for laboratory analysis. The enumeration of aerobic mesophiles, lactic acid bacteria (LAB) and yeasts populations were carried out on Plate Count Agar, de Man Rogosa Sharpe Agar and Oxytetracycline Glucose Yeast Extract Agar respectively. The LAB species were characterized using Gram Reaction, Catalase Reaction, Oxidase Test, Salt Tolerance Test, Growth at Different Temperatures and Growth at Different pH. The LAB and yeasts Isolates were tentatively identified by determining their pattern of carbohydrate fermentation using the API 50 CH and ID 32 C galleries respectively. The LAB were also screened for their technological properties on rate of acidification, production of exopolysaccharides (EPS), amylase and protease activity including their antimicrobial activity against some common enteric pathogens using the Agar Well Diffusion Assay. Starter culture trials were carried out using dominant strains of lactic acid bacteria and yeasts in singles and in combinations. Challenge testing with Escherichia coli (RM EC. 0157; 11Q-1411), Vibrio cholerae, Staphylococcus aureus (RM SA 1L-1304), and Salmonella typhimurium(RM ST 20B-1410), in a sterile millet dough was also carried out. The lactic acid bacteria identified were Lactobacillus fermentum (33.33%), Weissella confusa (20%), Lactobacillus brevis (16.67), Pediococcus acidilactici (13.33%), Lactococcus lactis ssp lactis 1 (10%) and Lactococcus rafinolactis (6.67%) whereas the yeasts were characterized and identified as Saccharomyces University of Ghana http://ugspace.ug.edu.gh xii cerevisiae (43.75%); Candida krusei (25%) Candida albicans (18.75%) and Candida membranifascians (12.5%); Mean pH values decreased from 6.47-6.38 to 4 .02-3.83 with corresponding increase in titratable acidity from 0.18-0.19 to 0.51-0.62 during all the fermentation trials. The population of LAB increased from 107 to1010 cfu/g whilst the population of yeasts increased from 105 to108 cfu/g during all the dough fermentation trials. Three LAB isolates (Lactobacillus fermentum, Lactobacillus brevis and Weissella confusa) exhibited the fastest rates of acidification with the least pH values and corresponding high percentage titratable acidity values and therefore have the potential to be used as starter cultures for Fura production. All the lactic acid bacteria isolates exhibited antimicrobial activity against all the pathogens tested in the present work (Salmonella typhimurium, E. coli, Vibrio cholerae and Staphylococcus aureus), with L. fermentum exhibiting the strongest inhibition against Staphylococcus aureus and Vibrio cholerae. In the challenge test, the microbial numbers of most of the pathogens reduced significantly in the course of the fermentation and were not detected after 12 hours in many of the mixed culture combinations. Fermented and unfermented Fura samples were given different treatments involving vacuum packaging and irradiation and stored at ambient temperature. Fermentation did not have an effect on shelf life because the unfermented samples also fermented during storage. The combination of irradiation and vacuum packaging had the most significant effect on Fura and samples were wholesome after six (6) weeks. Samples which were irradiated but not vacuum packaged were also wholesome but had higher microbial counts. Samples which were vacuum packed but not irradiated had shelf life of four (4) weeks. Samples which were packed in polyethylene bags and given no further treatment had a shelf life of two weeks University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION Fermentation involves the use of microorganisms and enzymes to produce foods with distinct quality attributes, quite different from the original agricultural raw material. The process depends on the biological activity of microorganisms to produce a range of metabolites which suppress the growth and survival of undesirable microflora in foodstuffs (Ross et al., 2002). It is one of the oldest and most economical methods of producing and preserving food (Billings, 1998; Chavan and Kadam, 1989), and provides a natural way to reduce the volume of the material to be transported; destroys undesirable components, enriches the nutritive value and appearance of the food, decreases the energy required for cooking and results in a safer product (Simango, 1997). Fermentation may be a useful strategy for reducing bacterial contamination of food. The number of harmful microorganisms (Staphylococci, Coliform bacteria "E.coli" and Salmonella) in sorghum significantly decreased with the increase of fermentation period (Adam et al., 2009) and could also reduce the prevalence of diarrheal diseases (Mensah et al., 1990). According to Egounlety et al, (2002), fermentation is a low-cost and the most economical technique of production and preservation of foods. It helps to preserve perishable foods and to improve their nutritional and organoleptic qualities. As of 1995, fermented food represented between one quarter and one third of food consumed in Central Europe (Holzapfel et al, 1995). According to Motarjemi and Nout (1996) and Oyewole (1997), the fermentation process prevents food spoilage and food-borne diseases with respect to consumers living in a climate, which favours the rapid deterioration of food. In addition, University of Ghana http://ugspace.ug.edu.gh 2 fermented foods are of particular importance in ensuring adequate intake of protein and/or calories in the diet. Food fermentation, and especially lactic acid fermentation, is an important technology in Africa, indigenous and adaptable to the culture of the people. There are many cereal based fermented foods in Africa, such as ogi and mahew in Benin, kenkey in Ghana, injera in Ethiopia, poto-poto in Congo, ogi and kunu-zaaki in Nigeria, uji and togwa in Tanzania, kisra in Sudan (Tomkins et al., 1988, Hounhouigan et al., 1993, Oyewole, 1997, and Blandino et al., 2003). The desirable changes of taste, flavor, acidity, digestibility, and texture in these gruels are contributed by fermentation. The cereals most commonly fermented are maize, sorghum, millet, tef and occasionally rice and wheat (Oyewole, 1997). Fura is one of the cereal-based fermented meals. It is a traditional staple food in West Africa mostly in Ghana, Nigeria and Burkina Faso (Jideani et al., 2001). It is generally produced from millet and blended with spices, water and compressed into dough balls and cooked (Kordylasi, 1990, Jideani et al., 2001). The cooked dough balls are broken up and made into porridge by mixing with yoghurt (nunu), fresh milk or water (Kordylasi, 1990). Sugar may be added to taste. Fura fermentation, like many fermented foods in Africa is spontaneous, mostly home-based and on a small scale production. In spite of the fact that Fura is a staple food for most West African countries, produced with inexpensive techniques and equipment applied in simple environments, it is processed without following any scientific principles. Product quality and safety is therefore difficult to predict and standardize, leading to products of inconsistent quality. University of Ghana http://ugspace.ug.edu.gh 3 1.1 RATIONALE OF THE STUDY A wide variety of microorganisms, notably lactic acid bacteria and yeasts, are associated with Fura production and these microorganisms spontaneously come from raw materials, the environment, processing equipment and persons involved in the production (Owusu- Kwateng et al., 2010). The use of LAB starter cultures for cereal fermentation in Africa has been a subject of increasing interest in trying to standardize and guarantee product quality and uniformity. Their use by small-scale processing units and small agro-food industrial enterprises is however still limited. The use of starter cultures has been suggested by Sanni (1993) and Kimaryo et al. (2000), as an appropriate approach for the control and optimization of the fermentation process in order to alleviate the problems relevant of variations in organoleptic quality and microbiological stability observed in African indigenous fermented foods. The development of starter cultures is however one of the pre-requisites for the establishment of small scale industrial production of fermented foods in Africa (Sanni, 1993). In order for Fura to obtain its optimum possible benefits and be able to contest satisfactorily with imported and industrially processed foods, there is the need to upgrade its processing technologies in order to add value and ensure the safety and stability of the final product. This may include irradiation with a proper dose to extend the shelf life or improve the technological properties of the product. The microbial load of irradiated Banku Mix Powder, Fermented Maize Powder and Cassava Dough Powder were very low, indicating high product quality and the possibility of using low doses of gamma radiation to improve the hygienic quality and extend the shelf-life of these food products (Adu-Gyamfi and Appiah, 2012). There is therefore the need to develop a University of Ghana http://ugspace.ug.edu.gh 4 starter culture to optimize the fermentation of millet into Fura and also ensure the overall safety of the final product with the use of gamma radiation. Owusu-Kwarteng et al., (2013) isolated, characterized and identified the lactic acid bacteria (LAB) and yeasts associated with Fura processing and also assessed the technological properties of these isolates and recommended their potential and basis for starter culture development. However, all efforts to have access to these isolates proved futile due to loss of the isolates. 1.2 MAIN OBJECTIVE The main objective of this work is to develop a starter culture for the fermentation of millet into Fura and extend the shelf life of the product by gamma radiation. 1.2.1 SPECIFIC OBJECTIVES: To isolate the Lactic acid bacteria and Yeasts involved in Fura processing To investigate the technological properties of Lactic Acid Bacterial including their antimicrobial properties during fermentation of millet into Fura. To evaluate the suitability of selected isolates as starter culture. To investigate the ability of gamma radiation to extend the shelf life of Fura. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER TWO LITERATURE REVIEW 2.1 THE PEARL MILLET GRAIN Pearl millet is believed to have originated from North Africa and has been consumed since pre-historic times. Pearl millet grain is a primary human food source in many regions of Africa, Asia (Burton et al., 1972), India and Pakistan (FAO, 1994). It has an excellent amino acid profile and higher crude protein than corn or sorghum (Burton et al., 1972; Smith et al., 1989). Pearl millet has a number of nutritional advantages over other cereals used as source of food. It possesses high phenolic content, moderate reducing ability and high free radical scavenging activity and therefore can serve as a source of antioxidants in our diets (Odusola, et al., 2013). The protein in millet consists of all varieties of essential amino acids including leucine. It is a good source of Tryptophan, an amino acid which can raise serotonin level and helps stress reduction (Odusola, et al., 2013). The grain is processed in so many ways for preparation of various food products. Some of the products include cooked whole grain, thin and thick porridges, steam cooked grits (couscous, burabosko), Kunun Zaki, Tuwo and Fura (Nkama and Ikwelle, 1997; Jideani et al., 1999, 2001, 2002). 2.2 FERMENTATION Fermentation is one of the oldest methods of food preparation and preservation (Pederson 1971; Steinkraus et al., 1983; Campbell-Platt, 1994), and has been defined in various ways by different authors. It involves the use of microorganisms and enzymes to produce foods with distinct quality attributes, quite different from the original agricultural raw material. The process depends on the biological activity of microorganisms for production of a range of metabolites to suppress the growth and survival of undesirable microflora in foodstuffs (Ross University of Ghana http://ugspace.ug.edu.gh 6 et al., 2002). According to Campbell-Platt (1987), fermented foods are those which have been subjected to the action of micro-organisms or enzymes so that desirable biochemical changes cause significant changes to the food. Adams (1990) on a microbiological point of view describes the term ‖fermentation‖ as a form of energy-yielding microbial metabolism in which an organic substrate, usually a carbohydrate, is partially oxidized, and an organic carbohydrate acts as the electron acceptor. Fermentation also has different meanings to biochemists and to the industrial microbiologists. On the biochemical point of view, it relates to the generation of energy by the catabolism of organic compounds, with the organic compounds acting as both electron donors and terminal electron acceptors, whereas its meaning in industrial microbiology has been extended to describe any process for the production of products by mass culture of a micro-organism (Anonymous). Whichever definition is used however, microorganisms, by virtue of their metabolic activities and/or enzymes endogenous to the raw materials may contribute to the development of characteristic properties such as taste, aroma, visual appearance, texture, shelf life, and safety (Hammes, 1990). However, if the products of enzyme activities have unpleasant odours or undesirable, unattractive flavours or the products are toxic or disease producing, the foods are described as spoiled (Steinkraus, 1997). Fermentation must therefore yield desirable products and so a spoiled food is rather different from a fermented food as explained above. Fermented foods constitute a substantial part of the diet in many African countries and are considered as an important means of preserving and introducing variety into the diet, which often consists of staple foods such as milk, cassava, fish and cereals (Steinkraus, 1995; Belton and Taylor, 2004). University of Ghana http://ugspace.ug.edu.gh 7 According to Hansen, (2002), it is possible to obtain a large variety of different conditions, and the raw materials traditionally used for fermentation are diverse and include fruits, cereals, honey, vegetables, milk, meat and fish. The microorganisms responsible for the fermentation may be the microbiota indigenously present on the substrate, or they may be added as starter cultures (Harlander, 1992). 2.2.1 Historical Perspective of Fermentation Fermented food production might have started as ‗natural‘ processes where nutrient availability and environmental conditions selected particular microorganisms, to modify and preserve the food. People then became familiar with particular fermented foods produced in their part of the world, and many of these foods became an integral part of the local diet, and were therefore regarded as essential. Migration of people then facilitated the technological transfer of fermented foods (Campbell-Plat, 1994). Preservation of food including the use of fermentation of otherwise perishable raw materials has been used by man since the Neolithic period (around 10000 years BC) (Prajapati and Nair, 2003). According to Gest, (2004) however, the scientific reason behind fermentation started with the identification of micro-organisms in 1665 by Van Leeuwenhoek and Hooke. Louis Pasteur revoked the ―spontaneous generation theory‖ around 1859 by fashionably designed experimentation (Wyman, 1862; Farley and Geison, 1974). The role of a sole bacterium, ―Bacterium‖ lactis (Lactococcus lactis), in fermented milk was shown around 1877 by Sir John Lister (Santer, 2010). Fermentation, from the Latin word fervere, was defined by Louis Pasteur as ―La vie sans l'air‖ (life without air). From a biochem-ical point of view, fermentation is a metabolic process of deriving energy from organic compounds without the involvement of an exogenous oxidizing agent. University of Ghana http://ugspace.ug.edu.gh 8 The fermentation process has been practiced for the millennium with the result that there is incredible selection of fermented foods ranging from those derived from meat and plant to those derived from milk and dairy products (Ray and Daeschel, 1992). The significant role of microorganisms in fermentation process was realized in 1861 AD during the development of pasteurization (Klaenhammer and Fitzgerald, 1994). According to Klaenhammer and Fitzgerald (1994); Hopzapfel; (1997), fermentation can be traced back thousands of years and has been used as a means of improving the keeping quality of food for more than 600 years. 2.2.2 Classification of Fermented Foods Fermented foods are produced worldwide using various manufacturing techniques, raw materials and microorganisms. According to Soni and Sandhu (1990), there are only four main fermentation processes namely, alcoholic, lactic acid, acetic acid and alkali fermentation. Alcoholic fermentation results in the production of ethanol with yeasts being the prime organisms (e.g. wines and beers), Acetic acid fermentation is performed by Acetobacter species which convert alcohol to acetic acid in the presence of excess oxygen. Lactic acid fermentation (e.g. fermented milks and cereals) is mainly carried out by lactic acid bacteria whiles Alkali fermentation often takes place during the fermentation of fish and seeds, popularly known as condiment (McKay and Baldwin, 1990). According to Dirar (1993); Iwuoha and Eke (1996); Steinkraus (1997) and Gadaga et al., (1999), however, classification of fermented foods can be in different ways depending on the desired focus, specifically: by the fermenting microorganisms -as bacteria, yeast or moulds; by classes beverages, cereal products or dairy products; by food group -as example, cereal, fruits or roots; by commodity -as example, alcoholic beverages or fermented vegetable proteins; by production method -as example, back-slopping, spontaneous fermentation or starter culture; by geographical location -as example, products from a specific country or region in a country. University of Ghana http://ugspace.ug.edu.gh 9 A traditional Sudanese classification based on the function of the food as presented by Dirar (1993) is illustrated in the table below Table 2.1 Different classification schemes of fermented foods Adapted from Dirar (1993) Yokotsuka(1982) Kuboye (1985) Campbell-Platt(1987) Odunfa (1988) Sudanese (Dirar, 1993) 1.alcoholic beverages (yeast) 1.cassava- based 1. beverages 1.starchy roots 1. kissar-staples 2.vinegar (acetobacter) 2. cereals 2. cereal products 2. cereals 2. milhat – sauces and relishes for staples 3.milk products (lactobacilli) 3. legumes 3. dairy products 3. alcoholic Beverages 3. marayiss – beers and alcoholic drinks 4.pickles (lactobacilli) 4. beverages 4. fish products 4. vegetable proteins 4. akilmunasabat – food for special occasions 5. fish or meat (enzymes and lactobacilli) 5.fruits and vegetable products 5. animal Proteins 6. plant proteins (moulds,with or 6. legumes 7. meat products University of Ghana http://ugspace.ug.edu.gh 10 without lactobacilli and yeast) 8. starch crop products 9. miscellaneous Products Source: Dirar, 1993 2.3 STARTER CULTURES A starter culture, according to Hopzapfel (1997) may be defined as a preparation which contains high numbers of viable microorganisms that may be added to accelerate the fermentation process in order to bring about desirable changes in a food substrate. It facilitates improved fermentation process and predictability of its product. According to (Wu et al., 2009; Mogra et al., 2008), starter cultures play a technological function in food manufacturing and are used as food ingredients at one or more stages in the process to develop the desired metabolic activity during the fermentation or ripening process. They contribute to the unique properties of a foodstuff especially with regard to taste, flavour, colour, texture, safety, preservation, nutritional value, wholesomeness and/or health benefits. Starter cultures are formed using a specific cultivation medium and a specific mix of fungal and bacterial strains (Dilip et al., 1991; Norman et al., 1999). Microorganisms used in starters include various bacteria, yeasts and moulds (Norman et al., 1999). 2.3.1 Bacteria Lactobacillus species are the most important bacteria in food manufacturing, and belong to the group of lactic acid bacteria. Owusu-Kwarteng et al., (2010) isolated and identified the Lactic Acid Bacteria (LAB) from Fura, based on morphological, physiological and biochemical characteristics as Lactobacillus University of Ghana http://ugspace.ug.edu.gh 11 spp. (51.42%), Pediococcus spp. (21.4%), Streptococcus spp. (14.3%), Leuconostoc spp. (8.5%), and Enterococcus spp. (4.3%). According to Aguirre and Collins (1993), the term ‗lactic acid bacteria‘ is a broad group of Gram-positive, catalase-negative, non-sporing rods and cocci, usually non-motile, that ferment carbohydrates to form lactic acid as the major end product. They are categorized into ‗homo‘ or ‗hetero‘ in relation to the metabolic routes they use (Embden-Meyerhof or Phosphoketolase pathways) according to the resulting end products. Lactic acid bacteria are reported as the basic starter cultures with widespread use in the dairy industry for cheese making, cultured butter milk, cottage cheese and cultured sour cream; and also widely used in cereal fermentation in Africa (Jay, 1986; Holzapfel 2002). Lactic acid is produced by the starter culture bacteria to prevent the growth of undesirable micro-organisms in common fermented products such as yogurt, (Ray and Daeschel 1992). 2.3.2 Yeasts According to Aidoo et al., (2006), a wide variety of yeasts are involved in traditional fermented foods and play vital roles in the production of these traditional fermented foods and beverages worldwide. The functions of yeasts in cereal fermented foods and beverages have been reported by several authors. These have been the production of aroma compounds through the conversion of carbohydrates into alcohols, esters, organic acids and carbonyl compounds, inhibition of mycotoxins producing moulds (nutrient completion), degradation of mycotoxins, production of tissue degrading enzymes (cellulases, pectinases) which make substrates available for other microorganisms and Probiotic properties (Jespersen, 2003; Kohajdova and Karovicova, 2007; Osmorio-Cadavid et al., 2008). Apart from Lactic Acid Bacteria, Saccharomyces cerevisiae is noted to be a predominant yeast species involved in food fermentation in Africa (Shetty et al., 2007). University of Ghana http://ugspace.ug.edu.gh 12 Species of yeast isolated during Fura fermentation were Issatchenkia orientalis (26%), Saccharomyces cerevisiae (22%), Pichia anomala (16%), Candida tropicalis (16%), Saccharomyces pastorianus (10%), Yarrowia lipolytica (6%), and Galactomyces geotricum (4%) Owusu-Kwarteng et al., (2010). Yeast species isolated from an ogi maize fermentation mix included Geotrichum fermentans, G. candidum, Rhodotorula graminis, Saccharomyces cerevisiae, Candida krusei, and C. tropicalis (Omemu et al., 2007). Kurtzman and Fell (1998), Pretorius (2000), Romano et al., (2006), and Tamang and Fleet (2009), have also reported about twenty one (21) major genera of functional yeasts species from fermented foods and beverages. 2.3.3 Moulds Moulds play a very minor role in fermented foods in Africa, but have however been found during fermentation of cereal based foods such as kenkey (Jespersen et al., 1994) and ogi (Banigo, 1993). Moulds of the genera Aspergillus, Rhizopus, Mucor, Actinomucor, Amylomyces, Neurospora and Monascus are used in the manufacture of fermented foods in Asia whiles in Europe, mould-ripened foods are primarily cheeses and meats, usually using a Penicillium-species (Leistner, 1990). Gari made by fermenting cassava slurry was found to contain Bacillus, Aspergillus and Penicillium spp. as the predominant organisms (Ofuya & Akpoti, 1988). Odunfa & Komolafe (1989) reported that the predominant micro-organisms present in dawadawa, a fermented condiment made in Ghana, after 24h of fermentation were Bacillus sp., with small numbers of Staphylococcus sp. (0.3%). After 36h of fermentation, Bacillus sp. (60%) and Staphylococcus sp. (34%) were isolated whiles after 48h fermentation 56% Bacillus sp. and 42% Staphylococcus sp. were isolated. University of Ghana http://ugspace.ug.edu.gh 13 2.4 FUNCTIONS OF STARTER CULTURES Starter cultures have been used to improve the quality and acceptability of many food products. The quality of sauerkraut was improved by the use of starter culture L. lactis ssp. Lactis and the organoleptic properties and expiration date of the final product of sauerkraut obtained by the use of lactic acid bacterium L. mesenteroides as a starter culture were also improved (Kristek et al., 2004). An improvement in the texture and quality of bread due to increase in the air cells, produced with Lactic Acid Bacteria as a starter culture, has been reported (Coda et al., 2008; Katina et al., 2002; Lavermicocca et al., 2000). New and better strains of A. oryzae introduced into soybean fermentation improved the process efficiency as well as the quality and consistency of the final product (Beuchart, 1995). Lactic acid bacteria, in particular Lactobacilli, is able to decrease pH, thus preventing the growth of pathogenic and spoilage microorganisms and therefore improve the hygienic safety and storage of meat products (Lucke, 1985; Samelis et al., 1994). The functions of Starter cultures for African fermented cereal products have been reported by several authors as enhancement in fermentation (Halm et al., 1996a & b; Hounhouigan et al., 1999; Mugula et al., 2003), improvement in the ability of reducing pathogens (Olukoya et al., 1994), reduction of anti-nutritional factors (Khetarpaul and Chauhan, 1989; Sharma and Kapoor, 1996; Murali and Kapoor, 2003), improve nutrition (Sanni et al., 1998 and 1999a,b), and the improvement of aroma properties (Annan et al., 2003 a,b). Holzapfel, (1997; 2002) reported the ability of Starter culture to: improve shelf-life; enhance inhibition or elimination of foodborne pathogens; improve sensory quality (taste, aroma, visual appearance, texture, consistency); reduce preparation procedures (reduction of cooking times and lower energy consumption); improve nutritional value (―upgrading‖) by University of Ghana http://ugspace.ug.edu.gh 14 degradation of antinutrition factors; improve protein digestibility and bio-availability of micronutrients as well as biological enrichment. 2.5 FACTORS TO CONSIDER IN SELECTING LACTIC ACID BACTERIA STARTER CULTURES FOR CEREAL FERMENTATION There are a number of technological properties that need to be measured when selecting Lactic Acid Bacteria strains for cereal fermentation depending on the desired characteristics of the final product, the desired metabolic activities, the characteristics of the raw materials and the applied technology (Soro-Yao et al. 2014). 2.5.1 Fast Acidification Food preservation by lactic fermentation depends on the removal of fermentable carbohydrates, the consumption of oxygen, the formation of organic acids in addition to a corresponding decrease in pH. Acidification may influence several quality characteristics of fermented product such as safety (Russell, 1992; Breidt and Fleming, 1997), reduction in fermentation time and organoleptic qualities (Mcfeeters, 2004). The immediate and rapid production of sufficient quantities of organic acids to reduce pH below 4.0 within 24 h of fermentation is an essential requirement of fermented cereal-based foods. The ability of L. fermentum to exhibit faster rates of acidification or pH reduction during spontaneous fermentation of many cereals has been confirmed (Sulma et al., 1991; Halm et al., 1993; Hounhouigan et al., 1993; Olsen et al., 1995; Sawadogo-Lingani et al., 2007). L. fermentum plays a major role in acidification by lowering pH, to create a favourable condition for the growth of yeasts during the alcoholic fermentation stage of dolo and pito wort fermentation (Sawadogo-Lingani et al., 2007). Acid production and decrease in pH results in an increase in sourness due to the metabolism of sugar leading to a probable decrease in sweetness. University of Ghana http://ugspace.ug.edu.gh 15 2.5.2 Good Antimicrobial properties The inhibitory properties of fermented foods are often considered based on their ability to reduce diarrhea and/or improve microbial quality and antimicrobial activity in vitro. The potential of fermented cereal gruels to reduce the incidence of diarrhoea in young children was demonstrated in Tanzania (Lorri and Svanberg, 1994). In a related studies, Motoho, a fermented sorghum porridge from Lesotho inhibited the survival of Shigella boydii, Salmonella typhi and Escherichia coli (Sakoane and Walsh, 1987). The ability of a fermented sorghum flour and porridge to inhibit the growth and survival of Salmonella typhimurium was also reported (Nout et al., 1987). The microbial antagonism of Lactic acid bacteria could be attributed to the production of organic acids, ethanol, diacetyl, hydrogen peroxide or carbon dioxide, alone or in combination, and could also result from the production of bacteriocins (De Vuyst and Vandamme 1994). The rapid production of these compounds may contribute to the inhibition of pathogenic or spoilage flora and therefore enhance the shelf life and microbial safety of the fermented product (Omemu & Faniran 2011; Okerere et al. 2012; Ekwem 2014). 2.7.3 Dominant population in the Indigenous Microbiota The ability of Lactic Acid Bacteria to dominate the indigenous microbiota during cereal dough fermentation has been related to its fast and predominant growth under fermentation conditions and/or its ability to produce antagonistic substances, such as bacteriocins. The use of molecular fingerprinting techniques such as Random Amplified Polymorphic DNA with Polymerase Chain Reaction (RAPD-PCR) and Pulsed-field Gel Electrophoresis (PFGE), to amplify the growth of a selected freeze-dried LAB starter culture during cassava fermentation for gari production has been reported (Huch et al., 2008). University of Ghana http://ugspace.ug.edu.gh 16 2.5.4 Good Probiotic Effects Microorganisms considered as feasible probiotics are mainly of the Lactobacillus genus with over one hundred species recognized, such as L. acidophilus, L. rhamnosus, L. reuteri, L. casei, L. plantarum, L. bulgaricus, L. delbrueckii, L. helveticus ( Krishnakumar and Gordon, 2001; Playne et al., 2003; Shah, 2007). Probiotic bacteria are very sensitive to many environmental stresses, such as acidity, oxygen and temperature (Heller, 2001; Parvez et al., 2006) and they must therefore be able to: adhere to the intestinal epithelium and colonize the lumen of the tract; stabilize the intestinal microbiota; counteract the action of harmful microorganisms; produce antimicrobial substances; stimulate host immune response (Parvez et al., 2006; Soccol et al., 2010). They prevent the growth of pathogenic microorganisms through competition, exclusion and the production of organic acid and antimicrobial compounds. Acid and tolerance are two fundamental properties that demonstrate the ability of probiotic microorganism to survive passage through the upper gastrointestinal tract (Soro-Yao et al. 2014). 2.5.5 Nutritional Quality of the Fermented Food The products made from millet, maize or/and sorghum dough contribute to the protein requirements of West African peoples and are particularly important as weaning foods for children and as dietary staples for adults (FAO 2012). Significant amounts of inositol hexaphosphates (IP6), known as phytic acid or phytates, anti-nutritional factors, are however found in the above mentioned cereals and therefore affect the bioavailability of minerals, leading to low bioavailability of minerals, a significant problem for child nutrition in West African countries (Camara and Amaro 2003). Tannins and α-galacto-oligo-saccharides (α-GOS) such as stachyose and raffinose are other anti-nutrients of importance in cereal grains. A phytase, α-galactosidase or tannase producing University of Ghana http://ugspace.ug.edu.gh 17 LAB is therefore useful during cereal dough fermentation to help decrease the amount of phytic acid or tannins and metabolise stachyose or raffinose, which have a greater influence on the nutritional quality of cereal grains. Moreover, the ability of Lactic Acid Bacteria strains to bind mycotoxins such as aflatoxin, which may form during the storage of cereal grains, should also be considered (Soro-Yao et al. 2014). Lactic acid fermentation also provides optimum pH conditions for enzymatic degradation of phytate, which is present in cereals in the form of complexes with polyvalent cations (such as iron, zinc, calcium, magnesium and proteins) (Coulibaly et al.2011). 2.5.6 Starch hydrolysis The energy density of cereal gruels could be increased with the use of amylolytic LAB to hydrolyse starch. (Songré-Ouattara et al. 2009). The level of carbohydrate, some non- digestible and oligosaccharides decrease during cereal fermentation ( Blandino et al., 2003). According to FAO/WHO (1995) amylolytic Lactic Acid Bacteria may reduce the viscosity of bulk starchy weaning gruel, to improve nutrient density and maintain an acceptable thickness for feeding young children. Amylolytic lactic acid bacteria have been isolated from cereal fermentation in tropical climates (Ga‖nzle et al., 2008, Sanni et al., 2002). Olasupo et al., (1996) isolated amylolytic lactic acid bacteria from Ghanaian kenkey (fermented maize dough) and nono (Nigeria). Agati et al., (1998), found amylolytic L plantarium strains from retted cassava in Nigeria and Congo respectively, while amylolytic L. fermentum strains were isolated from mawe and ogi in Benin. Hounhouigan et al., (1993b) reported some amylolytic lactic acid bacteria in mawe from Benin whiles Johansson et al., 1995 also indicated that amylolytic lactic acid bacteria accounted for 14 % of the total lactic acid bacteria isolated from Nigerian ogi. University of Ghana http://ugspace.ug.edu.gh 18 2.5.7 Exopolysaccharide Formation Many strains of Lactic Acid Bacteria produce exopolysaccharides (EPS) as capsules tightly attached to the bacterial cell wall, or as a loose slime (ropy polysaccharide) which is released into the substrate (Mayra-Makinen and Bigret, 1998). EPS could be composed of one type of sugar monomer (homopolysaccharides) or consist of multi type of monomers (heteropolysaccharides) and could be substituted organic or inorganic molecules (Broadbent et al., 2001). Heteropolysaccharides are produced by several species of Lactic Acid Bacteria (L. lactis ssp. lactis, Lb. delbrueckii ssp. bulgaricus, and S. thermophilus) whereas homopolysaccharides are produced by a few organisms such as Leu. mesenteroides. The production of expolysacharides (EPSs) have acquired a lot of attention due to their contribution to improvement of texture and viscosity of fermented food products (Savadogo et al., 2004). Since EPS have viscosity enhancing and stabilizing properties, exopolysaccharide-producing (EPS+) starter cultures are commonly used to enhance water binding and viscosity in yogurt and fermented milks. The ability of EPS+ starter pair, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to improve the moisture and melt properties of low fat Mozzarella cheese has been demonstrated (Broadbent et al., 2001). 2.6 STARTER CULTURES IN AFRICAN CEREAL FERMENTATION Recent production of fermented cereal based foods on a large scale depends almost entirely on the use of defined strains to replace the undefined strain mixtures traditionally used for the manufacture of these products (Klaenhammer and Fitzgerald, 1994). Lactic acid bacteria and yeasts strains have been used successfully as starter cultures in a number of indigenous cereal based fermented foods. These strains have been used as starter cultures in the various food products because of their desirable effects in such foods (Oyewole, 1990). These effects may be the ability to reduce fermentation times, minimize dry matter losses, avoid contamination University of Ghana http://ugspace.ug.edu.gh 19 with pathogenic and toxigenic bacteria and moulds, and reduce the risk of incidental micro- flora causing off-flavours in foods (Haard, 1999). The use of isolated strains during cereal dough fermentation has been reported to minimize dry matter losses, enhance the control over the fermentation step, enhance acid production or reduction in pH, contribute to aroma and taste formation, improve the overall acceptability of the product and enhance the nutritional quality of the product by producing preservative compounds or reduce mycotoxins (Hounhouigan et al. 1993; Halm et al., 1996; Annan et al., 2003b; Lardinois et al., 2003; Fandohan et al., 2005; Teniola et al., 2005; Agarry et al., 2010; Songré-Ouattara et al., 2010; Enwa et al., 2011; Ekwem 2014). Improvement in fermentations without losing other desirable traits or introducing accidentally, undesirable characteristics however remains the challenge (Annan et al., 2016). Below are some applications of starter cultures in some selected African foods. 2.6.1 Kisra [ Kisra is an indigenous staple food for the majority of Sudanese people. It is a pancake-like bread made from sorghum or millet flour. Kisra fermentation is a traditional process, whereby sorghum or millet flour is mixed with water in a ratio of about 1:2 (w/v), usually a starter is added by a back-slopping using mother dough from a previous fermentation as a starter at a level of about 10%. Fermentation is completed in about 12-19 hours by which time the pH drops from about six to less than four. Due to the tedious process of kisra preparation, most of the population abandoned kisra consumption and shifted to bread. A starter culture consisting of lactic acid bacteria (Lactobacillus fermentum, Lactobacillus brevis and Lactobacillus amylovorus) combined with Saccharomyces cerevisiae, on traditional fermentation of sorghum flour (variety dabar), was able to reduce fermentation time from 19 hours to 4 hours and the pH to 3.47(Asmahan and Muna, 2009). University of Ghana http://ugspace.ug.edu.gh 20 2.6.2 Ogi It is a fermented cereal gruel processed from maize, although sorghum and or millet are also employed as the substrate for fermentation. It is considered the most important weaning food for infants in West Africa although it is also consumed by adults (Banigo, 1993; Onyekwere et al., 1993). A mixed culture of Lactobacillus and Acetobacter improved the nutrient quality of ―Ogi‖ by increasing the concentrations of riboflavin and niacin beyond that found in both the unfermented grain and the traditionally spontaneous fermented ―Ogi‖ (Akinrele, 1970). Banigo et al., (1972) reported the ability of a combined inoculum of L. plantarum, Lactococcus lactis and Saccharomyces rouxii to increase the rate of souring of the dough in ―Ogi‖ production. Sanni et al., (1994), reported higher levels of ethanol in spontaneously fermented Nigerian ―Ogi‖ than those inoculated with lactic acid bacteria. Twelve and three- fold increases in lysine production were respectively observed in ―Ogi‖ when fifty mutants from L. plantarum and seven mutants from yeast strains selected from cultures capable of over producing lysine used were (Odunfa et al., 1994). Olukoya et al., (1994) demonstrated the potential of ―Dogik‖, an improved ―Ogi‖ produced from starter culture strains of lactobacilli isolated from local fermented foods with strong antibacterial activity to control diarrhea. A starter culture of L. plantarum reduced the pH from 5.9 to 3.4 within 12 h compared to 2-3 days required in the normal traditional process of ―Ogi‖ preparation (Sanni et al., 1994). Teniola and Odunfa (2001) observed high increases in levels of lysine and methionine in ―Ogi‖ prepared from dehulled maize grains inoculated with mixed starter cultures of Saccharomyces cerevisiae and Lactobacillus brevis. 2.6.3 Uji It is an East African sour porridge made from maize, millet or sorghum. Mbugua and Ledford, (1984) investigated the ability of pure lactic cultures isolated from naturally University of Ghana http://ugspace.ug.edu.gh 21 fermenting ―Uji‖ mash and pure cultures of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus delbruecki to ferment ―Uji‖. It was established that most bacterial strains failed to successfully ferment sterile or heat-treated ―Uji‖ slurries as demonstrated by poor acid formation. They ascribed this to the absence of symbiotic relationships in sterile media, usually present in mixed bacterial populations, as well as the destruction of thermolabile factors and changes in the isolated organisms during the sub-culturing process. ―Uji‖ fermented by mixed native ―Uji‖ bacteria was more organoleptically acceptable than isolated starter culture of L. bulgaricus or S. thermophilus. In a related study, Masha et al. (1998) studied the fermentation of ―Uji‖ using a starter culture of lactic acid bacteria (L. plantarum, L. brevis, L. buchneri, L. paracasei and Pediococcus pentosaceus), using backslopping and spontaneous fermentation at 30ºC and recorded a low pH of 3.5 with the lactic acid bacteria starter culture fermentation while viscosity of ―Uji‖ was only slightly affected by the spontaneous method of fermentation. They also found that the aroma profile of ―Uji‖ fermented with lactic acid bacteria recorded high concentrations of acids ( hexanoic, octanoic and nonanoic) and some alcohols ( 1-propanol, 1-hexanol, 1- nonanol and 2- undecenol), spontaneously fermented samples recorded high concentrations of esters ( ethyl butanoate, hexyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate and ethyl nonanoate), other alcohols ( ethanol, 1- butanol, 3-methyl- 1-butanol and 2-methyl- 1-propanol) and acids ( acetic and heptanoic acid), while the backslopping method of fermentation recorded low concentrations of all volatiles identified. Unfermented ―Uji‖ recorded mainly high levels of aldehydes (pentanal, hexanal, heptanal, nonanal, (E)-2- heptenal and (E)-2-octenal) and other compounds (2-heptanone, 2-pentyl furan, 1-octen-3-ol and isopropyl alcohol). University of Ghana http://ugspace.ug.edu.gh 22 2.6.4 Mawe Mawe is fermented dough made from maize and used to prepare several dishes such as koko. Hounhouigan et al., (1999) demonstrated the effectiveness of starter cultures of L. fermentum and L. brevis in fermenting sterile ―Mawe‖ suspensions to produce porridge with similar acidity levels as the naturally fermented ―Mawe‖. Starter cultures of only the yeasts, C. krusei and S. cerevisiae produced ―Mawe‖ with high pH (5.6 and 5.5 respectively) and low titratable acids expressed as percentage lactic acid (0.05 and 0.06, respectively). Results of sensory evaluation showed that the porridges produced with ―Mawe‖ fermented with starter cultures had less flavour than the traditional commercially produced ―Mawe‖ porridge. 2.6.5 Mahewu Mahewu (amahewu) is a non-alcoholic sour beverage made from corn meal, consumed in Africa and some Arabian Gulf countries (Chavan & Kadam, 1989). It is an adult-type of food, although it is commonly used to wean children (Shahani et al., 1983). It is prepared from maize porridge, which is mixed with water. Sorghum, millet malt or wheat flour is then added and left to ferment (Odunfa et al., 2001). The fermentation is a spontaneous process carried out by the natural flora of the malt at ambient temperature (Gadaga, et al., 1999). According to van der Merwe et al., (1964) the traditional spontaneously produced ―Mahewu‖ is considered undesirable since it involves a long fermentation time (about 36 h), proceeds too irregularly, permits the development of undesirable bacteria which results in undesirable off-flavours from secondary fermentations. A considerable research has therefore been carried out over the years on the use of starter cultures to produce ―Mahewu‖ of consistently high quality within relatively shorter times of 8 to 12 h (van der Merwe et al., 1964, Schweigart, 1970, Hesseltine, 1979). The most satisfactory acid producing starter culture was found to be L. delbruecki (van der Merwe et al., 1964). Schweigart (1971) observed the effectiveness of freeze or spray-dried ―Mahewu‖ cultures consisting mainly of L. delbruecki University of Ghana http://ugspace.ug.edu.gh 23 as starter culture for bulk fermentations. A lag phase of 8 h in contrast to 3 h with the use of fresh starter cultures was however observed. 2.7 LACTIC ACID BACTERIA AND THEIR USES IN FOOD Lactic acid bacteria are technologically important organisms recognized for their fermentative ability as well as their health and nutritional benefits (Gilliand, 1990) and are the most widespread of desirable microorganisms in food fermentation. They are found in fermented cereal products, milk, cheese and fermented meats (Campbell-Platt, 1987), converting the available carbohydrate to organic acids and lowering the pH of food, thereby making the food unfavourable for the growth of spoilage and pathogenic bacteria (Adams and Moss, 1995). They also produce various compounds such as organic acids, diacetyl, hydrogen peroxide, bacteriocins or bactericidal proteins during lactic fermentations (Lindgren and Dobrogosz, 1990; Pederson, 1971). Lactic acid bacteria species used for food fermentations belong to the genera Lactococcus, Streptococcus, Pediococcus, Leuconostoc, Lactobacillus, and the newly recognized Carnobacterium. Lactic acid bacteria have been applied commercially as starter cultures in the dairy, baking, meat, vegetable and alcoholic beverages industries, once used to retard spoilage and preserve foods through natural fermentations. In addition to their desirable effects on food taste, smell, color and texture, is the ability to inhibit undesirable microflora in the food. Lactic acid bacteria and their products therefore give fermented foods distinctive flavours, textures, and aromas while preventing spoilage, extending shelf-life, and inhibiting pathogenic organisms (Rattanachaikunsopon and Phumkhachorn, 2010). 2.8 CLASSIFICATION OF LACTIC ACID BACTERIA According to (Vandamme et al., 1996; Stiles and Holzapfel, 1997), Lactic Acid Bacteria fermentation are categorised as homofermentaters and heterofermentaters based on the products they form from glucose. The homofermenters convert glucose 1,6-diphosphate University of Ghana http://ugspace.ug.edu.gh 24 through Embden Meyerhof (EM) pathway. The enzyme aldolase cleaves fructose 1,6- diphosphate between C3 and C4 to give the phosphate esters dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The end product of this fermentation pathway is lactic acid (De Vries and Southamer, 1968). According to Hutkins (2006) however, LAB can be divided into three groups based on utilization of sugars as homofermentative, heterofermentative and facultatively heterofermentative. The Homofermentative genera (Pediococcus sp., Streptococcus sp., Lactococcus sp. and some lactobacilli) metabolize hexoses by enzymes of the glycolytic Embden-Mayerhoff pathway resulting in more than 90% of substrate being converted to lactic acid during anaerobic metabolism. The Heterofermentative genera (Weisella sp., Leuconostoc sp. and some Lactobacilli) metabolize hexoses via Warburg-Dickens (pentose phosphate) pathway resulting in the conversion of only 50 % of substrate to lactic acid, whiles the rest is metabolized to acetic acid, formic acid and ethanol. Facultative heterofermentative lactobacilli can metabolize hexoses through both pathways, with Warburg-Dickens pathway predominating in the deficiency of fermentable sugars. 2.9 CHARACTERIZATION AND IDENTIFICATION OF MICROORGANISMS IN FERMENTED FOODS 2.9.1 Phenotypic methods Phenotypic characterization of lactic acid bacteria includes morphological examinations as well as physiological and biochemical tests. Based on morphology, microscopic examination has been used as the first criteria that provide information about genus level, purity of lactic acid bacteria, whiles staining methods such as simple stain, gram stain, acid fast stain, endospore stain, capsule stain are used to differentiate the cells. The most important and widely used method is Gram staining. Bacteria can be divided into two large groups on the basis of the reaction to Gram stain, as Gram University of Ghana http://ugspace.ug.edu.gh 25 positive organisms and Gram negative organisms. Lactic acid bacteria belong to the Gram positive group. Rounded or spherical cells are called cocci; elongated rod shaped cells are called bacilli; ovoid cells, intermediate in shape between cocci and bacilli are called cocobacilli; cell division in two perpendicular directions in a single plane that lead to tetrad formation are called tetracocci (Garvie, 1984). With regards to Physiological and Biochemical Tests, lactic acid bacteria have been classified based on: Mode of glucose fermentation (homo or heterofermentation); Growth at certain cardinal temperatures (e.g. 10°C and 45°C); Range of sugar utilization (Stiles and Holzapfel, 1997); the methyl esters of fatty acids (Decallone et al., 1991) and the pattern of proteins in the cell wall (Gatti et al., 1997) or in the whole cell (Tsakalidou et al., 1994). In addition to the above, growth in different salt concentrations also provide differentiation, especially cocci shaped starter lactic acid bacteria. Furthermore, other characteristics which are arginine hydolysis, acetoin formation, bile tolerance, type of hemolysis, production of exopolysaccharides, growth factor requirements, presence of certain enzymes, growth characteristics in milk and serelogical typing are used for biochemical characterization. For example, L. lactis ssp. cremoris is distinguished from L. Lactis ssp. lactis by inability to grow at 40 ºC, growth in 4% salt, hydrolyse arginine, and ferment ribose (Axelson, 1998). Bottazzi (1988) classified LAB on six physiological tests which included production of gas from glucose, hydrolysis of arginine, growth and survival at 15, 45 48, 60 and 65 °C and tolerance of 4, 6, and 8 % NaCl. The use of rapid identification systems, such as the API (API systems S.A., La Balme Les Grotte, Montalieu, France) have been used to examine isolates based on different carbohydrate fermentation characteristics. University of Ghana http://ugspace.ug.edu.gh 26 According to William and Sandler (1971) and Morelli (2001) however, phenotypic methods are not completely accurate. Phenotypic methods of microbial identifications thus have essential limitations such as poor reproducibility, the ambiguity of some techniques (largely resulting from the plasticity of bacterial growth), the extensive logistics for large-scale investigations and their poor discriminatory power. Addition to the above is the fact that the whole information potential of a genome is never expressed. All these disadvantages poorly affect the reliability of phenotype-based methods for culture identification at the genus or species level. 2.9.2 Genotypic methods Genotypic microbial identification methods are broken into two broad categories as: pattern- or fingerprint based techniques and sequence-based techniques. In the Pattern-based techniques, a series of fragments are produced from an organism's chromosomal DNA by typically using a systematic method. The fragments are then separated by size to generate a profile, or fingerprint that is unique to that organism and its very close relatives. With enough of this information, researchers can create a library, or database, of fingerprints from known organisms, to which test organisms can be compared. When the profiles of two organisms match, they can be therefore considered very closely related, usually at the strain or species level. For instance, DNA fingerprints of thermophilic lactic acid bacteria generated by repetitive sequence based polymerase chain reaction have been applied (Uriaza et al., 2000). In the sequence-based techniques, the sequence of a specific stretch of DNA, usually, but not always, associated with a specific gene, is determined. In general, the approach is the same as for genotyping: a database of specific DNA sequences is generated, and then a test sequence is compared with it. The degree of similarity, or match, between the two sequences is a measurement of how closely related the two organisms are to one another. A number of University of Ghana http://ugspace.ug.edu.gh 27 computer systems have been created that can compare multiple sequences to one another and build a phylogenetic tree based on the results (Ludwig and Klenk, 2001). Traditionally, sequence-based methods, such as analysis of the 16S rRNA gene, have proved effective in establishing broader phylogenetic relationships among bacteria at the genus, family, order, and phylum levels, whereas fingerprinting-based methods are good at distinguishing strain- or species-level relationships, but are less reliable for establishing relatedness above the species or genus level (Vandamme et al., 1996). The combination of these methods with other phenotypic tests however creates a polyphasic approach that is the standard for describing new bacterial species (Gillis et al., 2001). For instance, Hebert et al., (2000) characterized natural isolates of Lactobacillus by respectively, physiological and biochemical test, SDS- PAGE of whole cell proteins, and sequencing of variable region (V1) of the 16S ribosamal DNA. In a related study, lactic acid bacteria from artisanal Italian cheese were characterized by combination of PCR 16S-23S rDNA and sequencing, coupled with phenotypic methods such as salt tolerance, growth at different temperatures and gas production from glucose (Ayad, et al., 2001). 2.10 ANTIMICROBIAL COMPOUNDS PRODUCED BY LACTIC ACID BACTERIA A range of antimicrobial metabolites are produced during the fermentation process which give rise to the preservative action of starter cultures in food and beverage systems(Ross et al., 2002). These consist of many organic acids such as lactic, acetic and propionic acids produced as end products responsible for an unfavourable acidic environment for the growth of many pathogenic and spoilage microorganisms. Jay, (1982); Piard and Desmazeaud, (1992) also reported that LAB produce various antimicrobial compounds, which can be classified as low-molecular-mass compounds such as hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione), University of Ghana http://ugspace.ug.edu.gh 28 uncharacterized compounds, and high-molecular-mass, HMM) compounds such as bacteriocins, all of which can antagonize the growth of some spoilage and pathogenic bacteria in foods. According to Buckenhuskes (1993); Brinkten et al., (1994) and Olasupo et al., (1995), the antimicrobial-producing LAB may be used as protective cultures to improve the microbial safety of foods and they also play an important role in the preservation of fermented foods, which is usually achieved by inhibition of contaminating spoilage bacteria such as Pseudomonas and pathogens such as Staphylococcus aureus, Salmonella spp. and Listeria monocytogenes. Reis et al., (2012) attributed the antimicrobial properties of LAB to competition for nutrients and the production of one or more antimicrobial active metabolites such as organic acids (mainly lactic and acetic acid), hydrogen peroxide and also other compounds, such as bacteriocins and antifungal peptides by the LAB. Breidt and Fleming (1997) attested the ability of Lactobacillus species to produce a range of metabolites, such as lactic and acetic acids which lower pH, and inhibit competing bacteria, including psychrotrophic pathogens. Adams (1990) also proposed that lactic acid bacteria are inhibitory to many other microorganisms when cultured together, and related it to the shelf life extension and improved microbiological safety of lactic-fermented foods. 2.10.1 Organic Acids and Low pH Lactic acid fermentation is characterized by the accumulation of organic acids and its associated reduction in pH (Berry et al., 1990). Daeschel, 1989, reported the production of lactic acid and reduction of pH as the primary antimicrobial effect exerted by LAB. The species of organisms, culture composition and growth conditions determines the levels and types of organic acids produced during the fermentation process (Lindgren and Dobrogosz 1990), whiles the antimicrobial effect of organic acids largely depends on the reduction of pH and the undissociated form of the molecules (Gould 1991, Podolak et al., 1996). University of Ghana http://ugspace.ug.edu.gh 29 According to Kashket (1987), the undissociated acid is lipophilic and therefore diffuses passively across the membrane as the low external pH causes acidification of the cell cytoplasm. Thus acids apply their antimicrobial effect by interfering with the maintenance of cell membrane potential, inhibiting active transport, reducing intracellular pH and inhibiting a variety of metabolic functions (Ross et al., 2002) For instance, according to Woolford, 1975, Lactic acid in equilibrium with its undissociated and dissociated forms, is the major metabolite of LAB fermentation and the extent of the dissociation depends on pH, where a large amount of lactic acid is in the undissociated form at low pH, and this is toxic to many bacteria, fungi and yeasts. Different microorganisms however vary considerably in their sensitivity to lactic acid. At pH 5.0 for instance, lactic acid was inhibitory toward spore-forming bacteria but was ineffective against yeasts and moulds. Acetic and propionic acids produced by LAB strains, may also interact with cell membranes, and cause intracellular acidification and protein denaturation (Huang et al., 1986) and are more antimicrobially effective than lactic acid due to their higher pKa values and higher percent of undissociated acids than lactic acid at a given pH (Earnshaw 1992). The inhibitory potential of acetic acid was demonstrated to be higher towards Listeria monocytogenes than that of lactic and citric acids (Richards et al., 1995) as well as towards the growth and germination of Bacillus cereus (Wong and Chen 1988). Organic acids have a very wide mode of action and inhibit both gram-positive and gram- negative bacteria as well as yeast and moulds (Caplice and Fitzgerald, 1999) 2.10.2 Hydrogen Peroxide Starter strains can also produce a range of other antimicrobial metabolites such as H2O2, produced using such enzymes as the flavo protein oxidoreductases NADH peroxidase, NADH oxidase and α-glycerophosphate oxidase, which can have a strong oxidizing effect on membrane lipids and cellular proteins (Codon, 1987) by destroying the basic molecular University of Ghana http://ugspace.ug.edu.gh 30 structure of bacteria cell protein (Lindgren and Dobrogosz 1990). The inhibition of Staphylococcus aureus, Pseudomonas sp. and various psychotrophic microorganisms in foods due to the production of H2O2, by Lactobacillus and Lactococcus strains has been reported by Davidson et al. 1989; and Cords and Dychdala, 1993. 2.10.3 Carbon dioxide Carbon dioxide is produced by heterolactic fermentation. Carbon Dioxide has an antimicrobial activity due to the fact that it creates partial pressure (Lindgren and Dodrogosz, 1990) and may also create an anaerobic environment which inhibits enzymatic decarboxylations, and it‘s accumulation in the membrane lipid bilayer may cause a dysfunction in permeability (Eklund, 1984; De Vuyst and Vandamme, 1994). The effectiveness of CO2 to inhibit the growth of many food spoilage microorganisms, especially Gram-negative psychrotrophic bacteria has been reported (Farber 1991, Hotchkiss 1999). The degree of inhibition by CO2 however varies considerably between the organisms. Wagner and Moberg (1989) reported the ability of CO2 at 10% to lower the total bacterial counts by 50% and also a strong antifungal activity at 20-50% CO2 (Lindgren and Dobrogosz 1990). 2.10.4 Diacetyl [ Diacetyl ((2, 3-butanedione) is a product of citrate metabolism (Lindgren and Dobrogosz, 1990; Cogan and Hill, 1993) via pyruvate where it is further metabolized anaerobically and aerobically to diacetyl and acetoin (De Vuyst and Vandamme 1994) and may be produced by strains of Leuconostoc, Lactococcus, Pediococcus and Lactobacillus (Jay, 1982; Cogan, 1986). According to Jay (1986), diacetyl reacts with the arginine-binding protein to disturb the arginine utilization and consequently inhibit the growth of Gram- negative bacteria. It was demonstrated by Jay (1982) that Gram-negative bacteria were more sensitive to diacetyl than Gram positive bacteria; whereas Gram negative were inhibited by diacetyl at 200 µg/mL, Gram positive bacteria were inhibited at 300 µg/mL. Strains of University of Ghana http://ugspace.ug.edu.gh 31 Listeria, Salmonella, Yersinia, Escherichia coli, and Aeromonas were however inhibited by diacetyl at 344 µg/mL. 2.10.5 Bacteriocins Several strains of LAB associated with foods produce bacteriocins, which are proteinaceous compounds with activity against related species. They are ribosomally-synthesized peptides or proteins secreted by certain strains of bacteria. The growth rate and/or survival of pathogens may be affected by the antagonistic activity of LAB depending on the type and the concentration of bacteriocin. Most bacteriocins kill target cells by permealization of the cell membrane, and the activity is often very specific, since they employ specific receptors on the target cell surfaces (Kjos et al., 2011). De Vuyst and Vandamme (1994) define bacteriocins as bioactive peptides or proteins, active against Gram-positive bacteria and usually against species closely related to the producer strain. A number of bacteriocin producing LAB have been isolated from various traditional spontaneous fermented foods (Todorov and Dicks, 2006; Sanni et al., 1999; Olsen et al., 1995 and Olukoya et al., 1993). Both Gram-positive and Gram-negative bacteria were inhibited by bacteriocins produced by strains of Lb. reuteri and Pd. acidilactici isolated from fura; the former were however generally more susceptible than the latter (Owusu-Kwarteng et al., 2012). University of Ghana http://ugspace.ug.edu.gh 32 Table 2.2 Classes of bacteriocins Produced by Lactic Acid Bacteria. Class Subclass Description Class I Labntibiotics Class II Small( <10kDa),heat stable, non-lanthionine containing membrane-active peptides II a Listeria- active peptides II b Two- peptide bacteriocins II c Thiol-activated peptides Class III Large (>30 kda) heat-labileproteins Class IV Complex bacteriocin:protein with lipid and /or Carbohydrate Table 2.3 Antimicrobial substances produced by lactic acid bacteria Antimicrobial substance Main target organism Organic acids Lactic acid Acetic acid Putrefactive and Gram-negative bacteria, some fungi Putrefactive bacteria, clostridia, some yeasts and some fungi Hydrogen peroxide Pathogens and spoilage organisms, especially in protein rich foods Enzymes Lactoperoxidase system with hydrogen peroxide Lysozyme (by recombinant DNA) Pathogens and spoilage bacteria (milk and diary products). Undesired Gram-positive bacteria Low-molecular-weight metabolites Reuterin Wide spectrum of bacteria, yeasts, and molds University of Ghana http://ugspace.ug.edu.gh 33 Diacetyl Fatty acids Gram-negative bacteria Different bacteria Bacteriocins Nisin Some LAB and Gram-positive bacteria, notably endospore-formers Other Gram-positive bacteria, inhibitory spectrum according to producer strain and bacteriocin type Source: Breidt & Fleming (1997) 2.11 FOOD IRRADIATION Food irradiation according to the Codex Alimentarius Commission (2003), is the processing of food products by the use of ionising radiation in order to control foodborne pathogens, reduce microbial load and insect infestation, inhibit the germination of root crops, and extend the durable life of perishable produce. Further applications include delay of ripening, increase of juice yield, sprout inhibition and improvement of rehydration. Irradiation is also effective on non-food items, such as medical hardware, plastics, tubes for gas pipelines, houses for floor heating, shrink-foils for food packaging, automobile parts, wires and cables (isolation), tires, and even gemstones. Irradiation process also helps in reduction of spoilage bacteria, insects and parasites. The Food and Drug Administration has approved irradiation as an effective food quality technique for preservation and increasing storage life of meat, fresh fruits, vegetables and spices. Irradiation process is also used in certain fruits and vegetables for delaying and inhibiting sprouting and ripening processes. The effects of irradiation on the food and on animals and people eating irradiated food have been studied extensively for more than 40 years and clearly demonstrates that irradiation process is approved for application on foods. The process has proved to be very efficient in the prevention of many food borne diseases and University of Ghana http://ugspace.ug.edu.gh 34 intoxications, and also provides consumers with wholesome and nutritious food items (Ganguly et al, 2011). 2. 12 SOURCES OF IONISING RADIATION The Codex General Standard for Irradiated Foods has recommended three major sources of ionizing radiations for use in food processing. These are gamma rays produced from cobalt- 60 (60Co) and cesium-137 (137Cs); machine sources generated electron beams having a maximum energy of 10 MeV; and X-ray with a maximum level of 5 MeV(Codex Alimentarius Commission, 2003). Both Cobalt-60 and Cesium-137 emit highly penetrating gamma rays that can be used to treat food in bulk or in its final packaging. Whereas Cobalt-60 is produced in a nuclear reactor via neutron bombardment of highly refined cobalt-59 (59 Co) pellets, cesium- 137 is produced as a result of uranium fission. Cobalt-60 is, at present, the most commonly employed radioisotope for gamma irradiation of food. 2.12.1 Gamma rays [ According to Suresh et al., (2005), Cobalt-60 emits Gamma rays with energies of 1.17 and 1.33 MeV whiles energy of 0.66 MeV is emitted by Caesium-137. The 60Co is a radioactive metal that decays with a half-life of around 5.3 years whiles Caesium-137 has a half-life of around 30.1 years. Few commercial gamma facilities however use 137Cs as a gamma ray source in spite of the fact that it has a longer half-life, due to the fact that it emits gamma rays that are approximately half the energy of those emitted by 60Co. 2.12.2 Electron-beam machines Electron-beam machines are powered by electricity and use linear accelerators to produce accelerating electron beams to near the speed of light. The high-energy electron beams have limited penetration power and are suitable only for foods of relatively shallow depth. They do not make use of any radioactive substance in the processing system (Stewart, 2001). University of Ghana http://ugspace.ug.edu.gh 35 2.12.3 X-rays X-rays caused by atomic transition are generated by machines through bombardment with a metallic target into various energies. X-rays have been shown to be more penetrating than gamma rays from cobalt-60 and cesium-137(Stewart, 2001). However, the efficiency of conversion from electrons to X-rays is generally less than 10% and for that matter the use of machine sourced radiation is minimal (ICGFI, 1999). Table 2.4 Major irradiation technologies—advantages and disadvantages FACTORS Electron beam X ray Gamma SOURCE Electric power Electric power Radioisotopes Electrons are generated using electronics and accelerated to high energy using magnetic fields, 10MeV. When accelerator is powered off, no radiation is emitted Created when high- energy electrons (up to 5MeV) strike a metal plate (e.g., tungsten or tantalum alloys); typical conversion efficiency is 5-10%. When accelerator is powered off, no radiation is emitted Radioactive decay of Cobalt-60 (2.5 MeV) or Cesium 137 (0.51 MeV). Radioisotope source is always emitting radiation— shielding of source must be the default position. MECHANISM High energy electrons High energy photons High energy photons cleave water molecules, creating oxygen and hydroxyl radicals that damage Stimulate atoms within target to release high- energy electrons, which cleave water molecules into radicals. Direct cleavage of DNA also stimulate atoms within target to release high- energy electrons, which cleave water molecules into University of Ghana http://ugspace.ug.edu.gh 36 DNA, membranes. Direct cleavage of DNA also occurs occurs. radicals. Direct cleavage of DNA also occurs. SPEED Seconds Seconds Minute(depending on source strength ) PENETRABILITY 6-8cm, suitable for relatively thin or low-density products 30-40cm, suitable for all Products 30-40cm, suitable for all products INFRASTRUCTURE REQUIRED Shielding: > 2m concrete or < 1m steel/iron! lead Cooling: extensive for high-voltage electronics and accelerator Ventilation: for ozone removal while unit is operating Shielding:>2m m concrete or < 1m steel/iron/lead Cooling: extensive for high-voltage electronics and accelerator additional cooling systems required for plate target Ventilation: for ozone removal while unit is operating Shielding: Depending on design, > 5m water or > 2m concrete or < 1m steel/iron/lead. Cooling: moderate for control equipment Ventilation: at all times for ozone removal when source is exposed to air Niemira and Fan (2009). 2.13 APPLICATION OF FOOD IRRADIATION 2.13.1 Reduction of pathogenic microorganisms Irradiation is given the term cold pasteurization due to the fact that it does not substantially raise the temperature of food under irradiation. It is therefore used for the control of food- borne illnesses in seafood, fresh produce, and frozen meat products. Ionising radiation has University of Ghana http://ugspace.ug.edu.gh 37 been shown to reduce the number of disease-causing bacteria such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, Clostridium botulinum, Vibrio parahaemolyticus, in various food commodities and allow food to be irradiated in its final packaging. Irradiation alone may however not be sufficient to reduce the number of food poisoning outbreaks, it is essential to adhere to good manufacturing practice to prevent subsequent contamination during processing (Centre for Food Safety, 2009). 2.13.2 Decontamination Spices and condiments naturally contain a great number of microorganisms which originate in developing countries where harvest and storage conditions are insufficiently controlled. Accordingly, spices and condiments could be contaminated by a high level of mesophylic, sporogenic, and asporogenic bacteria, hyphomycetes, and faecal coliforms. Microorganisms of public health significance such as Salmonella, Escherichia coli, Clostridium perfringens, Bacillus cereus, can also be present (Bendini et al., 1998). Most spices and herbs were fumigated until the early 1980s, usually with sterilizing gases such as ethylene oxide and methyl bromide to destroy contaminating microorganisms. The use of these fumigants has however been banned in a number of countries due to their safety and environmental concerns. The use of ethylene oxide has been banned in many countries whiles methyl bromide is being phased out globally (Marcotte, 2005). Irradiation has therefore emerged as a feasible alternative method widely used in the food industry for the decontamination of dried food ingredients by considering its antimicrobial activity and relatively minor effects on quality (Sádecká et al., 2005; Farkas, 2001). The effectiveness of radiation treatment against bacteria has been confirmed to be more than thermal treatment, coupled with the fact that it is also less harmful to the spices than heat sterilization, which involves the loss of thermo labile University of Ghana http://ugspace.ug.edu.gh 38 aromatic volatiles and/or causes additional thermally induced change (Loaharanu, 1994; Alam Khan, 2010). 2.13.3 Extension of shelf-life Irradiation treatment can be used to considerably extend the shelf-life of many fruits and vegetables, meat, poultry, fish and seafood (ICGFI, 1999). Low doses of radiation may be used to extend the shelf life of fruits and vegetables by delaying ripening, inhibiting the growth of mould and preventing sprout (CDC, 2007; Niemira and Fan, 2006). The application of a low dose radiation to slow down the ripening of bananas, mangoes and papaya, control fungal rot in strawberries and inhibit sprouting in potato tubers, onion bulbs, yams and other sprouting plant foods has been demonstrated (Thomas, 2001a; Thomas, 2001b). This is achieved by modifying the normal biological changes associated with ripening, maturation, sprouting, and aging (WHO and FAO, 1988). 2.13.4 Disinfestation Insect infestation is the major problem encountered in preservation of grains and grain products. Irradiation has been shown to be an effective pest control method for these commodities and a good alternative to methyl bromide, the most widely used fumigant for insect control, which is being phased out due to its ozone depleting properties. Disinfestation is intended for preventing losses caused by insects in store grains, pulses, flour, cereals, coffee beans, fresh and dried fruits, dried nuts, and other dried food products including dried fish. It is however important to note that proper packaging of irradiated products is necessary for preventing re-infestation of insects (ICGFI, 1999; Ahmed, 2001). Irradiation (as a pest control method) has some advantages such as the absence of undesirable residues in the foods treated, no resistance development by pest insects and few significant changes in the physicochemical properties or the nutritive value of the treated products (Ahmed, 2001). Studies on the use of irradiation (as an approved method) to control stored- University of Ghana http://ugspace.ug.edu.gh 39 product pests in wheat, flour and dry legume seeds in many countries have been reported (Azelmat et al., 2005; Boshra & Mikhaiel, 2006). Table 2.5 Uses of Various Doses of Irradiation for Food Safety and Preservation Purpose Effective Dose range(KGy) Product Low dose(up to 1 KGy) (a) Inhibition of sprouting 0.06-0.2 Potatoes, onions, garlic, gin ger root, chestnut, etc. (b) Insect disinfestation (including quarantine treat ment) 0.15-1.0 Cereals and legumes, fresh and dried fruits, dried fish and meat, etc. (c) Parasite disinfection 0.3-1.0 Fresh pork, freshwater fish, fresh fruits. (d) Delay of ripening 0.5-1.0 Fresh fruits. Medium Dose (1-10 kGy) (a) Extension of shelf-life 1-3 Raw fish and seafood, fruits and vegetables. (b) Inactivation of spoilage and pathogenic bacteria 1-7 Raw and frozen seafood, meat and poultry, spices and dried vegetable seasonings. (c) Improving technical 3-7 Increasing juice yield University of Ghana http://ugspace.ug.edu.gh 40 Source: Loaharanu, 2003. properties of foods (grapes), reducing cooking time (dehydrated vegeta bles) High Dose (above 10 kGy) (a) Decontamination of certain food additives and Ingredients 10-50 Spices, enzyme preparations, natural gum, gel, etc. (b) Industrial sterilization (in combination with mild heat) 30-50 Meat, poultry, seafood, sausages, prepared meals, hospital diets, etc. University of Ghana http://ugspace.ug.edu.gh 41 CHAPTER THREE MATERIALS AND METHODS 3.1 Study area and design Two processing sites each from Nima and Dome in the Accra metropolis were used in the present study. A brief field study involving informal interaction with producers, consumers and vendors was carried out. Processing procedures were also observed and two experienced processors were selected from each area for collection of samples. All data was statistically analyzed using the SPSS Software. A survey was carried out to confirm the processing steps documented by literature. 3.2 Sample Collection and Preparation Samples were aseptically collected from the four processors at the various stages of processing millet into Fura. The samples collected were steep water at interval of 0, 6 and 12 h of steeping and fermenting dough at 0, 6 and 12 h of dough fermentation. Samples were transported in an ice chest with ice packs to the CSIR-Food Research Institute‘s microbiology and chemistry laboratories in Accra for microbiological and chemical analyses respectively. 3.3 Chemical Analysis 3.3.1 Determination of pH The pH of steep water was determined directly using a pH meter (Radiometer pHM 92. Radiometer Analytical A/S, Bagsvaerd, Denmark) after calibration using standard buffers, and fermenting dough was determined after blending with distilled water in a ratio of 1:1. University of Ghana http://ugspace.ug.edu.gh 42 3.3.2 Determination of Titratable Acidity [For each sample (steep water and fermenting dough) 10 ml or 10 g of sample was made up to 200 ml with distilled water and 80 ml titrated against 0.1 m NaOH using 1 % freshly prepared phenolphthalein as indicator as described by Amoa-Awua et al., (2006). 3.4 MICROBIOLOGICAL ANALYSIS 3.4.0 Enumeration of microorganisms 3.4.1 Homogenization and Serial Dilution Ten grams (10 g) of sample was added to 90.0 ml sterile Salt Peptone Solution (SPS), which was prepared with 0.1% peptone and 0.85% NaCl, pH adjusted to 7.2 and homogenized in a stomacher (Lad Blender, Model 4001, and Seward Medical). For liquid samples, 1ml was added to 9ml SPS. After homogenizing for 30 s at normal speed, ten-fold dilutions were prepared. The homogenate was serially diluted (1:10) and 1 ml aliquots of each dilution were directly inoculated into Petri dishes and the appropriate isolation media added. All analyses were done in duplicate. 3.4.2 Enumeration of Aerobic Mesophiles In accordance with the Nordic Committee on Foods Analysis Method (NMKL. No. 86, 2006), aerobic mesophiles were enumerated by the pour plate method using Plate Count Agar (Oxiod CM325; Oxoid Ltd., Basingstoke, Hampshire, UK). Plates were incubated at 30°C for 72 h. 3.4.3 Enumeration and Confirmation of Total Coliforms Total coliforms were enumerated by the pour plate method using Trypton Soy Agar (Oxoid CM131), pH 7.3 overlaid with Violet Red Bile Agar (Oxoid CM107), pH 7.4 and incubated at 37°C for 24 h. Confirmation of colonies was done using Brilliant Green Bile Broth (Oxoid CM31), pH 7.4 and incubated at 37°C for 24 h as described by NMKL. No. 44, ( 2004). University of Ghana http://ugspace.ug.edu.gh 43 3.4.4 Enumeration of Lactic Acid Bacteria Enumeration of Lactic acid bacteria was done by the pour plate method using deMan, Rogosa and Sharpe medium (MRS, Oxoid CM361) agar (De Man et al., 1960) with pH 6.2. 0.1% cycloheximide supplement was added to suppress yeast growth and Cystein HCl to achieve anaerobic conditions during incubation without having to use Anaerocult A. The plates were incubated anaerobically in an anaerobic jar at 30°C for 120 h. 3.4.5 Enumeration of Yeasts Enumeration of Yeasts and Moulds was done by the pour plate method using Oxytetracycline-Glucose Yeast Extract Agar (Oxoid CM545; Oxoid Ltd., Basingstoke, Hampshire, UK) to which OGYEA supplement was added to inhibit bacteria growth. The pH was adjusted to 7.0 and incubated at 25°C for 120 h in accordance with ISO 7954 (1987). 3.4.6 Isolation of Lactic Acid Bacteria About 20 colonies of lactic acid bacteria were selected from a segment of the highest dilution or suitable MRS agar plate. The colonies were sub-cultured into the corresponding broth medium and streaked repeatedly on agar until pure colonies were obtained. 3.4.7 Isolation of Yeasts About 15 colonies were selected from a segment of the highest dilution or suitable plate of yeast colonies on OGYEA and examined by microscopy, purified by successive sub culturing in Malt Extract Broth (Oxoid CM57) and streaking on Malt Extract Agar (Oxoid CM59) pH 5.4 until pure colonies were obtained. 3.5 CHARACTERISATION OF LAB ISOLATES 3.5.1 Characterization of Lactic Acid Bacteria Isolates by Gram Reaction Gram reaction was determined using 3% freshly prepared potassium hydroxide solution as described by Gregersen (1978). The tip of a cover slip was used to pick a pure colony of LAB University of Ghana http://ugspace.ug.edu.gh 44 and added to a drop of potassium hydroxide solution on a slide. The colony was mixed thoroughly with the solution using the cover slip and drawn for the production of slime. Formation of a slime indicated Gram negative reaction and non-slimy reaction indicated Gram positive reaction. 3.5.2 Characterisation of Lactic Acid Bacteria Isolates by Catalase Reaction A drop of 3% freshly prepared hydrogen peroxide solution was placed on a clean glass slide and a single colony of the pure culture picked and emulsified. This was then observed for bubbles or effervescence resulting from the liberation of free oxygen as gas bubbles. This indicated the presence of the enzyme catalase in the culture and vice versa. 3.5.3 Oxidase Test Oxidase test was done using Identification Sticks (Oxoid Ltd., Basingstoke, Hampshire, UK) by smearing the sticks on pure colonies and observing for colour change. Positive results were achieved by purple colouration. 3.5.4 Microscopic Examination Cell shape and arrangements were determined using the phase contrast microscope and the wet mount technique. A drop of sterile distilled water was placed on a clean slide and a small amount of the pure culture emulsified in it. A cover slip was placed on it and examined under the microscope using the X40 magnification and oil immersion using the X100. 3.5.5 Growth at Different Temperatures Two tubes containing MRS broth (Oxoid CM359) were inoculated with pure colony mass of the test organism and incubated at 10°C and 45°C respectively for 72-96 h. Growth in the tubes were determined by visual turbidity after the incubation period. This was done for all the isolates University of Ghana http://ugspace.ug.edu.gh 45 3.5.6 Salt Tolerance Test Two tubes with MRS broth (Oxoid CM359) containing 6.5% and 18% (w/v) NaCl were inoculated with pure colony mass of the test organism and incubated at 30°C for a period of 4 days. This was done for all the isolates and the tubes observed for growth of the inocula after the incubation period. 3.5.7 Growth at Different pH MRS broth (Oxoid CM359) with pH adjusted to 4.4 and 9.6 were inoculated with pure colony mass of the test organism and incubated at 30°C for 72-96 h. Growth was determined by visual turbidity after the incubation period. 3.5.8 Identification of Lactic Acid Bacteria Isolates were tentatively identified by determining their pattern of carbohydrate fermentation using the API 50 CH (BioMérieux, Marcy-l‘Etoile, France) and compared to the API database. 3.6.1 Macroscopic and Microscopic Examination of Yeast Colonies on solid media were examined macroscopically for colonial morphology. Characteristics described included colour, surface, size, form, margin, and elevation. Cultures were also observed microscopically as wet mounts for cellular morphology. 3.6.2 Identification of Yeast Isolates [ Isolates were identified by determining their pattern of fermentation and assimilation of various carbohydrates using ID 32 C galleries (BioMérieux, Marcy-l‘Etoile, France). 3.7 Antimicrobial Studies [ The inhibitory potential of lactic acid bacteria cultures was investigated using the Agar Well Diffusion method as described by Schillinger and Lücke (1989) and Olsen et al., (1995). The University of Ghana http://ugspace.ug.edu.gh 46 appropriate agar was poured into Petri dishes and allowed to solidify and dry for 1-2 days. Circular wells were made in the agar using sterile cork borers. Seven cultures of lactic acid bacteria isolated at different stages of Fura fermentation were each cultured in MRS broth (Oxoid CM359) at 30°C for 24 h. A volume of 0.1 ml of the cultures was transferred into wells and left to diffuse into the agar for approximately 4-5 h. The wells were overlaid with about 10 ml of the appropriate soft agar (0.7% agar) containing the indicator strains which were prepared by adding 0.25 m1 of 10-1 dilution of an overnight culture of the indicator organism to 10 ml of MRS agar (MRS, Oxoid CM361), for lactic acid bacteria, and malt extract agar for the yeast isolates. 3.8 TECHNOLOGICAL PROPERTIES OF IDENTIFIED LACTIC ACID BACTERIA 3.8.1 Rate of Acidification of Millet Dough by LAB Fermentation trials were carried out using six dominant LAB cultures identified earlier during steeping and dough fermentation of millet. Dried millet grains were milled into flour, sealed in clear polyethylene bags and irradiated with a dose of 10kGy. Hundred grams (100g) of the flour was then mixed with 75 ml (1:0.75w/v) sterile water of pH 7.0 and kneaded into dough. The lactic acid bacteria cultures used as inoculum was prepared from a 16 h culture incubated at 30ºC and 0.1ml of the culture transferred into sterile SPS and diluted to a concentration of 107cfu/ml. This was checked by microscopic counting using a Thomas counting chamber and by plating out on MRS agar. Six different batches of flour were kneaded into dough and fermented respectively, with one isolate inoculated into each batch. The mixture was shaken to obtain uniform distribution, and left at room temperature to ferment for 12h. 10 g of dough was aseptically collected for determination of pH and titratable acidity at 0-4h, 4-8h and 8-12h designated as 0h, 4h, 8h and 12h respectively. One batch of the dough was not inoculated and was used as control (spontaneous fermentation). University of Ghana http://ugspace.ug.edu.gh 47 3.8.2 Production of Exopolysaccharides (EPS) by LAB Isolates Screening of isolates for EPSs production was carried out according to Guiraud (1998). Isolates cultured on MRS agar were streaked onto LTV agar [0.5 % (w/v) tryptone (Difco), 1 % (w/v) meat extract (Fluka, Biochemika, Chemie GmbH, Buchs, Switzerland), 0.65 % (w/v) NaCl (Sigma), 0.8 % (w/v) potassium nitrate (Merck, KgaA), 0.8 % (w/v) sucrose (PA Panreac Guimica SA, Barcelona, Espana), 0.1 % (v/v) Tween 80 (Merck), 1.7 % (w/v) agar (Sigma), pH 7.1±0.2] and incubated at 30ºC for 48 h. The colonies were tested for slime formation using the inoculated loop method (Knoshaug et al., 2000). Isolates were considered positive for slime production if the length of slime was above 1.5 mm. Positive isolates were confirmed using MRS- Sucrose Broth without glucose and peptone as described by Pidoux et al., (1990) [1 % (w/v) meat extract, 0.5 % (w/v) yeast extract (Fluka, Biochemika), 5 % (w/v) sucrose (PA Panreac Guimica), 0.2 % (w/v) K2HPO4.3H2O (Merck), 0.5 % (w/v) sodium acetate trihydrate (Merck), 0.2 % (w/v) triammonium citrate anhydrous (Fluka, Biochemika), 0.02 % (w/v) MgSO4.7H2O (Merck), 0.005 % (w/v) manganese (II) sulphate monohydrate (Merck), 0.1 % (v/v) Tween 80, pH 5.0 ± 0.2)]. The isolates were cultured in MRS- sucrose broth and incubated at 30 ºC for 24 h. A volume of 1.5 ml of the 24 h culture was centrifuged at 4000 g for 10 min (4 ºC) and 1 ml of the supernatant put in a glass tube and an equal volume of 95 % ethanol added. In the presence of EPSs, an opaque link is formed at the interface. The positive isolates were noted according to the intensity of the opaque link. 3.8.3 Tests for Amylase Secretion by LAB Isolates The LAB isolates were streaked on Nutrient Agar (Oxiod CM3; Oxoid Ltd., Basingstoke, Hampshire, UK) made up of 2 % soluble starch (with pH adjusted to 7.2) and incubated in an anaerobic jar at 30 °C for 3 days. The plates were then flooded with iodine solution after incubation. Production of amylase was indicated by the formation of a clear zone around the University of Ghana http://ugspace.ug.edu.gh 48 colonies with the remaining parts of the plates staining blue-black as described by Almeida et al., (2007). 3.8.4 Test for Protease Secretion by LAB Isolates [ The LAB isolates were streaked on Plate Count Agar (Oxiod CM325; Oxoid Ltd., Basingstoke, Hampshire, UK) supplemented with 0.5 % casein and incubated at 30oC for 3 days. The plates were then flooded with 1M HCl. Protease positive was indicated by a clear zone around the colonies as described by Almeida et al., (2007). 3.9 DEVELOPMENT OF STARTER CULTURE 3.9.1 Irradiated Millet Flour Millet grains were purchased from the open market at Madina in Accra. The grains were milled and packaged in polyethylene pouches at 1kg per pouch. The flour was then decontaminated with a radiation dose of 10KGy. 3.9.2 Starter Cultures Three cultures of lactic acid bacteria (L. fermentum, W. confusa and L.brevis; and two yeast cultures (C. krusei and S. cerevisiae) isolated earlier from Fura fermentation were used. The cultures were stored in 50 % glycerol at – 20o C. 3.9.3 Inoculation Trials Fermentation experiments were conducted using irradiated flour and the starter cultures. The trials were conducted in duplicates and the results therefore represent duplicate measurements. University of Ghana http://ugspace.ug.edu.gh 49 3.9.3.1 Fermentation with Single Starter Culture For each of the fermentation trials, 100g of irradiated flour was kneaded with 75ml sterilized distilled water (4:3 w/v) into a dough. The water was spiked with either 107 cfu/ml of lactic acid bacteria or 106 cfu/ml of yeast as single starter culture (L. fermentum, W. confusa, L.brevis, C. krusei and S. cerevisiae). The dough was left to ferment at ambient temperature (28-300C) for 12h and sampled at 0h, 4h, 8h and 12h for determination of pH, titratable acidity and microbiological analysis. Five batches of dough were inoculated whiles one batch was not inoculated and served as control. 3.9.3.2 Fermentation with Combined Starter Culture 100g of irradiated flour was kneaded with 75ml sterilized distilled water (4:3 w/v) into a dough. Seven separate batches were prepared by adding to the dough, combinations of cultures of L. fermentum, W. confusa, L.brevis, C. krusei and S. cerevisiae as: Con (control/spontaneous): no starter culture; FK: L. fermentum + C. Krusei; FS: L. fermentum + S. cerevisiae, CK: W.confusa + C. Krusei; BS: L.brevis + C. krusei + S. cerevisiae; CS:W. confusa +S. cerevisiae and BK: L. brevis + C. krusei. Samples of fermenting dough were collected for analyses as described above. 3.10 Survival of Enteric Pathogens in Fermenting Dough The ability of different enteric pathogens to survive in fermenting dough was studied by the method described by Mante et al., (2003). The enteric pathogens used were Escherichia coli (RM EC. 0157; 11Q-1411), Vibrio cholerae, Staphylococcus aureus (RM SA 1L-1304), and Salmonella typhimurium(RM ST 20B-1410), all obtained from the Food Research Institute Microbiology laboratory. Pure cultures of each pathogen in nutrient broth at a concentration of 107 cfu/ml, were each inoculated into a fermenting batch containing the starter cultures. For the different fermentation periods, 10 ml was collected at intervals and the population of University of Ghana http://ugspace.ug.edu.gh 50 surviving pathogens enumerated by the pour plate method and incubated at the appropriate temperatures of the pathogens and the count of each pathogen determined. 3.11 SHELF LIFE STUDIES 3.11.1 Dose Optimization Thirty grammes of fermented Fura samples were packaged in poly- ethylene vacuum pouches and sealed using a vacuum sealer. The pouches were treated with irradiation doses of 0, 2.5 5.0, 7.5, and 10.0 kGy at the RTC of GAEC using a 60Co source (SLL-515, Hungary) at a dose rate of 1.43 kGy/hr in air. The absorbed dose was confirmed by Fricke‘s dosimetry. The microbiological quality (microbial load and profile) of each sample, estimated by enumeration of aerobic mesophiles on plate count agar and viable Moulds and Yeasts count by enumeration on OGYEA before and after irradiation. 3.11.2 Storage Two samples used for the study were fermented and unfermented Fura with eight treatments as: 1. Unfermented, Non-Irradiated, Non-Vacuum Packed Fura – UNV0 (Control) 2. Unfermented, Non- Irradiated, Vacuum packed Fura –UNV 3. Fermented, Non Irradiated, Non-Vacuum Packed Fura –FN V0 4. Fermented, Non- Irradiated,, Vacuum Packed Fura -FNV 5. Unfermented, Irradiated Non Vacuum Packed Fura –UI V0 6. Unfermented Irradiated, Vacuum Packed Fura - UIV 7. Fermented, Irradiated, Non-Vacuum Packed Fura - FI V0 8. Fermented, Irradiated, Vacuum Packed Fura -FIV The samples were treated with irradiation dose of 10.0 kGy at the Radiation Technology Centre of Ghana Atomic Energy Commission, using a 60Co source (SLL-515, Hungary) at a University of Ghana http://ugspace.ug.edu.gh 51 dose rate of 1.43 kGy/hr in air. The samples were then stored at ambient temperature for six weeks. The absorbed dose was confirmed by Fricke‘s dosimetry. The microbiological quality (microbial load and profile) of each sample was estimated by enumeration of aerobic mesophiles on plate count agar and viable Moulds and Yeasts count by enumeration on OGYEA before and after irradiation and at weekly intervals during storage. University of Ghana http://ugspace.ug.edu.gh 52 CHAPTER FOUR RESULTS 4.1 Field Study Fura is produced by women of all ages and mostly of Islamic origin. The production is carried out at the family level involving about three to four women on a small scale. Most producers have little or no formal education and engaged in the traditional processing as family business handed down from within the family from one generation to the other. Fura is produced from pearl millet and spices such as ginger, pepper, mint and cloves. Out of the twenty five (25) processors interviewed, only one mentioned that she knew about fermentation and that occasionally prepared some on demand whilst twenty four (24) said Fura is not fermented during processing. They explained that mostly they are not able to sell all their produce on the same day and therefore the product becomes too sour if they already fermented it during processing. 4.2 Acidification of steep water and dough during spontaneous fermentation The study on the change in pH and Titratable Acidity in Fura was confined to four (4) processors who were instructed to steep the millet grains for 12h and also ferment the subsequent dough for 12h. The pH and Titratable Acidity of steep water and dough of sample from the four production sites are shown in Figures 4.1 (a-d). At the start of steeping the pH was between 6.05 and 5.89 which decreased to 4.94 and 4.89 at the end of steeping. The initial pH of freshly prepared dough was in the range of 5.22 and 4.83 but decreased to a range of 3.98 and 3.69 at the end of dough fermentation. At the end of steeping, processor 2 recorded the lowest pH value of 4.89 followed by precessors 1 and 3 with equal value of 4.92 followed by processor 4 recording the highest pH of 4.94. Consequently, at the end of dough fermentation, University of Ghana http://ugspace.ug.edu.gh 53 processor 2 recorded the lowest pH value followed by processors 1 and 3 with processor 4 recording the highest pH. TTA in % lactic acid obtained during steeping ranged from 0.1 and 0.2% at the start of steeping to 0.21-0.27% at the end of steeping. Similar results were observed for dough fermentation with TTA increasing from between 0.13 and 0.23 % at the start of dough fermentation to between 0.50 and 0.81 % at the end of dough fermentation. The highest % TTA was recorded by processor 3 with a value of 0.27 at the end of steeping followed by processors 3 and 4 whiles the lowest value was recorded by processor 2 with a value of 0.04. At the end of dough fermentation however, the highest value was recorded by proceesor 3 at 0.38 followed by processors 2 and 4 while processor 3 recorded the lowest value at 0.27. Fig. 4.1. (a-d) Acidification during fermentation of millet into Fura University of Ghana http://ugspace.ug.edu.gh 54 4.3 Changes in Microbial Population during Steeping and Dough Fermentation 4.3.1 Population of Lactic Acid Bacteria (LAB) Table 4.1 shows the counts of Isolates on MRS, considered to be Lactic Acid Bacteria. They were Gram positive, catalase negative, mainly rods and cocci and nonsporing. The counts ranged between a level of 3 log CFU/ml and 4 log CFU/ml at the beginning of steeping to 8-10 log CFU/ml after 12 h of steeping. The LAB count at the beginning of dough fermentation was between 6-7 log CFU/ml but increased to 10 log CFU/ml at the end of fermentation. The highest lactic acid bacteria population was recorded by processor 2 with a value of 1010 cfu/ml followed by processors 3 and 4 with a population of 7 log CFU/ml whiles processor 1 recorded the lowest population with a value of 4 log CFU/ml at the end of steeping. Processor 2 at the end of dough fermentation consequently recorded the highest population of 10 log CFU/g whiles all the other processors recorded concentrations of 8 log CFU/g. Table 4.1 Population of Lactic Acid Bacteria Sample Mean LAB counts (log CFU/g or ml) Steep water Processor 1 Processor 2 Processor 3 Processor 4 0hr 4.89 ± 0.46c 4.99 ± 0.35d 3.85 ± 0.4b 3.26 ± 0.33a 6hr 5.20 ± 0.11a 8.15 ± 0.19d 6.60 ± 0.5c 5.60 ± 0.2b 12hr 7.21 ± 0.21a 10.26 ± 0.30 c 7.63 ± 0.20b 7.63 ± 0.32b Fermenting Dough 0hr 6.27 ± 0.45a 7.52 ± 0.09c 6.15 ± 1.10a 7.30 ± 0.53b University of Ghana http://ugspace.ug.edu.gh 55 Means with same letters in a row are not significantly different (p<0.05) 4.3.2 Population of Yeasts The population of yeasts at all production sites is shown in Table 4.2. The counts of yeasts at the start of steeping at all production sites were at a range of 3 -4 log CFU/ml and increased to 7 and 8 log CFU/ml after 12 h of steeping. During dough fermentation the yeast counts increased from between 4 log CFU/g to 8 log CFU/g after 12 h. Processor 2 recorded the highest population of yeasts of 8 log CFU/ml followed by processor 3 and 4 with a population of 7 log CFU/ml whiles processor 1 recorded the lowest population with a value of 5 log CFU/ml at the end of steeping. At the end of dough fermentation, processor 2 consequently recorded the highest population of 8 log CFU/g whiles processors 4, 1 and 3 recorded concentrations of 7, 6 and 5 log CFU/g respectively. 6hr 6.67 ± 0.11a 7.53 ± 0.11b 7.95 ± 0.20c 8.28 ± 0.37d 12hr 8.78 ± 0.35a 10.04 ± 0.33b 8.70 ± 0.30a 8.51 ± 0.33a University of Ghana http://ugspace.ug.edu.gh 56 Table 4.2 Population of Yeasts Means with same letters in a row are not significantly different (p<0.05) 4.3.2. Population of aerobic mesophiles The population of aerobic mesophiles during steeping and dough fermentation of samples from the four production sites during the production of fura is shown in Table 4.3. The population was made up of Gram positive catalase-negative rods and cocci, Gram positive catalase positive cocci and Gram negative bacteria. At the inception of steeping the aerobic mesophilic population was in the range of 5 to 6 log CFU/ml and increased to a range of 6 to 8 log CFU/ml after 6 h and finally to 9 log CFU/ml at the end of steeping after 12h. Processor 1 recorded the highest population at 9 log CFU/ml at the end of steeping whiles all the other processors recorded a population of 8 log CFU/ml. The microbial Sample Mean Yeast counts (log CFU/g or ml) Steep water Processor 1 Processor 2 Processor 3 Processor 4 0hr 3.04 ± 0.99a 3.94 ± 0.23b 3.85 ± 0.6b 4.78 ± 0.39c 6hr 5.76 ± 0.07b 5.09 ± 0.48a 5.70 ± 0.7b 5.90 ± 0.71c 12hr 7.72 ± 0.78b 8.69 ± 0.42c 7.20 ± 0.2a 7.11 ± 0.78a Fermenting dough 0hr 5.880± 0.71d 5.61 ± 0.42c 5.48 ± 0.3b 4.97 ± 0.40a 6hr 6.89 ± 0.28c 7.71 ± 0.57d 6.09 ± 0.5b 5.95 ± 0.61a 12hr 7.85 ± 0.35b 8.57 ± 0.42c 7.85 ± 0.8b 7.23 ± 0.64a University of Ghana http://ugspace.ug.edu.gh 57 population at the start of dough fermentation was between 6 and 8 log CFU/g which increased to 9 log CFU/g at the end of 12h fermentation. At the end of dough fermentation, processor 2 recorded the highest population with a value of 9 log CFU/g whiles all the other processors recorded a value of 8 log CFU/g Table 4.3 Population of aerobic mesophiles Means with same letters in a row are not significantly different (p<0.05) 4.3.4 Population of total coliforms The population of total coliforms during steeping and dough fermentation from the four production sites during the production of Fura is shown in Table 4.4. The mean microbial load of total coliforms at the start of steeping was 5 log CFU/ml and remained the same within 12h at two of the production sites during the 12h of steeping. Sample Mean Mesophilic Counts (log CFU/g or ml) Steep water Processor 1 Processor 2 Processor 3 Processor 4 0hr 5.34 ± 0.07a 5.86 ± 0.14c 5.48 ± 0.03b 6.28 ± 0.3d 6hr 6.57 ± 0.14a 8.27 ± 0.07d 7.51 ± 0.01c 7.41 ± 0.2b 12hr 9.86 ± 0.07d 8.68 ± 0.01 c 8.43 ± 0.01b 8.00 ± 0.21a Fermenting dough 0hr 6.08 ± 0.02a 7.49 ± 0.2c 7.08 ± 0.2b 8.16 ± 0.1d 6hr 5.60 ± 0.14a 7.33 ± 0.1b 8.64 ± 0.3d 8.38 ± 0.7c 12hr 8.96 ± 0.07b 9.41 ±0.1c 8.91 ± 0.2b 8.66 ± 0.21a University of Ghana http://ugspace.ug.edu.gh 58 At production site 3 and 4 a tenfold increase in the coliforms population was recorded despite a decrease in pH by one unit. During the dough fermentation, the population of total coliforms decreased drastically to between 1 and 2 log CFU Table 4.4 Population of total coliforms Means with same letters in a row are not significantly different (p<0.05) 4.4 Phenotypic characterization of Lactic Acid Bacteria A total of ninety (90) Lactic Acid Bacteria colonies were isolated from steeped water and dough fermentation during Fura processing. The phenotypic characteristics of the isolates are shown in Table 4.5. They were all Gram positive catalase negative, oxidase negative, non- spore forming rods and cocci devoid of cytochromes, acid tolerant, and facultative anaerobe, that produce lactic acid as the major end-product during fermentation of carbohydrates and were considered to be lactobacillus spp. Sample Mean Coliform Counts (log CFU/g or ml) Steep water Processor 1 Processor 2 Processor 3 Processor 4 0hr 5.90 ± 0.14b 5.72 ± 0.21a 5.71 ± 0.1a 5.90 ± 0.7b 6hr 5.69 ± 0.21a 5.71 ± 0.21a 6.97 ± 0.2c 6.75 ± 0.21b 12hr 5.51 ± 0.14a 5.62 ± 0.2b 6.52 ± 0.1d 6.47 ± 0.8c Fermenting dough 0hr 6.28 ± 0.07c 5.67 ± 0.07b 5.72 ± 0.4d 5.41 ± 0.14a 6hr 4.92 ± 0.21c 3.90 ± 0.21c 5.72 ± 0.3a 3.23 ± 0.07b 12hr 1.51 ± 0.21a 1.85 ± 0.42b 2.76 ± 0.1c 2.51 ± 0.14d University of Ghana http://ugspace.ug.edu.gh 59 The most dominant strains were rods in singles and pairs and grew at pH 4.4 and 9.6 as well as in 6.5% NaCl but not at 45oC, 10oC and 18 % NaCl. They fermented L-arabinose, Ribose, D-xylose, Galactose, D-Glucose, D-fructose, D-mannose, N acethyl glucosamide, Arbutin, Salicin, Cellobiose, Maltose, Lactose, Melibiose, Saccharose, Trehalose, D-raffinose, β gentiobiose, D-lyxose, Gluconate and 5 cetoglunate in the API 50 CHL galleries (Appendix) and were tentatively identified as Lactobacillus fermentum. The second most dominant species were cocci in pairs and were tentatively identified as Weisella confusa because they grew at pH 4.4 and 9.6 as well as in 6.5% NaCl but not at 45o C, 10oC and 18 % NaCl. Moreover, by mode of Carbohydrate fermentation using the API CHL 50, they were able to utilize L-arabinose, Ribose, D-xylose, Galactose, D-Glucose, D-fructose, D-mannose, L- sorbose, Rhamnose, mannitol, sorbitol, N acethyl glucosamide, Amygdaline, Arbutin, Salicin, Cellobiose, Maltose, Lactose, Melibiose, Saccharose, Trehalose, D-raffinose, β gentiobiose and Gluconate. [ The third most dominant species which were identified as Lacobacillus brevis were short rods and grew at pH 4.4 and 9.6 and at 45oC but not at 10oC and in 6.5% and 18% NaCl. They were able to ferment L-arabinose, Ribose, D-xylose, Galactose, D-Glucose, D-fructose, Maltose, Melibiose, Saccharose, Trehalose, Melezitose, D-raffinose, D-turanose, Gluconate, and 5 cetoglunate. The fourth dominant species were cocci in pairs and grew at pH 4.4 and 9.6 and at 45oC as well as in 6.5% NaCl but not 10oC and 18 % NaCl. They utilized L-arabinose, Ribose, β methyl-xyloside, Galactose, D-Glucose, D-fructose, L-sorbose, N acethyl glucosamide, Amygdaline, Arbutin, Esculin, Salicin, Cellobiose, Maltose, Trehalose and β gentiobiose and were identified as Pediococcus acidilactici. University of Ghana http://ugspace.ug.edu.gh 60 Table 4.5 Phenotypic characteristics of lactic acid bacteria isolated from steeping water and fermenting dough + = present; - = absent Group 1 2 3 4 5 6 Cell form Rods Rods Cocci Cocci Cocci Cocci Cellular arrangement Singles /pairs Singles /pairs Singles Pairs Pairs Tetrads Grams reaction + + + + + + Catalase reaction - - - - - - Anaerobic growth + + + + + + Oxidase test - - - - - - Growth at pH 4.4 + + + + + - Growth at pH 9.6 + + + + + + Growth in 6.5% NaCl + - - + + - Growth in 18% NaCl - - - - - - Growth in Growth at 100C - - - - - - Growth at 450C - + + + - - Isolate Identified L. fermentum 1 L . brevis Lactococcu s rafinolactis P. acidilactici W. confusa lactococcus lactis ssp lactis 1 % isolate 33.33 16.67 6.67 13.33 20.00 10.00 University of Ghana http://ugspace.ug.edu.gh 61 4.5 Characterisation and Identification of Yeasts A total of 32 yeast colonies were isolated from steep water and fermenting dough from the four production sites. Colony and cell morphology was initially used to characterize and group the isolates. This was` followed by tentative identification with fermentation of sugars in ID 32C galleries. The most dominant yeasts (43.75 %) isolated from all the processing stages utilized galactose, glucose, sucrose, raffinose, maltose, DL-lactate, trehalose, α-metyl- D-glucoside, melibiose and were identified as Saccharomyces cerevisiae. The second dominant yeast (25%) utilized glucose, N-acetyl- glucosamide and DL-lactate and was identified as Candida krusei. The third yeasts isolates (18.75%) were identified as Candida albicans whilst the last group (12.5%) was identified as Candida membranifascians 4.6 Technological properties of Lactic acid Bacteria Isolates 4.6.1 Rate of Acidification by Lactic Acid Bacteria Isolates The rate of acidification during dough fermentation was evaluated using pH and titratable acidity. Figure 4.2 shows the rate of acidification during dough fermentation by Lactic Acid Bacteria isolates as obtained by changes in pH during different periods of fermentation. At 0-4 h the rate of acidification ranged from 0.07 to 0.85 units with L. brevis 2 showing the highest rate of acidification and L. rafinolactis showing the lowest rate of acidification after the control (Fig.4.2). At 4-8 h fermentation however, the rate ranged from between 0.8 to 1.6 units with W.confusa recording the highest whiles L. lactis ssp lactis 1 and L. brevis 2 recorded the lowest rates of acidification after the control. The rate at 8-12 h ranged from between 0.1 to 0.6 units with L. lactis ssp lactis having the highest rate of acidification. The fastest rate of acidification was recorded during the fourth to the eighth hour whilst the lowest was recorded during the zero to fourth hour followed by the eighth to the twelfth hour during the fermentations. There was a corresponding increase in the Titratable acidity expressed as percentage lactic acid, during steeping and dough fermentation period as shown in figure 4.3. University of Ghana http://ugspace.ug.edu.gh 62 The titratable acidity at the start of dough fermentation was between a range of 0.07 and 0.12 which increased to a range of 0.22 and 0.44 at the end of dough fermentation. The highest %TTA was recorded by L. brevis with a value of 0.44% followed by L. fermentum and W. confusa with values of 0.42% and 0.41% respectively. The lowest %TTA was recorded by the spontaneous fermentation and L. rafinolactis with percentages of 0.26 and 0.36 respectively. Fig.4.2. Changes in pH during dough fermentation by Lactic Acid Bacteria isolates 0 0.5 1 1.5 2 0-4h 4-8h 8-12h L. rafinolactis L.fermentum 1 W. confusa L.lactis ssp lactis 1 P. acidilactici L. brevis 2 CONTROL pH Time/h University of Ghana http://ugspace.ug.edu.gh 63 Fig.4.3. Titratable acidity during acidification of fermenting dough by lactic acid bacteria 4.6.2 Amylase Secretion exopolysaccharide production and protease secretion by Lactic Acid Bacteria Isolates The lactic acid bacteria isolates were screened for their ability to secrete amylase by growing them on a modified Nutrient agar containing 2 % starch and the result is shown in Table 4.6 below. The isolates consisted of 30 L. fermentum, 15 L. brevis. 18 W. confusa and 12 P. acidilactici. Out of these isolates 13.33 % each of L. fermentum, and L .brevis, 16.67% of W. confusa and 8.33% of P. acidilactici produced clear zones ranging from 1mm to 2 mm, with 11.11% W. confusa producing clearing zones from 3mm to 4mm indicating amylase secretion. For exopolysaccharride, 46.67% of L. fermentum, 20% of L. brevis, 38.89% of W. confusa and 66.67% P. acidilactici produced a slime between 1mm and 2mm. 40% L. fermentum 60% of L. brevis, 61.11% and 25% of W.confusa produced a slime of 3- 4mm whiles of 13.33% of L. fermentum, 20% L. brevis and 8.33% produced a slime above 5mm as shown in the table. Only 3.33% L. fermentum and 5.56% W. confusa secreted protease with clearing zones of 1-2mm. University of Ghana http://ugspace.ug.edu.gh 64 Table 4.6 Amylase Secretion, exopolysaccharide (EPS) production and protease secretion by Lactic Acid Bacteria Isolates ND: no clearing zone; +: 1-2mm clearing zone, ++: 3-4mm clearing zone, +++:5mm clearing zone. For exopolysaccharaide production, ND: no slime; 1-2mm length of slime, ++: 3-4mm length of slime, +++:5mm length of slime. ISOLATE TEST ND + ++ +++ % of Isolate L. fermentum (n=30) Amylase secretion EPS production Protease secretion 86.67 0.00 96.67 13.33 46.67 3.33 0.00 40.00 0.00 0.00 13.33 0.00 L. brevis 2(n=15) Amylase secretion EPS production Protease secretion 86.67 0.00 100.00 13.33 20.00 0.00 0.00 60.00 0.00 0.00 20.00 0.00 W. confusa (n=18) Amylase secretion EPS production Protease secretion 72.22 0.00 94.44 16.67 38.89 5.56 11.11 61.11 0.00 0.00 0.00 0.00 P. acidilactici (n=12) Amylase secretion EPS production Protease secretion 75.00 0.00 100.00 8.33 66.67 0.00 16.67 25.00 0.00 0.00 8.33 0.00 University of Ghana http://ugspace.ug.edu.gh 65 4.6.3 Antimicrobial Interaction between Lactic Acid Bacteria isolates There was no microbial interaction between the lactic acid bacteria isolates as shown in table 4.7 below Table 4.7 Antimicrobial Interaction between Lactic Acid Bacteria isolates ISOLATES (LAB) INDICATOR STRAINS (LAB) L. rafinolactis L. fermentum W. confusa L. lactis ssp lactis 1 P. acidilactici L. brevis 2 L. rafinolactis - - - - - - L. fermentum - - - - - - W. confusa - - - - - - L. lactis ssp lactis 1 - - - - - - P. acidilactici - - - - - - L. brevis 2 -: no inhibition zone 4.6.4 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates There was neither a microbial interaction between the lactic acid bacteria isolates and Saccharromyces cerevisiae nor C. krusei (Table 4.8). There was however a weak interaction University of Ghana http://ugspace.ug.edu.gh 66 between L. fermentum, W.confusa and L. brevis against C. albicans and C. membranifascians as shown. Table 4.8 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates -: no inhibition zone, +: 1-2mm inhibition zone, ++: 3-4mm inhibition zone. 4.6.5 Antimicrobial Activity of Lactic Acid Bacteria against Some Common Enteric Pathogens Table 4.9 shows the Antimicrobial activity of lactic acid bacteria against pathogen indicator- strains. All the isolates exhibited antimicrobial activity against all the pathogens tested (Salmonella typhimurium, E. coli, Vibrio cholerae and Staphylococcus aureus), except for P. acidilactici against E. coli. L. fermentum exhibited the strongest inhibition against Staphylococcus aureus and Vibrio cholerae with inhibition zones exceeding 5 mm while Salmonella typhimuruim and E. coli showed inhibition zones of less than 3 mm as shown in the table. This was followed by L. brevis which exhibited a strong inhibition zone of 3-4mm against all the tested strains. W. confusa also exhibited a strong inhibition zone of 3-4mm against Salmonella typhimurium, E. coli and Staphylococcus aureus but 1-2mm inhibition zone against Vibrio cholera. ISOLATES (LAB) INDICATOR STRAINS (YEASTS) Saccharomyce s Cerevisiae Candida Krusei Candida Albicans Candida membranifascians L. rafinolactis L. fermentum W. confuse L. lactis ssp lactis 1 P. acidilactici L. brevis 2 - - - - - - - - - - - - - + ++ + - + - + + - + + University of Ghana http://ugspace.ug.edu.gh 67 Table 4.9 Antimicrobial activity of lactic acid bacteria against pathogen indicator- strains -: no inhibition zone, +: 1-2mm inhibition zone, ++: 3-4mm inhibition zone, +++:5mm inhibition zone 4.7 Starter culture trials 4.7.1 Changes in Microbial Population Changes in microbial population of lactic acid bacteria and yeasts as a result of the enrichment addition of different single starter cultures are displayed in Table 4.10. L. fermentum, L. brevis and W. confusa (LAB) and S. cerevisiae and C. krusei (Yeasts) were the isolates used for the trials. The counts of lactic acid bacteria were significantly higher throughout the tests with regards to the addition of the LAB isolates than the spontaneous fermentation. At the start of fermentation, the lactic acid bacteria population was 5 log CFU/g which increased to a final count of 9 log CFU/g in LAB inoculum enrichment (L. ISOLATES (LAB) INDICATOR STRAINS (PATHOGENS) Staphylococcus Aureus E- coli Salmonella Typhi Vibrio cholera L. rafinolactis ++ + + ++ L. fermentum +++ ++ ++ +++ W. confuse ++ + ++ + L. lactis ssp lactis 1 ++ + + + P. acidilactici + - + ++ L. brevis 2 ++ ++ ++ ++ University of Ghana http://ugspace.ug.edu.gh 68 fermentum, L. brevis and W. confusa) fermentations. In the case of the spontaneous fermentation however, the highest count was 7 log CFU/g as shown in Table 4.13. Consequently, high Yeast counts were significantly recorded in fermentations with added S. cerevisiae or C. krusei compared to the spontaneous fermentation. The yeasts counts were 5 log CFU/g at the start of fermentations with added S. cerevisiae and C. krusei, which finally increased to 8 log CFU/g after 12 h, in contrast to the spontaneous fermentation, which recorded a maximum count of 7 log CFU/g. Table 4.10 mean microbial counts (log CFU/g) for fermentations with single starter cultures LACTIC ACID BACTERIA Fermentation Time(h) Fermentation Types Control/ Spontaneous L. fermentum L. brevis W. confusa 0 3.11a 5.91d 5.67c 5.08b 4 3.38a 6.12b 6.53c 6.61c 6 4.23a 7.33b 7.50b 8.18c 12 7.40a 9.33b 9.30b 9.45b Means with same letters in a row are not significantly different (p<0.05) YEASTS Fermentation time(h) Fermentation types Control/spontaneous Saccharomyces cerevisiae Candida krusei 0 2.37a 5.75b 5.72b University of Ghana http://ugspace.ug.edu.gh 69 4 4.41a 5.09c 5.37b 8 5.27a 7.45c 7.19b 12 7.70a 8.37c 8.13b Means with same letters in a row are not significantly different (p<0.05) 4.7.2 Microbial Counts during Dough Fermentation with combined Starter Cultures The microbial populations during Fura dough fermentation using different starter cultures are displayed in Table 4.16. The microbial population of lactic acid bacterial and yeast, with regards to the combination of various starter cultures was higher, compared to the spontaneous fermentation. The population of lactic acid bacteria for the starter cultures at the start of fermentation was 7 log CFU/g, which rose to 10 log CFU/g at the end of fermentation. The lactic acid population for the spontaneous fermentation however started with 4 log CFU/g and increased to 7 log CFU/g at the end of fermentation. Similarly, the population of yeasts with regards to the combined starter cultures began with 5 log CFU/g at the initial level of fermentation and ended at 8 log CFU/g after 12h of fermentation. Compared to spontaneous fermentation however, the initial yeast population was 4 log CFU/g which increased to 6 log CFU/g after 12h of fermentation. Comparatively, the population of lactic acid bacteria resulting from the combination of starter cultures was higher than the population of yeasts. University of Ghana http://ugspace.ug.edu.gh 70 Table 4.11 Mean microbial counts (log CFU/g) during dough fermentation with combined starter cultures Time Control/ Spontaneous L. fermentum + C. krusei L. fermentum + S. cerevisiae W. confusa + C. krusei W. confusa + S. cerevisiae L. brevis + S. cerevisiae L. brevis + Candida krusei LAB 0h 4.85a 7.53b 7.36b 7.43b 7.40b 7.36b 7.63b 4h 6.75a 7.67e 7.52d 7.36b 7.38b 7.96e 7.43c 8h 6.17a 10.27c 10.33c 9.35b 10.44d 10.55e 10.54e 12h 7.54a 10.33d 10.14c 9.72b 10.37d 10.10c 10.61e YEASTS 0h 4.68a 5.00b 5.11b 5.48c 5.60d 5.15b 5.15b 4h 4.70a 5.70d 5.78d 5.48c 5.85d 5.90e 5.30b 8h 5.48a 6.60c 7.18d 6.00b 8.60f 8.00e 8.57f 12h 6.61a 7.54c 7.51c 7.48b 8.64f 8.30d 8.48e Means with same letters in a row are not significantly different (p<0.05) 4.7.3 Acidification of Fermenting Dough in Fermentation Trials with Starter Cultures The acidification of fermenting dough in fermentation trials with starter cultures are shown in Figure 4.4 (a-b). At the start of fermentation, the spontaneous (control) fermentation recorded a pH of 6.44 which finally dropped to 5.44 after 12h of fermentation. The pH values for the starter cultures at the start of dough fermentation were in the range of 6.44-6.32 which finally dropped to a range of 4.5 and 3.96 at the end of fermentation. L.brevis recorded the lowest pH among the starter cultures, followed by L. fermentum and W. confusa with 4.44., whiles University of Ghana http://ugspace.ug.edu.gh 71 C. krusei recorded the highest pH 4.6 at the end of fermentation (Figure 4.4a). There was a corresponding increase in Titratable acidity in all the fermentations. The TTA values recorded for the spontaneous fermentation ranged from 0.11 to 0.22 within twelve (12) hours of fermentation whereas the values recorded for the starter cultures ranged from 0.11 to 0.46 with L.brevis recording the highest and C. krusei having the least as shown in Figure 4. 4b. Fig.4.4. (a-b) pH and Titratable Acidity of Fermenting Dough in Fermentation Trials with Starter Cultures 4.7.4 Acidification of Fermenting Dough in Fermentation Trials with combined Starter cultures The acidification of fermenting dough in fermentation trials with combined starter cultures are displayed in Figure 4.5 (a-b). At the start of fermentation, the spontaneous (control) fermentation recorded a pH of 6.47 which finally dropped to 5.92 after 12h of fermentation. The pH values for the starter culture combinations at the start of dough fermentation were in the range of 6.47-6.38 which finally dropped within a range of 4.02 and 3.83 at the end of fermentation. L. brevis +Saccharomyces cerevisiae recorded the least pH among the starter culture combinations, followed by Lactobacillus fermentum +Saccharomyces cerevisiae with 3.83 and 3.93 respectively whiles Lactobacillus fermentum +Candida krusei and W. confusa University of Ghana http://ugspace.ug.edu.gh 72 + Candida krusei recorded the highest pH of 4.02 after the spontaneous fermentation, which also recorded a pH of 5.93 at the end of fermentation (Figure 4.5b). There was a corresponding increase in Titratable acidity in all the fermentations. The TTA values recorded for the spontaneous fermentation ranged from 0.18 to 0.27 within twelve (12) hours of fermentation whereas the values recorded for the starter cultures ranged from 0.18 to 0.62 with Lactobacillus brevis+ Saccharomyces cerevisiae and Lactobacillus fermentum +Saccharomyces cerevisiae recording the highest TTA and Lactobacillus fermentum +Candida krusei, Lactobacillus brevis+ Candida krusei and W. confusa + Candida krusei having the least TTA as expressed in Figure 4.5b. Fig. 4.5 (a-b) pH and Titratable Acidity of Fermenting Dough in Fermentation Trials with combined Starter Cultures. 4.7.5 Survival of Enteric Pathogens Table 4.12 displays the survival of four enteric pathogens during millet dough fermentation with different starter cultures. The pathogens were inoculated into kneaded dough at a concentration of 107 cfu/ml. Table 4.17 displays the survival of four enteric pathogens during millet dough fermentation with different starter cultures. The pathogens were inoculated into kneaded dough at a concentration of 107 cfu/ml. University of Ghana http://ugspace.ug.edu.gh 73 The population of Vibro cholerae was not detected after 12h except in the control/spontaneous and W. confusa + S. cerevisiae fermentation which recorded 3 and 2 log CFU/g respectively. The population of Salmonella typhimurium significantly reduced from a range of 7-8 to 2 log CFU/g with the control /spontaneous recording 4 log CFU/g at the end of fermentation. Even though the populations of E.coli and Staphylococcus aureus were not completely eliminated at the end of fermentation, their counts were significantly lower than that of the control/spontaneous fermentation. Table 4.12 Count (log CFU/g) for survival of enteric pathogens inoculated into spontaneous and mixed culture fermentation of millet dough Fermentation types Ferm. Time Control L. fermentum + C. krusei L. fermentu m + S. cerevisiae W. confusa + C. krusei W. confusa + S. cerevisiae L. brevis + S. cerevisiae L. brevis + Candida krusei Vibrio cholera 0h 7.30b 7.36b 6.70a 6.70a 6.95ab 7.85c 6.60c 4h 7.70c 6.52b 6.60a 5.48b 6.60b 6.90b 5.95b 8h 5.49e 3.30b 3.00a 2.30cd 3.48b 3.70d 2.90cd 12h 3.78b Nd Nd Nd 2.00b Nd Nd Staphylococcus aureus 0h 8.76d 7.32b 8.08c 7.38c 5.95a 8.70c 7.08c 4h 7.53c 7.85c 7.48c 6.18b 5.60a 7.78c 6.49c University of Ghana http://ugspace.ug.edu.gh 74 Means with same letters in a row are not significantly different (p<0.05) 4.8 STORAGE OF FURA SAMPLES 4.8.1 Dose optimization The microbial counts for the determination of the irradiation dose for storage of Fura samples are displayed in Table 4.13. The microbial counts before irradiation for aerobic mesophiles as well as yeasts and moulds were both high at 109 CFU/g. The population of yeasts and moulds for the control samples were already high at the count of 108 cfu/g at day zero (0), which was maintained to the 2nd day, finally increasing to 109 CFU/g during the 4th day, maintaining it through to the 8th day. The Total Plate Counts (TPC) were also high initially and was maintained throughout at 109 CFU/g. 8h 5.90e 4.70b 5.60d 4.36a 4.78b 5.41cb 4.60c 12h 4.30e 2.00a 2.38b 2.48c 2.60d 2.48d 2.30c Salmonella typhimurium 0h 7.70a 7.95b 7.08c 7.69a 8.86e 8.18d 8.00cd 4h 6.78c 6.90bc 7.11bc 7.48e 7.23cd 6.26a 7.49ab 8h 5.18c 4.95b 4.60a 5.30cd 5.38cd 5.60c 6.41d 12h 4.78d 3.26a 3.40b 3.60c 3.60d 3.40c E. Coli 0h 8.38e 7.30a 8.30d 7.53b 7.90c 8.78f 8.60f 4h 8.60b 7.45a 7.51a 7.30a 7.48a 7.60a 6.48a 8h 7.57e 5.20b 5.28b 6.70d 4.90a 5.95c 5.20c 12h 6.70e 4.70d 4.78b 4.30c 3.85a 4.00b 4.38b University of Ghana http://ugspace.ug.edu.gh 75 The Total Plate Counts (TPC) as well as yeasts and mould counts for 2.5KGy both started at 106cfu/g with the TPC increasing to 108cfu/g at the end of the 8th day whiles the yeast and mould were maintained at 107CFU/g Whiles the initial counts for the 10.0KGy was 101cfu/ml and ended at 103cfu/ml, the counts for both 5.0 and 7.5KGy started at 103CFU/g and ended at 105CFU/g. The above results therefore gave the 10.0KGy advantage over the other doses for the extension of shelf life for fura during its storage. Table 4.13.The microbial counts (CFU/g) for dose optimization for storage of Fura samples TIME(DAY) RADIATION DOSE(KGy) Total Viable Counts (TVC) 0 2.5 5.0 7.5 10.0 0 3.0 x109 1.9 x106 3.0 x104 4.0 x103 6.0 x101 2 5.0 x109 2.3 x106 3.5 x104 6.0 x103 7.0 x101 4 6.0 x109 3.0 x107 1.0 x105 2.0 x104 9.0 x101 6 8.0 x109 4.0 x107 4.0 x105 1.0 x105 1.0 x102 8 8.0 x109 2.0 x108 1.0 x106 3.0 x105 5.0 x103 YEASTS AND MOULDS 0 5.0 x108 3.0 x106 4.0 x103 1.3 x103 5.0 x101 University of Ghana http://ugspace.ug.edu.gh 76 2 5.3 x108 5.0 x106 5.0 x103 2.1 x103 5.2 x101 4 2.0 x109 8.3 x106 3.0 x104 3.2 x103 6.0 x101 6 4.0 x109 2.0 x107 3.1 x105 5.0 x104 4.0 x102 8 8.0 x109 3.0 x107 5.2 x105 2.0 x105 4.0 x103 4.8.2 Shelf Life Studies The total plate count (TPC) and moulds and yeasts count (MYC) during the ambient storage of Fura samples are displayed in Table 4.19 and Figures 4.6 a and b below. Before irradiation, the TPC was 8 log CFU/g and 7 log CFU/g for the fermented and unfermented Products respectively, whiles the MYC were 8 log CFU/g and 6 log CFU/g for both samples. After irradiation (week 0), the TPC for the irradiated samples ( UIV0 UIV, FIV0 and FIV) drastically fell to between 1 log CFU/g and 2 log CFU/g whiles the population of yeasts and moulds (MYC) fell to 0 and 3 log CFU/g, with the unfermented, irradiated and vacuum packed sample(UIV) recording zero. The microbial counts for the Non-Irradiated Samples (UNV0, UNV, FNV0 and FNV) were maintained between 8 log CFU/g and 9 log CFU/g until the second week when it begun to develop unpleasant flavor and texture. The microbial populations for the irradiated samples however increased progressively between 1 log CFU/g and 8 log CFU/g until the end of the sixth week when it begun to show signs of spoilage (unpleasant odour and texture). It was observed that the microbial populations for the vacuum packed samples were mostly slightly lower than that of the Non- vacuum packed samples and stayed on the shelf longer than the Non-Vacuum packed samples. Moreover, the populations of the fermented samples were mostly higher than that of the unfermented samples (Figure 4.6 a and b). University of Ghana http://ugspace.ug.edu.gh 77 Table 4.14 Population of Aerobic Mesophiles and Yeast and Moulds before irradiation of Fura samples Fig. 4.6a Population of Aerobic Mesophiles during the storage of Fura samples Fig. 4.6b Population of Yeasts and Moulds during the storage of Fura samples 0 2 4 6 8 10 12 WEEK 0 WEEK1 WEEK2 WEEK3 WEEK4 WEEK5 WEEK6 UNV0 UNV FNV0 FNV UIV0 UIV FIV0 FIV log g CFU/g Time/Week 0 1 2 3 4 5 6 7 8 9 10 WEEK0 WEEK1 WEEK2 WEEK3 WEEK4 WEEK5 WEEK6 UNV0 UNV FNV UIV0 UIV FIV0 FIV log g CFU/g Time/Week MICROBIAL COUNTS BEFORE IRRADIATION TEST FERMENTED UNFERMENTED Aerobic mesophiles 1.5 x108 2.4 x107 Yeast and moulds 2.0 x108 2.0 x106 University of Ghana http://ugspace.ug.edu.gh 78 CHAPTER FIVE DISCUSSION 5.1 Processing of Fura Fura is produced by women of all ages and mostly of Islamic origin. The production is carried out at the family level involving about three to four women on a small scale. Most producers have little or no formal education and engaged in the traditional processing as family business handed down from within the family from one generation to the other. The production of Fura is similar to several other African traditional products. It is however more similar to the production of Kenkey in the sense that most of the processors had little or no formal education and also more of Islamic origin (Obodai et al., 2014). Most processors do not ferment their produce due to the fact that they are not able to sell all their produce on the same day and therefore the product becomes too sour if they already fermented it during processing. The fermentation in Fura like many other traditional processes, is caused spontaneously by the natural flora of the raw materials, process utensils, water and the environment (Owusu-Kwarteng et al., 2010), making it difficult to control. 5.2 Acidification during spontaneous fermentation There was a decrease in pH with a corresponding increase in acidity during spontaneous steeping and dough fermentation. A corresponding increase in total titratable acidity was recorded during the unit operations. The decrease in pH was indicative of the fermentation of the product as reported for other natural fermented foods (Sulma et al., 1991; Jespersen et al., 1994; Halm et al., 1996; Kalui et al; 2009; Olukoya et al., 1993). The decrease in pH and increase in TTA could be attributed to the metabolic activities of Lactic Acid Bacteria. Lactic acid bacteria, in particular Lactobacilli, is able to decrease pH, thus preventing the growth of pathogenic and spoilage microorganisms and therefore improving the hygienic safety and storage of meat products (Lucke, 1985; Samelis et al., 1994). University of Ghana http://ugspace.ug.edu.gh 79 5.3 Lactic acid bacteria involved in Fura fermentation The present study showed a drastic increase in the population of lactic acid bacteria between103 CFU/ml and 104 CFU/ml at the beginning of steeping to 1010 CFU/ml after 12 h of steeping. The population of lactic acid bacteria at the beginning of dough fermentation was between 106 CFU/g and also increased to 1010 CFU/g at the end of fermentation. These observations confirm the presence and importance of lactic acid bacteria during steeping of millet grains and dough fermentation. The increase in population resulted in the acidification of the product. The increase in lactic acid bacteria was also facilitated by the presence of high yeast counts which resulted in the increase in amino acid concentration from yeasts synthesis and excretion by yeast cell autolysis (Gobbetti et al., 1994). Other studies have confirmed LAB as the predominant microorganisms involved in the fermentation of Fura (Owusu- Kwarteng et al., 2010) and other fermented cereal products such as ogi from maize (Odunfa, 1985), kenkey from maize (Halm et al., 1993; Hayford and Jakobsen, 1999), togwa from a mixture of maize and sorghum (Mugula et al., 2003), koko from millet (Lei and Jakobsen, 2004) and gowé from sorghum (Vieira-Dalodé et al., 2007). Lactic acid bacteria are technologically important organisms recognized for their fermentative ability as well as their health and nutritional benefits (Gilliand, 1990) and are the most widespread of desirable microorganisms in food fermentation. They have been found in fermented cereal products, milk, cheese and fermented meats (Campbell-Platt, 1987). They convert the available carbohydrate to organic acids and lower the pH of food, thereby making the food unfavourable for the growth of spoilage and pathogenic bacteria (Adams and Moss, 1995). They also have the ability to inhibit undesirable microflora in the food. Lactic acid bacteria and their products therefore give fermented foods distinctive flavours, textures, and aromas while preventing spoilage, extending shelf-life, and inhibiting pathogenic organisms (Rattanachaikunsopon and Phumkhachorn, 2010). University of Ghana http://ugspace.ug.edu.gh 80 The lactic acid bacteria identified in the present work were Lactobacillus fermentum (33.33%), Weissella confusa (20%), Lactobacillus brevis (16.67%), P. acidilactici (13.33%), Lactococcus lactis ssp lactis 1 (10%) and Lactococcus rafinolactis (6.67%). The dominant lactic acid bacteria identified in the present work as responsible for Fura fermentation was Lactobacillus fermentum. L. fermentum has been reported by several workers as the dominant lactic acid bacteria responsible for the fermentations of other cereal products including Brukutu (Atter et al., 2014), pito/dolo (Sawadogo-Lingani et al., 2007); opaque sorghum beer (Kayode et al. 2006), koko (Lei and Jakobsen, 2004) and kenkey (Hayford et al., 1999). Lactobacillus fermentum and W. confusa were isolated by Lei and Jacobson (2004) as the dominant lactic acid bacteria for the fermentation of millet into koko from five production sites in Northern Ghana. L. fermentum has also been associated with the fermentation of fufu from cassava and the flavour typical of the product (Adekoge and Babaola, 1988). The role of L. fermentum in aroma formation has also been demonstrated during fermentation of maize dough by Annan et al., (2003), and this could be equally exploited for Fura production since flavour is an important quality characteristic of Fura. Sawadogo-Lingani et al., (2007) reported Lactobacillus fermentum as the dominant lactic acid bacteria responsible for souring of dolo. The other lactic acid bacteria identified in the fermentation of Fura in the present work were Weissella confusa, Lactobacillus brevis, P. acidilactici, Lactococcus lactis ssp lactis 1 and Lactococcus rafinolactis. The isolation of Weisella confusa is in agreement with Owusu-Kwarteng, (2013), who reported it as the second predominant species identified with molecular methods during Fura processing in Northern Ghana. The presence of Weisella confusa has also been reported as responsible for some other African cereal-based fermented foods such as millet koko in Ghana (Lei and Jakobsen, 2004), University of Ghana http://ugspace.ug.edu.gh 81 togwa in Tanzania (Mugula et al., 2003), bushera in Uganda (Muyanja et al. 2002) and gowé in Benin (Vieira-Dalode et al., 2007). The presence of L. brevis has been reported in other Ghanaian fermented foods including kenkey by Olsen et al., (1995), Annan et al., (2016) and in agbelima cassava dough by Amoa-Awua et al., (1996). Lactococcus lactis ssp lactis 1 and Lactococcus rafinolactis have been isolated from acid coagulating cheese samples (Radovanovic and Katic, 2009). 5.4 Yeasts involved in fura Fermentation The yeast population increased during the fermentation of millet for Fura production. The co- existence and symbiotic association between lactic acid bacteria and yeasts have been reported by many authors for several African traditional fermented products (Jespersen et al., 1994; Hounhouigan et al., 1993; Omemu et al., 2007). The yeasts identified as responsible for Fura fermentation in the present work were Saccharomyces cerevisiae (37.5%); Candida membranifascians (18.75); Candida krusei (25%); Candida albicans (18.75%). Saccharomyces cerevisae and C. krusei were the dominant yeast species associated with Fura fermentation in the present work. Owusu Kwarteng et al., (2013) however identified Candida krusei and K. maxiamus as the predominant yeasts in Fura processing. Saccharomyces cerevisiae is noted to be a predominant yeast species besides Lactic acid bacteria involved in food fementation in Africa (Shetty et al., 2007). The presence of Saccharomyces cerevisiae and Candida krusei have been reported in other cereal fermentations (Hayford and Jesperson, 1999; Halm et al., 1993), with S. cerevisiae considered as the yeast species most often reported in African indigenous fermented foods and beverages (Jespersen 2003). Jesperson et al., (1994) isolated S. cerevisiae and C. krusei as the dominant yeast in maize dough fermentation and suggested that since yeasts are known to produce a wide range of aromatic University of Ghana http://ugspace.ug.edu.gh 82 compounds including organic acids, esters, aldehydes, alcohols, lactones and terpenes, they are likely to influence the organoleptic and structural quality of fermented maize dough. The dominance of Saccharomyces cerevisiae has been confirmed in other millet based fermented products such as Traditional Opaque Beer (Misihairabgwi et al., 2015) and Tchoukoutou (Kayodé et al., 2011). The functions of yeasts in cereal fermented foods and beverages have been reported by several authors as the production of aroma compounds through the conversion of carbohydrates into alcohols, esters, organic acids and carbonyl compounds, inhibition of mycotoxins producing moulds (nutrient completion), degradation of mycotoxins, production of tissue degrading enzymes (cellulases, pectinases) which make substrates available for other microorganisms and Probiotic properties (Jespersen, 2003; Kohajdova and Karovicova, 2007; Osmorio-Cadavid et al., 2008). Additionally, yeasts have been reported to display amylolytic, protease and phytase activities and this may contribute to breaking down maize starch and also allow better access to nutritionally essential minerals (Amoa-Awua et al., 1997, 2006; Omemu et al., 2007). 5.5 Antimicrobial activity of Lactic Acid Bacteria against Common Enteric Pathogens All the lactic acid bacteria isolates exhibited antimicrobial activity against all the pathogens tested in the present work, i.e Salmonella typhimurium, E. coli, Vibrio cholerae and Staphylococcus aureus, except for P. acidilactici against E. coli. L. fermentum however exhibited the strongest inhibition against Staphylococcus aureus and Vibrio cholerae. This result is in agreement with the work of Annan et al., (2016), which reported antimicrobial activity of Lactobacillus fermtentum against Staphylococcus aureus and Vibrio cholerae during nsiho fermentation. University of Ghana http://ugspace.ug.edu.gh 83 Sawadogo-Lingani et al., (2008) also reported a high level of antimicrobial activity by L. fermentum against Staphylococcus aureus but weak activity against E. coli and Listeria innocua during the production of dolo. With respect to the survival of enteric pathogens using a challenge test, addition of starter cultures improved the antimicrobial activity of Fura against the enteric pathogens. Vibrio cholerae was only detected in one of the starter cultures sample at 12h of dough fermentation from the initial counts at concentration 106- 10 CFU/g. In the spontaneous fermentation the count was 103 CFU/g after the fermentation. None of the other pathogens were eliminated at the end of the 12h fermentation except in one instance. However there was a 3-4 log reduction in Staphylococcus aureus, 4-7 log reduction in Salmonella typhimurium count and 3-6 log reduction in E.coli count in the starter culture inoculated samples. Salmonella typhimurium was however eliminated entirely at the end of fermentation with regards to L. fermentum and C. krusei combination. Generally, the population of pathogens at the end of starter culture fermentation was significantly lower as compared to spontaneous fermentation. The microbial activity of the fermenting Fura on the pathogens could be due to the accumulation of organic acids and its associated reduction in pH (Berry et al., 1990) as well as the production of other antimicrobial compounds. In related works, the antimicrobial activity of lactic acid bacteria isolated from African fermented foods, against some common enteric pathogens was investigated (Kostinek et al., 2005; Savadogo et al., 2004; Mante et al., 2003; Olsen et al., 1995; Mensah et al., 1991). High production of hydrogen peroxide and a bacteriocin by a heterofermentative strain of lactic acid bacteria isolated from fermented cassava was reported (Kostinek et al., 2005). In Burkina–Faso, the production of a bacteriocin by a strain of L. fermentum isolated from dolo was also reported (Savadogo et al., 2004). In Ghana Mante et al., (2003) demonstrated the antimicrobial activity of fermenting University of Ghana http://ugspace.ug.edu.gh 84 cassava dough against Vibrio cholera, Salmonella typhimurium, Salmonella enteritidis, E. coli, and Shigella dysenteriae. LAB produce various antimicrobial compounds, which can be classified as low-molecular- mass compounds such as hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3- butanedione), uncharacterized compounds, and high-molecular-mass, HMM) compounds such as bacteriocins, all of which can antagonize the growth of some spoilage and pathogenic bacteria in foods (Piard and Desmazeaud, 1992). Daeschel, 1989, reported the production of lactic acid and reduction of pH as the primary antimicrobial effect exerted by LAB. 5.6 Microbial Interactions during Fura Fermentation The antimicrobial interactions observed between the different species of microorganisms isolated in the present work were very minimal. This could be due to the fact that the different species of lactic acid bacteria were not delibrately selected from different stages during the fermentation, example at the beginning and end of fermentation. Lactic acid bacteria to lactic acid bacteria interaction can be typified by antagonism where bacteriocin produced by one species or strain inhibits or eliminates another species. Olsen et al., (1995) demonstrated that isolates at the advanced stages of fermentation showed inhibition against isolates from the early stages of maize dough fermentation during kenkey production. In the present work, there were no antimicrobial interactions observed between the different species of lactic acid bacteria and the yeast Saccharomyces cerevisiae or Candida krusei. There were however some amount of interactions between different species of lactic acid bacteria against Candida albicans and Candida membranifascians, which were isolated during steeping. University of Ghana http://ugspace.ug.edu.gh 85 The association between yeast and lactic acid bacteria in different fermented foods have been reported by many authors (Jesperson 2003; Iwuoha and Eke 1996; Jesperson et al., 1994; Oyewole and Odunfa 1990; Halm et al., 1993). In a co-metabolism between yeasts and lactic acid bacteria, the bacteria provide the acidic environment, which select for the growth of yeasts, whereas the yeasts provide vitamins and other growth factors to the bacteria (Gobbetti et al., 1994). Saccharomyces cerevisiae have also been known to stimulate the growth of LAB, by providing essential metabolites such as pyruvates, amino acids and vitamins, whiles the Saccharomyces cerevisiae exploit certain bacterial metabolites as carbon sources (Leroi and Pidoux, 1993; Gadaga et al., 2001) from the bacteria. The association between lactic acid and yeasts has also been suggested as symbiotic (Saunders et al., 1972; Gobbeti et al., 1994). 5.7 Technological Properties The different lactic acid bacteria isolates from Fura showed different rates of acidification during dough fermentation. The fastest rate of acidification during the dough fermentation was demonstrated by L. brevis with a value of 0.44% followed by L. fermentum and W. confusa with values of 0.42% and 0.41% respectively, which gave them an advantage over the other species in starter culture selection. Acidification may influence several quality characteristics of fermented products such as safety (Russell, 1992; Breidt and Fleming, 1997), reduction in fermentation time and organoleptic qualities (Mcfeeters, 2004). The immediate and rapid production of sufficient quantities of organic acids to reduce pH below 4.0 within 24 h of fermentation is an essential requirement of fermented cereal-based foods. Amylolytic lactic acid bacteria have been isolated from cereal fermentation in tropical climates. However, the LAB isolates from Fura exhibited low amylase secretion in the University of Ghana http://ugspace.ug.edu.gh 86 present work. Olasupo et al., (1996) isolated amylolytic lactic acid bacteria from Ghanaian Kenkey (fermented maize dough) and Nono (Nigeria). Agati et al., (1998), found amylolytic L plantarium strains from retted cassava in Nigeria and Congo respectively, while amylolytic L. fermentum strains were isolated from mawe and ogi in Benin. Hounhouigan et al., (1993b) reported some amylolytic lactic acid bacteria in mawe from Benin whiles Johansson et al., 1995 also indicated that amylolytic lactic acid bacteria accounted for 14 % of the total lactic acid bacteria isolated from Nigerian ogi. Amylolytic LAB may reduce the viscosity of bulk starchy weaning gruel to improve nutrient density and maintain an acceptable thickness for feeding young children (FAO/WHO, 1995). Most of the isolates from Fura demonstrated exopolysaccharide production in the present work. Many strains of LAB produce exopolysaccharides (EPS) as capsules tightly attached to the bacterial cell wall, or as a loose slime (ropy polysaccharide) which is released into the substrate (Mayra-Makinen and Bigret, 1998). The production of expolysacharides (EPSs) have acquired a lot of attention due to their contribution to improvement of texture and viscosity of fermented food products (Patricia et al., 2002; Savadogo et al., 2004). Exopolysaccharide-producing (EPS+) starter cultures are commonly used to enhance water binding and viscosity in yogurt and fermented milk since they have viscosity enhancing and stabilizing properties. 5.8 Starter culture selection Several factors need to be considered when selecting LAB strains for cereal fermentation depending on the desired characteristics of the final product, the desired metabolic activities, the characteristics of the raw materials and the applied technology (Soro-Yao et al. 2014). Lactic acid bacteria and yeasts strains have been used successfully as starter cultures in a number of indigenous cereal based fermented foods, due to their desirable effects in such foods (Oyewole, 1990), including the ability to reduce fermentation times, minimize dry University of Ghana http://ugspace.ug.edu.gh 87 matter losses, avoid contamination with pathogenic and toxigenic bacteria and moulds, and reduce the risk of incidental micro-flora causing off-flavours in foods (Haard, 1999). Food preservation by lactic acid fermentation also depends on the removal of fermentable carbohydrates, the consumption of oxygen, the formation of organic acids in addition to a corresponding decrease in pH. In the present work, there was a significant decrease in pH with a corresponding increase in acidity during dough fermentation with respect to inoculation with the starter cultures, with the trend in total titratable acidity directly opposite that observed for pH. Similar findings were obtained by Farahat (1998) where different strains of Lactobacillus were used to ferment dabar, resulting in decrease in pH to 3.7. In a related work, a starter culture consisting of lactic acid bacteria (Lactobacillus fermentum, Lactobacillus brevis and Lactobacillus amylovorus) combined with Saccharomyces cerevisiae, on traditional fermentation of sorghum flour (variety dabar), was able to reduce fermentation time from 19 hours to 4 hours and the pH to 3.47(Asmahan and Muna, 2009). Halm et al., (1996) were able to reduce maize steeping time from 48 to 24h at a semi- commercial kenkey plant using a mixed culture of Lactobacillus fermentum and Saccharomyces cerevisiae. A starter culture of L. plantarum also reduced the pH from 5.9 to 3.4 within 12 h compared to 2-3 days required in the normal traditional process of ―Ogi‖ preparation (Sanni et al., 1994). Masha et al. (1998) compared in laboratory trials, the fermentation of ―Uji‖ with a starter culture of lactic acid bacteria (L. plantarum, L. brevis, L. buchneri, L. paracasei and Pediococcus pentosaceus), using backslopping and spontaneous fermentation at 30ºC and found a decrease in pH from over 5.0 in the unfermented sample to final pH levels of 3.5 with the pure cultures of lactic acid bacteria whiles a pH of 4.1was recorded in the spontaneous fermented Uji. University of Ghana http://ugspace.ug.edu.gh 88 5.9 Shelf life studies Irradiation of the Fura resulted in a reduction in counts of aerobic mesophiles and also yeasts and moulds. Irradiation has been used to extend the shelf life of several other products and has been used for the control of postharvest quality of fresh produce (Niemira and Fan, 2006). Due to fura being a fermented product which has not been given any prior preservation treatment it took a high dose of gamma radiation to reduce the microbial load substantially. Exposure to 2.5kGy of gamma radiation reduced the population of aerobic mesophiles from 109 to 106 CFU/g and 10.0 kGy from 109 to 10 CFU/g. Effect on yeasts and moulds was similar except that counts were about one log unit lower including the initial counts. Fura with an aerobic mesophilic population of 107 to 109 CFU/g and yeasts and moulds population of 106-108 CFU/g has a shelf life of only a few days. Though refrigeration is able to prolong the shelf life it results in a hardening of the surface of the Fura rendering the texture unacceptable by most consumers. With irradiation of 10kGy the aerobic mesophilic population in the sample was reduced from 109 to 10 CFU/g and the yeasts and moulds from 109 to between 0 and 10 CFU/g. Irradiation extended the shelf life of the product by four weeks whereas vacuum packaging extended the shelf life for two weeks. University of Ghana http://ugspace.ug.edu.gh 89 CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions Though the survey showed that Fura was not fermented by the processors, this work shows that it is possible to ferment Fura and has confirmed the fermentation to be lactic acid fermentation during steeping and dough fermentation as reported in literature. The fermentation is characterized by reduction in pH and its corresponding increases in titratable acidity to improve the safety of the product and also give it better antimicrobial properties. The work has been able to isolate the dominant lactic acid bacteria and yeasts responsible for Fura fermentation and combined them as starter cultures, the use of which will help standardize the product. With irradiation of 10kGy, the microbial load on Fura was reduced from 9 to between 0 and 1 log CFU/g. Gamma radiation also extended the shelf life of Fura for four extra weeks while vacuum packaging extended it for two extra weeks. 6.2 Recommendation(S) It is recommended that a sensory evaluation be conducted using the isolates and the various combinations to come out with an optimal product. The mycotoxin (aflatoxin) content of both the raw material (millet) and the final product should be investigated. Good methods of storing the cultures isolated in the present work should be ensured to forestall their loss. University of Ghana http://ugspace.ug.edu.gh 90 CHAPTER SEVEN REFERENCES Adam E. Y., Abdel, M.E.S. and Warda S. A. G. (2009): Effect of fermentation on the nutritional and microbiological quality of dough of different sorghum varieties. Journal of Science and Technology. 10 (3). Adams M.R. (1990): Topical aspects of fermented foods. Trends in Food Science and Technology 1, 141-144. Adams, M.R. and Moss, M.O. (1995): Fermented and Microbial foods. 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Pot, B. Karel, K., Kalantzopouloset, G., (1994): Thecombined use of whole-cell protein extracts for the identification (SDS- PAGE) and enzyme activity screening of lactic acid bacteria isolated from traditional Greek dairy products.Systematics and Applied Microbiology 17,444–58. Uriaza, P., Gomez ñZavaglıa, A., Lozano, M.,Romanowski, V., and Graciella, A. DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence based polymerase chain reaction, Journal of Dairy Reseach, 67, 381-392 Van der Merwe, A. le R., Schweigart, F. and Cachia, V.A. (1964): Mahewu- it‟s industrial production and its value as a nutrient. Vitalsoffe zivilsationskrankenheiten.pp.7 Vandamme, P., Pot, B., Gill, M., de Vos P., Kerster, K., Swings, J. (1996): Polyphastic taxonomy, a consensus approach to bacteria systematic. Microbiology. Rev. 60: 407-438. Vieira-Dalodé, G., Jespersen, L., Hounhouiganl, J., Moller, P.L., Nago, C.M., Jakobsen, M., (2007): Lactic acid bacteria and yeasts associated with gowe production from sorghum in Benin. Journal of Applied Microbiology 103, 342-349. Wagner, M.K., and Moberg, L.J. (1989): Present and future use of traditional antimicrobials. Food Technology 1, 143-147. William, R.A.D., Sandler, S.A., (1971): Electrophoresis of glucose-6-phosphate dehydrogenase, cell wall composition and the taxonomy of heterofermentative lactobacilli. Journal of General Microbiology 65, 351–358. Wong, H.-C., and Chen, Y.-L. (1988): Effects of lactic acid bacteria and organic acids on growth and germination of Bacillus cereus. Appl. Environ. Microbiol. 54, 2179-2184. University of Ghana http://ugspace.ug.edu.gh 118 Woolford, M.K. (1975): Microbiological screening of food preservatives, cold sterilants and specific antimicrobial agents as potential silage additives. J. Sci. Food Agric. 26, 229- 237. World Health Organization, WHO, and Food and Agriculture Organization of the United Nations, FAO, (1988): Food irradiation: A technique for preserving and improving the safety of food. Geneva. Wu, Z.Y., Zhang, W.X., Zhang, Q.S., Hu, C., Wang, R., Liu, Z.H. (2009): Developing New Sacchariferous Starters for Liquor Production Based on Functional Strains Isolated from the Pits of Several Famous Luzhou-flavor Liquor Brewers. Journal of the institute of brewing 115 (2): 111–115 Wyman, J. (1862): "Spontaneous generation". British Medical Journal 2(90): 311–312. University of Ghana http://ugspace.ug.edu.gh 119 APPENDIX APPENDIX 1 Carbohydrate Fermentation Profile of Lactic Acid Bacteria CARBOHY DRATES ISOLATES L. ferme ntum 1 L. brevis 2 W. confusa P. acidilactic i Lactococcus lactis spp lactis 1 Lactococcus rafinolactis 1. Glycerol - - - - - - 2. Erythritol - - - - - - 3. D-arabinose - - - - - - 4. L-arabinose + + + + + + 5. Ribose + + + + + + 6. D-xylose + + + - + + 7. L-xylose - - - - - - 8. Adonitol - - - - - - 9. β methyl- xyloside - - - + - - 10. Galactose + + + + + + 11. D-Glucose + + + + + + 12. D-fructose + + + + + + 13. D-mannose + - + - + + 14. L-sorbose - - + + - - 15. Rhamnose - - + - - - 16. Dulcitol - - - - - - 17. Inositol - - - - - - 18. Mannitol - - + - + - 19. Sorbitol - - + - - - 20. α methyl-D- mannose - - - - - - 21. α methyl-D- glucoside - - - - + - 22. N acethyl glucosamide + - + + + + 23. Amygdaline - - + + + + 24. Arbutin + - + + + + 25. Esculin - - + - + 26. Salicin + - + + + + 27. Cellobiose + - + + + + 28. Maltose + + + + + + 29. Lactose + - + - - - 30. Melibiose + + - - - + 31. Saccharose + + + - + + University of Ghana http://ugspace.ug.edu.gh 120 32. Trehalose + + + + + + 33. Inulin - - - - - - 34. Melezitose - + - - - - 35. D-raffinose + + + - + - 36. Amidon - - - - - + 37. Glycogen - - - - + + 38. Xylitol - - - - - + 39. β gentiobiose + - + + - + 40. D-turanose - + - - + - 41. D-lyxose + - - - - 42. D-tagatose - - - - - 43. D-fucose - - - - - - 44. L-fucose - - - - - - 45. D-arabitol - - - - - - 46. L-arabitol - - - - - - 47. Gluconate + + + - + - 48. 2 ceto- gluconate - - - - - - 49. 5 cetoglunate + + - - - - University of Ghana http://ugspace.ug.edu.gh 121 APPENDIX 2 A. Mean pH values during fermentation of millet into “Fura”. Means with same letters in a row are not significantly different (p<0.05) B. Mean Titratable Acidity (%) during fermentation of millet into “fura” Means with same letters in a row are not significantly different (p<0.05) Sample pH Values Steep water Processor 1 Processor 2 Processor 3 Processor 4 0hr 6.05±0.01b 5.92±0.01a 5.90±0.01a 5.89±0.01a 6hr 5.60±0.14c 5.49±0.01bc 5.46±0.02b 5.32±0.01a 12hr 4.92±0.01b 4.89±0.01b 4.92±0.01b 4.94±0.01b Fermenting dough 0hr 5.22±0.01b 4.83±0.01a 5.10±0.07b 5.00±0.03b 6hr 4.45±0.04c 4.28±0.03a 4.55±0.02d 4.35±0.01b 12hr 3.75±0.07a 3.69±0.06a 3.84±0.01ab 3.98±0.01a Sample Mean Titratable Values Steep water Processor 1 Processor 2 Processor 3 Processor 4 0h 6h 12h 0.02±0.01a 0.05±0.01a 0.12±0.02a 0.01±0.01a 0.04±0.01a 0.15±0.01b 0.03±0.01a 0.16±0.01d 0.27±0.01d 0.04±0.01b 0.09±0.01c 0.21±0.01c Fermenting dough 0h 6h 12h 0.13±0.02a 0.21±0.01a 0.27±0.02a 0.20±0.14b 0.26±0.01b 0.35±0.01b 0.27±0.01d 0.36±0.02d 0.38±0.03c 0.23±0.02c 0.32±0.01c 0.36±0.01b University of Ghana http://ugspace.ug.edu.gh 122 C. Changes in Titratable acidity during acidification of fermenting dough by lactic acid bacteria Means with same letters in a row are not significantly different (p<0.05) Fermentation time(h) Mean titrable acidity of samples Spontane ous L. rafinolacti s L. fermentu m 1 W. confusa L. fermentu m 1 L.lactis ssp. Lactis P. acidilactic i L. brevis 0 0.07±0.01 0.09±0.01 0.11±0.01 0.09±0.01 0.09±0.01 0.09±0.01 0.09±0.01 0.12±0.01 4 0.11±0.03 0.14±0.01 0.18±0.02 0.16±0.01 0.15±0.01 0.13±0.02 0.14±0.01 0.19±0.01 8 0.18±0.02 0.22±0.01 0.29±0.01 0.27±0.02 0.24±0.01 0.24±0.01 0.25±0.01 0.30±0.07 12 0.22±0.03 0.36±0.01 0.42±0.01 0.41±0.01 0.35±0.01 0.37±0.04 0.40±0.01 0.44±0.01 University of Ghana http://ugspace.ug.edu.gh 123 D. Acidification of Fermenting Dough in Fermentation Trials with Starter cultures Fermentation types Fermentation time(h) Control/sp ontaneous L. fermentum L.brevis W. confusa S. cerevisiae C. krusei pH of fermenting dough 0 6.44±0.01d 6.43±0.02c 6.43±0.02c 6.34±0.03ab 6.34±0.01b 6.32±0.02a 4 6.53±0.02a 6.01±0.03a 6.01±0.01a 5.8±0.02a 6.09±0.02a 5.91±0.02a 8 6.44±0.02b 5.01±0.06c 4.94±0.08 a 4.71±0.02c 4.89±0.02c 4.6±0.02c 12 5.44±0.02e 4.44±0.02b 4.41±0.01bc 4.44±0.02c 4.50±0.01d 3.98±0.02a Titratable acidity of fermenting dough 0 0.11±0.02a 0.14±0.02a 0.13±0.01a 0.11±0.02a 0.12±0.03a 0.14±0.02a 4 0.12±0.02a 0.15±0.01b 0.150.01±b 0.17±0.01b 0.15±0.01b 0.16±0.02b 8 0.20±0.02 0.27±0.01b 0.29±0.01b 0.28±0.01b 0.27±0.03b 0.3±0.01ba 12 0.22±0.02a 0.4±0.01c 0.46±0.01d 0.43±0.01d 0.37±0.01b 0.35±0.03b Means with same letters in a row are not significantly different (p<0.05) University of Ghana http://ugspace.ug.edu.gh 124 E. Acidification of Fermenting Dough in Fermentation Trials with combined Starter cultures Fermenation Type Time Spontaneous L. fermentum + C. krusei L. fermentum + S. cerevisiae W. confusa + C. krusei W. Confusa + S. cerevisiae L. brevis + S. cerevisiae L. Brevis + Candida krusei pH of fermenting dough 0 6.47±0.03a 6.4±0.03 a 6.42±0.02a 6.38±0.06a 6.44±0.02a 6.42±0.02a 6.44±0.03a 4 6.36±0.04d 6.22±0.09c 6.16±0.03b 6.36±0.03d 6.34±0.01d 6.08±0.02a 6.19±0.03b 8 6.05±0.07d 4.3±0.02c 4.06±0.01a 4.29±0.01c 4.17±0.01b 4.11±0.03b 4.06±0.02a 12 5.92±0.02d 4.02±0.06c 3.93±0.02b 4.02±0.01c 4.01±0.02c 3.83±0.05a 3.95±0.02b Titratable acidity of fermenting dough 0 0.18±0.03a 0.18±02a 0.18±0.01a 0.19±0.02a 0.18±0.01a 0.19±0.01a 0.19±0.02a 4 0.2±0.03a 0.2±0.02a 0.26±0.01b 0.33±0.01d 0.22±0.02a 0.3±0.01c 0.29±0.02c 8 0.24±0.01a 0.34±0.02b 0.39±0.02c 0.36±0.03bc 0.44±0.03d 0.51±0.02d 0.47±0.01e 12 0.27±0.03a 0.45±0.01b 0.53±0.02b 0.51±0.01c 0.48±0.02d 0.62±0.02c 0.45±0.02d Means with same letters in a row are not significantly different (p<0.05) University of Ghana http://ugspace.ug.edu.gh 125 APPENDIX 3 ANOVA TABLES 1.0 ANOVA TABLES FOR ACIDIFICATION DURING SPONTANEOUS FERMENTATION A. ANOVA table for pH of steep water during spontaneous fermentations Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.043 3 0.014 10.121 0.004 Within Groups 0.011 8 0.001 Total 0.055 11 6h Between Groups 0.128 3 0.043 15.410 0.001 Within Groups 0.022 8 0.003 Total 0.150 11 12h Between Groups 0.005 3 0.002 9.783 0.005 Within Groups 0.001 8 0.000 Total 0.006 11 Significant difference P˂0.05 B. ANOVA table for pH of fermenting dough during spontaneous fermentations Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.288 3 0.096 5.523 0.024 Within Groups 0.139 8 0.017 Total 0.427 11 6h Between Groups 0.107 3 0.036 37.252 0.000 Within Groups 0.008 8 0.001 Total 0.115 11 12h Between Groups 1.198 3 0.399 4.544 0.039 Within Groups 0.703 8 0.088 University of Ghana http://ugspace.ug.edu.gh 126 B. ANOVA table for pH of fermenting dough during spontaneous fermentations Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.288 3 0.096 5.523 0.024 Within Groups 0.139 8 0.017 Total 0.427 11 6h Between Groups 0.107 3 0.036 37.252 0.000 Within Groups 0.008 8 0.001 Total 0.115 11 12h Between Groups 1.198 3 0.399 4.544 0.039 Within Groups 0.703 8 0.088 Total 1.901 11 Significant difference P˂0.05 C. ANOVA table for TTA of steep water during spontaneous fermentations Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.002 3 0.001 8.167 0.008 Within Groups 0.001 8 0.000 Total 0.002 11 6h Between Groups 0.028 3 0.009 65.784 0.000 Within Groups 0.001 8 0.000 Total 0.029 11 12h Between Groups 0.044 3 0.015 103.608 0.000 Within Groups 0.001 8 0.000 Total 0.045 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 127 D. ANOVA table for TTA of fermenting dough during spontaneous fermentations Sum of Squares df Mean Square F P-Value TIME0 Between Groups 0.028 3 0.009 69.500 0.000 Within Groups 0.001 8 0.000 Total 0.029 11 TIME6 Between Groups 0.033 3 0.011 55.611 0.000 Within Groups 0.002 8 0.000 Total 0.035 11 TIME12 Between Groups 0.018 3 0.006 24.356 0.000 Within Groups 0.002 8 0.000 Total 0.020 11 Significant difference P˂0.05 2.0 ANOVA TABLES FOR MICROBIAL POPULATION DURING SPONTANEOUS FERMENTATION A. ANOVA table for population of LAB during spontaneous steeping Sum of Squares Df Mean Square F P-Value 0h Between Groups 5.327 3 1.776 275.995 0.000 Within Groups 0.051 8 0.006 Total 5.378 11 6h Between Groups 16.804 3 5.601 944.039 0.000 Within Groups 0.047 8 0.006 Total 16.851 11 12h Between Groups 16.233 3 5.411 483.857 0.000 Within Groups 0.089 8 0.011 University of Ghana http://ugspace.ug.edu.gh 128 A. ANOVA table for population of LAB during spontaneous steeping Sum of Squares Df Mean Square F P-Value 0h Between Groups 5.327 3 1.776 275.995 0.000 Within Groups 0.051 8 0.006 Total 5.378 11 6h Between Groups 16.804 3 5.601 944.039 0.000 Within Groups 0.047 8 0.006 Total 16.851 11 12h Between Groups 16.233 3 5.411 483.857 0.000 Within Groups 0.089 8 0.011 Total 16.323 11 Significant difference P˂0.05 B. ANOVA table for population of LAB during spontaneous dough fermentation Sum of Squares Df Mean Square F P-Value 0h Between Groups 4.776 3 1.592 509.492 0.000 Within Groups 0.025 8 0.003 Total 4.801 11 6h Between Groups 4.194 3 1.398 315.370 0.000 Within Groups 0.035 8 0.004 Total 4.230 11 12h Between Groups 4.534 3 1.511 280.712 0.000 Within Groups 0.043 8 0.005 Total 4.577 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 129 C. ANOVA table for population of yeasts during spontaneous steeping Sum of Squares Df Mean Square F P-Value 0h Between Groups 3.981 3 1.327 419.048 0.000 Within Groups 0.025 8 0.003 Total 4.006 11 6h Between Groups 0.860 3 0.287 60.480 0.000 Within Groups 0.038 8 0.005 Total 0.898 11 12h Between Groups 4.247 3 1.416 285.998 0.000 Within Groups 0.040 8 0.005 Total 4.287 11 Significant difference P˂0.05 D. ANOVA table for population of yeasts during spontaneous dough fermentation Sum of Squares Df Mean Square F P-Value 0h Between Groups 1.501 3 0.500 125.878 0.000 Within Groups 0.032 8 0.004 Total 1.533 11 6h Between Groups 5.697 3 1.899 1.245E3 0.000 Within Groups 0.012 8 0.002 Total 5.709 11 12h Between Groups 2.470 3 0.823 318.761 0.000 Within Groups 0.021 8 0.003 Total 2.491 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 130 E. ANOVA table for population of Aerobic Mesophiles during spontaneous steeping Sum of Squares Df Mean Square F P-Value 0h Between Groups 1.495 3 0.498 623.000 0.000 Within Groups 0.006 8 0.001 Total 1.502 11 6h Between Groups 4.302 3 1.434 2.325E3 0.000 Within Groups 0.005 8 0.001 Total 4.307 11 12h Between Groups 4.872 3 1.624 276.431 0.000 Within Groups 0.047 8 0.006 Total 4.919 11 Significant difference P˂0.05 F. ANOVA table for population of Aerobic Mesophiles during spontaneous dough fermentation Sum of Squares Df Mean Square F P-Value 0h Between Groups 6.798 3 2.266 3.675E3 0.000 Within Groups 0.005 8 0.001 Total 6.803 11 6h Between Groups 16.733 3 5.578 8.472E3 0.000 Within Groups 0.005 8 0.001 Total 16.738 11 12h Between Groups 1.020 3 0.340 537.066 0.000 Within Groups 0.005 8 0.001 Total 1.025 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 131 G. ANOVA table for population of Total Coliforms during spontaneous steeping Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.056 3 0.019 7.599 0.010 Within Groups 0.020 8 0.002 Total 0.076 11 6h Between Groups 3.988 3 1.329 1.173E3 0.000 Within Groups 0.009 8 0.001 Total 3.997 11 12h Between Groups 2.571 3 0.857 1.870E3 0.000 Within Groups 0.004 8 0.000 Total 2.574 11 Significant difference P˂0.05 H. ANOVA table for population of Total Coliforms during spontaneous dough fermentation Sum of Squares Df Mean Square F P-Value 0h Between Groups 3.136 3 1.045 2.727E3 0.000 Within Groups 0.003 8 0.000 Total 3.139 11 6h Between Groups 9.613 3 3.204 5.268E3 0.000 Within Groups 0.005 8 0.001 Total 9.618 11 12h Between Groups 1.655 3 0.552 367.709 0.000 Within Groups .012 8 0.001 Total 1.667 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 132 3.0 ANOVA TABLES FOR CULTURE TRIALS A. ANOVA table for Microbial Population for fermentation with Single Starter Cultures (lactic acid bacteria) Sum of Squares Df Mean Square F P-Value 0h Between Groups 14.561 3 4.854 3.426E3 0.000 Within Groups 0.011 8 0.001 Total 14.572 11 4h Between Groups 21.136 3 7.045 765.800 0.000 Within Groups 0.074 8 0.009 Total 21.210 11 8h Between Groups 27.736 3 9.245 853.425 0.000 Within Groups 0.087 8 0.011 Total 27.823 11 12h Between Groups 8.702 3 2.901 54.051 0.000 Within Groups 0.429 8 0.054 Total 9.132 11 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 133 A. ANOVA table for Microbial Population for fermentation with Single Starter Cultures (yeasts) Significant difference P˂0.05 Sum of Squares Df Mean Square F P-Value 0h Between Groups 22.693 2 11.347 2.431E4 0.000 Within Groups 0.003 6 0.000 Total 22.696 8 4h Between Groups 1.457 2 0.728 59.926 0.000 Within Groups 0.073 6 0.012 Total 1.530 8 8h Between Groups 8.545 2 4.272 362.412 0.000 Within Groups 0.071 6 0.012 Total 8.616 8 12h Between Groups 0.690 2 0.345 91.076 0.000 Within Groups 0.023 6 0.004 Total 0.713 8 B. ANOVA table for Microbial Population for fermentation with Combined Starter Cultures Sum of Squares Df Mean Square F P-Value 0h Between Groups 1.823 6 0.304 93.557 0.000 Within Groups 0.045 14 0.003 Total 1.868 20 4h Between Groups 2.952 6 0.492 649.883 0.000 Within Groups 0.011 14 0.001 Total 2.963 20 8h Between Groups 31.361 6 5.227 110.072 0.000 Within Groups 0.665 14 0.047 Total 32.026 20 12h Between Groups 9.315 6 1.553 1.274E3 0.000 Within Groups 0.017 14 0.001 Total 9.332 20 University of Ghana http://ugspace.ug.edu.gh 134 4.0 ANOVA TABLES FOR ACIDIFICATIION DURING STARTER CULTURE TRIALS A. ANOVA table for pH of dough fermentation with single starter cultures Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.125 5 0.025 102.241 0.000 Within Groups 0.003 12 0.000 Total 0.128 17 4h Between Groups 0.930 5 0.186 608.538 0.000 Within Groups 0.004 12 0.000 Total 0.933 17 8h Between Groups 6.823 5 1.365 1.861E3 0.000 Within Groups 0.009 12 0.001 Total 6.832 17 12h Between Groups 3.488 5 0.698 2.854E3 0.000 Within Groups 0.003 12 0.000 Total 3.491 17 Significant difference P˂0.05 B. ANOVA table for TTA of dough fermentation with single starter cultures Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.003 5 0.001 1.305 0.325 Within Groups 0.006 12 0.000 Total 0.009 17 4h Between Groups 0.004 5 0.001 4.094 0.021 Within Groups 0.002 12 0.000 Total 0.006 17 University of Ghana http://ugspace.ug.edu.gh 135 8h Between Groups 0.020 5 0.004 20.378 0.000 Within Groups 0.002 12 0.000 Total 0.023 17 12h Between Groups 0.164 5 0.033 140.229 0.000 Within Groups 0.003 12 0.000 Total 0.166 17 Significant difference P˂0.05 C. ANOVA table for pH of dough fermentation with combined starter cultures Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.125 5 0.025 102.241 0.000 Within Groups 0.003 12 0.000 Total 0.128 17 4h Between Groups 0.930 5 0.186 608.538 0.000 Within Groups 0.004 12 0.000 Total 0.933 17 8h Between Groups 6.823 5 1.365 1.861E3 0.000 Within Groups 0.009 12 0.001 Total 6.832 17 12h Between Groups 3.488 5 0.698 2.854E3 0.000 Within Groups 0.003 12 0.000 Total 3.491 17 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 136 D. ANOVA table for TTA of dough fermentation with combined starter cultures Sum of Squares Df Mean Square F P-Value 0h Between Groups 0.001 6 0.000 0.886 0.530 Within Groups 0.003 14 0.000 Total 0.003 20 4h Between Groups 0.046 6 0.008 27.582 0.000 Within Groups 0.004 14 0.000 Total 0.050 20 8h Between Groups 0.150 6 0.025 69.132 0.000 Within Groups 0.005 14 0.000 Total 0.155 20 12h Between Groups 0.245 6 0.041 76.411 0.000 Within Groups 0.007 14 0.001 Total 0.252 20 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 137 5.0 ANOVA TABLES FOR MICROBIAL POPULATIONS DURING CHALLENGE TESTING A. ANOVA table for count for survival of Vibrio cholerae inoculated into spontaneous and mixed culture fermentation of millet dough Sum of Squares Df Mean Square F P-Value 0h Between Groups 3.220 6 0.537 7.213 0.001 Within Groups 1.042 14 0.074 Total 4.262 20 4h Between Groups 7.669 6 1.278 29.741 0.000 Within Groups .602 14 0.043 Total 8.270 20 8h Between Groups 17.187 6 2.865 93.992 0.000 Within Groups .427 14 0.030 Total 17.614 20 Significant difference P˂0.05 A. ANOVA table for count for survival of Staphylococcus aureus inoculated into spontaneous and mixed culture fermentation of millet dough Sum of Squares Df Mean Square F P-Value 0h Between Groups 17.280 6 2.880 23.045 .000 Within Groups 1.750 14 0.125 Total 19.030 20 4h Between Groups 13.537 6 2.256 28.472 0.000 Within Groups 1.109 14 0.079 Total 14.647 20 8h Between Groups 5.378 6 0.896 28.687 0.000 University of Ghana http://ugspace.ug.edu.gh 138 Within Groups 0.437 14 0.031 Total 5.815 20 12h Between Groups 9.988 6 1.665 1.079E3 0.000 Within Groups 0.022 14 0.002 Total 10.010 20 Significant difference P˂0.05 B. ANOVA table for count for survival of Salmonella typhimurium inoculated into spontaneous and mixed culture fermentation of millet dough Sum of Squares Df Mean Square F P-Value 0h Between Groups 2.800 6 .467 302.472 .000 Within Groups .022 14 .002 Total 2.822 20 4h Between Groups 2.892 6 0.482 6.690 0.002 Within Groups 1.009 14 0.072 Total 3.900 20 8h Between Groups 3.124 6 0.521 16.664 0.000 Within Groups 0.437 14 0.031 Total 3.561 20 12h Between Groups 11.886 6 1.981 1.040E3 0.000 Within Groups 0.027 14 0.002 Total 11.912 20 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 139 C. ANOVA table for count for survival of E.coli inoculated into spontaneous and mixed culture fermentation of millet dough Sum of Squares Df Mean Square F P-Value 0h Between Groups 5.891 6 0.982 636.417 0.000 Within Groups 0.022 14 0.002 Total 5.913 20 4h Between Groups 3.823 6 0.637 10.666 0.000 Within Groups 0.836 14 0.060 Total 4.659 20 8h Between Groups 16.037 6 2.673 99.788 0.000 Within Groups 0.375 14 0.027 Total 16.412 20 12h Between Groups 17.039 6 2.840 412.985 0.000 Within Groups 0.096 14 0.007 Total 17.135 20 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh 140 Significant difference P˂0.05 ANOVA for count of Aerobic Mesophiles during the storage of Fura samples Sum of Squares Df Mean Square F P-Value wk0 Between Groups 345.042 7 49.292 1.593E3 0.000 Within Groups 0.495 16 0.031 Total 345.537 23 wk1 Between Groups 202.247 7 28.892 612.129 0.000 Within Groups 0.755 16 0.047 Total 203.003 23 wk3 Between Groups 54.017 7 7.717 187.774 0.000 Within Groups 0.658 16 0.041 Total 54.675 23 wk4 Between Groups 287.842 7 41.120 1.490E3 0.000 Within Groups 0.442 16 0.028 Total 288.283 23 wk5 Between Groups 355.712 7 50.816 1.260E3 0.000 Within Groups 0.645 16 0.040 Total 356.357 23 wk6 Between Groups 376.278 7 53.754 3.392E3 0.000 Within Groups 0.254 16 0.016 Total 376.531 23 wk7 Between Groups 404.298 7 57.757 3.286E3 0.000 Within Groups 0.281 16 0.018 Total 404.579 23 University of Ghana http://ugspace.ug.edu.gh 141 ANOVA for count of Aerobic Yeasts and Moulds during the storage of Fura samples Sum of Squares Df Mean Square F P-Value wk0 Between Groups 241.379 7 34.483 879.943 0.000 Within Groups 0.627 16 0.039 Total 242.006 23 wk1 Between Groups 134.140 7 19.163 456.985 0.000 Within Groups 0.671 16 0.042 Total 134.811 23 wk3 Between Groups 62.999 7 9.000 157.501 0.000 Within Groups 0.914 16 0.057 Total 63.913 23 wk4 Between Groups 155.494 7 22.213 5.065 0.003 Within Groups 70.171 16 4.386 Total 225.665 23 wk5 Between Groups 365.327 7 52.190 2.306E3 0.000 Within Groups 0.362 16 0.023 Total 365.689 23 wk6 Between Groups 383.078 7 54.725 2.875E3 0.000 Within Groups 0.305 16 0.019 Total 383.383 23 wk7 Between Groups 393.277 7 56.182 3.010E3 0.000 Within Groups 0.299 16 0.019 Total 393.576 23 Significant difference P˂0.05 University of Ghana http://ugspace.ug.edu.gh