UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES SCHOOL OF BIOLOGICAL SCIENCES PROCESS OPTIMIZATION OF ZOOM KOOM BY EMMANUEL TEI-MENSAH (10875457) THIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN FOOD SCIENCE DEGREE. APRIL, 2023 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh i DECLARATION University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh ii DEDICATION I dedicate this work to my mentor Mr. Ebenezer Nartey for making the completion of this MPhil. programme successful. I also thank Mr. Francis Cosmos Baiden for his support both in prayers and finances. My special thanks also go to my lovely wife Akua Nana Safo, also go to Emmanuel Tei-Mensah Junior and daughter Adwoa Akyaa Narkie Mensah for their care and understanding throughout this programme. A special dedication to my late mum, Faustina Yaa Amanfo who really sacrificed for me. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEGEMENT I wish to honestly thank my supervisors, Dr. Bennett Dzandu, Dr. Idolo Ifie and Prof. John Owusu for their constructive criticisms and guidance which have brought this work to a successful completion. My special thanks also go to Prof. F. K. Saalia, Prof. Esther Sakyi-Dawson, Dr. Angela Parry-Hanson Kunadu, Dr. Maame Yaakwah Blay Adjei, Dr. Joycelyn Quansah and Dr. Newlove Afoakwa for their advice and guidance towards the success of this work. I also thank Mr. Ebenezer Nartey and Miss Araba Dhailly for their constructive criticisms, guidance and mentorship which have brought this work to a successful completion. I am grateful to the department of Food and Postharvest Technology of Koforidua Technical University-Ghana and Leeds University, UK for allowing me use their laboratories for some of my analysis. I am also appreciative to Mr. Eric Tetteh and Mr. Daniel Quaye for assisting me with some of the laboratory analysis. I am highly indebted to Christian Ofosu, Lucy Asare, Samuel Frimpong, Sandra Darko, Priscilla Barnes, Peggy Dadzie, Beatrice Tagoe, Jerusha Tabiri, Lisa Boahemaa, Portia Owusu, Desmond Karikari and Frank Dwomfoh for their massive support during my entire M.Phil. study. Special thanks to all Zoom-koom sellers at Nima, Mallam Atta market, Koforidua, Ashaiman- Tulaku and Amansaman for their willingness to participate in the survey work. I am grateful to Prof. John Owusu, Mr. William Odoom, Mr. Charles Adomako, Dr. Mrs. Vida Edusei, Mrs Regina Ofori Asante, Ryan Osei-Kusi Assibey and Mr. Yaw Gyau Akyereko , all of Food and Postharvest Technology Department, at Koforidua Technical University for their support and encouragement throughout my M.Phil. programme. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh iv Finally, I am most grateful to Mr. Francis Cosmos Baiden, my wife, son and daughter for their support and encouragement. I am above all most grateful to God almighty for giving me strength and wisdom to carry out this work. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS DECLARATION............................................................................................................................ i DEDICATION............................................................................................................................... ii ACKNOWLEGEMENT.............................................................................................................. iii LIST OF ACRONYMS ............................................................................................................. xiv ABSTRACT ................................................................................................................................ xvi CHAPTER ONE ........................................................................................................................... 1 Introduction ..................................................................................................................................... 1 1.1 General Overview ..................................................................................................................... 1 1.2 Rational for the Study ............................................................................................................... 5 1.3 Main objective .......................................................................................................................... 6 Specific Objectives ......................................................................................................................... 6 CHAPTER TWO ............................................................................................................................ 7 2.0 LITERATURE REVIEW ......................................................................................................... 7 2.1 Introduction ............................................................................................................................... 7 2.2 ‘Zoom-Koom’ (millet beverage) ............................................................................................... 8 2.2.1 Processing of Zoom-koom ...................................................................................................... 9 2.2.1.1 Steeping/Soaking ................................................................................................................ 9 2.2.1.2 Wort extraction ................................................................................................................. 10 2.2.1.3 Addition of sweeteners and storage .................................................................................. 10 2.3 Other Traditional millet-based beverages ............................................................................... 11 2.3.1 Sur ........................................................................................................................................ 11 2.3.2 Madua .................................................................................................................................. 11 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh vi 2.3.3 Kunnu-zaki ........................................................................................................................... 12 2.3.4 Oshikundu ............................................................................................................................ 13 2.3.5 Koozh ................................................................................................................................... 13 2.4 Millet and its nutritional value ................................................................................................ 14 2.4.1 Structure and Chemical Composition of Millet ................................................................... 15 2.4.2 Millet Production and consumption in Ghana ..................................................................... 18 2.5 Nutrient Profile of millet – based beverages ........................................................................... 20 2.5.1 Phenolic compounds and Antioxidant activity .................................................................... 20 2.5 Physicochemical and Microbial Quality of “Zoom-koom” .................................................... 22 2.5.1 Physicochemical Quality of “Zoom-koom” and millet-based beverage .............................. 22 2.5.2 Microbial Quality of “Zoom-koom” and millet-based beverage ......................................... 24 2.6. Microbial Safety of Beverages ............................................................................................... 25 2.6.1 Enterobacteriaceae ............................................................................................................... 25 2.6.2 E. coli ................................................................................................................................... 26 2.6.3 Staphylococcus aureus ......................................................................................................... 26 2.6.4 Yeast and Mould .................................................................................................................. 27 CHAPTER THREE ...................................................................................................................... 28 3.0 Materials and Methods ............................................................................................................ 28 3.1.1 Sampling site and procedure ................................................................................................ 28 3.1.2 Microbiological analysis ...................................................................................................... 29 3.1.2.1 Sample preparation ........................................................................................................... 29 3.1.2.2 Media preparation ............................................................................................................. 29 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh vii 3.1.2.3 Enumeration of Total bacteria, yeasts and moulds, total coliform, Escherichia coli and Staphylococcus aureus .................................................................................................................. 30 3.2 Physicochemical Analysis ...................................................................................................... 30 3.2.1 pH ......................................................................................................................................... 30 3.2.2 Titratable Acidity ................................................................................................................. 30 3.2.3 Brix ...................................................................................................................................... 31 3.2.4 Colour .................................................................................................................................. 31 3.3 Chemical Analysis .................................................................................................................. 32 3.3.1 Determination of total phenol compounds ........................................................................... 32 3.3.1.1 Sample preparation ........................................................................................................... 32 3.3.2 Antioxidant Activity ............................................................................................................ 33 3.4 Optimization of process for zoom-koom using Response Surface Methodology ................... 33 3.4.1 Materials .............................................................................................................................. 33 3.4.2 Design of optimization ......................................................................................................... 33 3.4.3 Preparation of zoom-koom ................................................................................................... 36 3.4.4 Consumer sensory acceptance of zoom-koom...................................................................... 37 3.5 Proximate composition of optimized zoom-koom................................................................... 37 3.5.1 Moisture content determination ........................................................................................... 38 3.5.2 Crude fat determination by goldfish apparatus method ....................................................... 38 3.5.3 Ash content determination ................................................................................................... 39 3.5.4 Crude fiber content determination ....................................................................................... 39 3.5.5 Crude protein content determination ................................................................................... 40 3.5.6 Carbohydrate /nitrogen free extract ..................................................................................... 40 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh viii 3.5.7 Energy .................................................................................................................................. 41 3.6 Data and Statistical analysis.................................................................................................... 41 CHAPTER FOUR ......................................................................................................................... 42 4.0 Results and Discussion ........................................................................................................... 42 4.1 Physicochemical properties of commercially processed zoom-koom ..................................... 42 4.2 Microbial quality of commercially processed zoom-koom ..................................................... 45 4.3 Bioactive Properties of commercially processed zoom-koom ................................................ 48 4.4 Influence of process variables on the physicochemical properties of laboratory-produced zoom- koom. ............................................................................................................................................. 49 4.4.1 Contour plots of the physicochemical properties of zoom-koom ......................................... 54 4.5 Influence of process variables on the microbiological quality of laboratory-produced zoom- koom. ............................................................................................................................................. 61 4.6 Influence of process variables on bioactive properties of laboratory-produced zoom-koom. . 67 4.7 Consumer acceptability of laboratory produced zoom-koom ................................................. 71 4.8: Modelling of the sensory attributes of laboratory produced zoom-koom .............................. 72 4.8.1 Appearance .......................................................................................................................... 74 4.8.2 Aroma .................................................................................................................................. 75 4.8.3 Flavour ................................................................................................................................. 76 4.8.4 Mouthfeel ............................................................................................................................. 77 4.8.5 Aftertaste .............................................................................................................................. 78 4.8.6 Overall Acceptability ........................................................................................................... 79 4.9 Selection of optimum conditions ............................................................................................ 80 4.10 Verification of optimized conditions .................................................................................... 82 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh ix 4.11. Physicochemical, microbiological and Bioactive properties of commercially processed zoom-koom and optimised zoom-koom ......................................................................................... 86 4.12 Proximate composition of optimized zoom-koom................................................................. 91 CHAPTER 5 ................................................................................................................................. 93 5.0 Conclusion and Recommendation .......................................................................................... 93 5.1 Conclusion .............................................................................................................................. 93 5.2 Recommendations ................................................................................................................... 94 APPENDICES .............................................................................................................................. 95 APPENDIX 1: PHYSICOCHEMICAL PROPERTIES OF ZOOM-KOOM SAMPLES ............ 95 APPENDIX 2: MICROBIOLOGICAL QUALITY OF ZOOM-KOOM SAMPLES .................. 96 APPENDIX 3: BIOACTIVITY OF ZOOM-KOOM SAMPLES ................................................. 97 APPENDIX 4: SENSORY EVALUATION SCORES OF FIFTEEN (15) ZOOM-KOOM EXPERIMENTAL SAMPLES ..................................................................................................... 98 APPENDIX 5: CONSENT FOR SENSORY EVALUATION .................................................... 99 REFERENCE .............................................................................................................................. 107 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh x LIST OF TABLES Table 1: Production of Millet in Ghana from 2006 to 2020 ......................................................... 19 Table 2: Phenolic compound content (μg/g defatted meal) in different types of millets ............. 20 Table 3: Analysis of zoom-koom from commercial processors .................................................... 29 Table 4: Variables and their levels used in the Box-Behnken design........................................... 34 Table 5: Box-Behnken Design matrix of variables (k=3) for optimization of the zoom-koom .... 35 Table 6: Analysis of zoom-koom samples ..................................................................................... 37 Table 7: Physicochemical properties of commercially processed zoom-koom ............................. 43 Table 8: Microbial Quality of commercially processed zoom-koom ............................................ 46 Table 9: Bioactivity of commercially processed zoom-koom ....................................................... 48 Table 10: Physicochemical quality laboratory produced zoom-koom .......................................... 50 Table 11: Microbiological quality of laboratory produced zoom-koom ....................................... 62 Table 12: Bioactivity of fifteen (15) zoom-koom experimental samples ..................................... 68 Table 13: Attribute liking scores of laboratory produced zoom-koom ......................................... 72 Table 14: Regression parameters of the model ............................................................................. 73 Table 15: Process combinations for verification of optimum region ........................................... 84 Table 16: Predicted and validated ratings for sensory attributes in the optimum region ............. 85 Table 17: Physicochemical properties of zoom-koom (commercial vs optimised) ....................... 88 Table 18: Microbiological quality of zoom-koom (commercial vs optimised) ............................. 89 Table 19: Bioactivity of zoom-koom (commercial vs optimised) ................................................. 90 Table 20: Proximate composition of optimized zoom-koom ........................................................ 92 Table 21: Physicochemical quality of fifteen (15) zoom-koom experimental samples ................ 95 Table 22: Microbiological quality of fifteen (15) zoom-koom experimental samples ................. 96 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xi Table 23: Bioactivity of fifteen (15) zoom-koom experimental samples ..................................... 97 Table 24: Attribute liking scores of fifteen (15) zoom-koom experimental samples .................... 98 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xii LIST OF FIGURES Figure 1: Typical Structure of Millet Seed (Singh & Raghuvanshi, 2012). ................................. 16 Figure 2: Cross section diagram of millet grain (Singh & Raghuvanshi, 2012). .......................... 17 Figure 3: Process for commercial production of zoom-koom. ...................................................... 28 Figure 4: Process flow showing Laboratory Processing of zoom-koom ....................................... 36 Figure 5: Contour plots of pH of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 54 Figure 6: Contour plots of TTA of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2) ........................................................................................................................................ 55 Figure 7: Contour plots of TSS of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 56 Figure 8: Contour plots of L* of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 57 Figure 9: Contour plots of a* of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 58 Figure 10: Contour plots of b* of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 59 Figure 11: Contour plots of ∆E * of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 60 Figure 12: Contour plots of Aerobic plate Count of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ................................................................................................................. 64 Figure 13: Contour plots of S. aureus of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ....................................................................................................................................... 65 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xiii Figure 14: Contour plots of Yeast & moulds of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ........................................................................................................................ 66 Figure 15: Contour plots of Total phenolic compounds of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2).......................................................................................................... 69 Figure 16: Contour plots of Antioxidant activity (%Inhibition) of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ...................................................................................... 70 Figure 17: Contour plots for the appearance of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ........................................................................................................................ 74 Figure 18: Contour plots for the aroma of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ........................................................................................................................ 75 Figure 19: Contour plots for the flavour of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ........................................................................................................................ 77 Figure 21: Contour plots for the aftertaste zoom-koom as a function of Blend ratio (X1) and Steeping time(X2). ........................................................................................................................ 79 Figure 22: Contour plots for the overall acceptability of zoom-koom as a function of Blend ratio (X1) and Steeping time(X2).......................................................................................................... 79 Figure 23: Overlaid contour plot of appearance, aroma, flavour, mouthfeel, aftertaste and overall acceptability of zoom-koom. ......................................................................................................... 83 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xiv LIST OF ACRONYMS CACS College of Agriculture and Consumer Sciences GSA Ghana Standards Authority TTA Titratable Acidity cfu Coliform forming unit Y&M Yeasts and moulds E. Escherichia Hrs Hours Min Minutes N.D Not detected SD Standard deviation Fig. Figure FAO Food and Agricultural Organization SRID-MOFA Statistics Research and Information Directorate-Ministry of Food and Agricultural w/w weight by weight CGIAR Consultative Group on International Agricultural Research USDA United State Department of Agriculture University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xv GDP Gross Domestic Product USCDC United States Centre for Disease Control µg/ml Microgram per millilitres mg/ml Milligram per millilitres P.E. T Polyethylene terephthalate ANOVA Analysis of Variances DPPH 2,2-Diphenylpicrylhydrazyl UV-Vis Ultraviolet Visible H Hours oC Degree Celsius CECMA Committee on the Development of Microbiological Criteria TPC Total phenolic compounds University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh xvi ABSTRACT The production process traditional beverages such as zoom-koom are usually characterized by lack of standardization, inconsistency, inefficiency and unsanitary conditions. A detailed analysis of zoom-koom and its production can help to optimize the process that will ensure the production of the product with reliable quality to meet consumer demand. This study aimed at determining zoom- koom product and its consumer acceptability to guide the standardization and optimization of the traditional production process. A Box - Behnken design was used to optimize the production process. Blend ratio of spices and steeped millet (700:50, 700: 100 and 700:150) and steeping time ( 2, 7 and 12 hrs) and steeping temperature (25, 35 and 45OC) was considered for this study. An optimum region of blend ratio of spices and steeped millet (700:50 to 700:100), steeping time between (2 to 6 hrs) and steeping temperature of 35OC was determine as optimize parameters obtain. Low counts of aerobic bacteria, yeast and mould and the absence of coliform, E. coli and Staphylococcus aureus in the experimental samples can be attributed to the sufficient hygienic measures implore during the processing. The commercially processed zoom-koom were acidic with pH ranging from 3.08 to 3.59. The acidity, TSS, colour (L*, b* and a*) were from 0.04 to 0.09, 6.80 to 9.63, 21.27 to 26.49, 0.53-4.29 and 3.78 to 11.4, respectively. The total colour change (∆E) ranged from 20.22 to 21.46 for the commercially produced zoom-koom. E. coli, total coliform S. aureus were not detected in the fifteen experimental zoom-koom samples. There was significant difference in the Enterobacteriaceae and E. coli counts of the commercial and experimental samples. Different experimental combinations should be explored to further optimize and standardize the traditional beverage. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 General Overview Consumer awareness of the impact of diet on their health has developed a rising concern across the world. This is becoming more and more popular, especially in Africa where the majority of people want foods that reflect their traditional identities, cultural traditions and religious beliefs. There are several such foods in Ghana, depending on the location, traditional group, and tribal environment. In addition to the diversification of meals brought about by urbanization, it is challenging to designate any one particular dish as the national delicacy due to numerous preparation techniques and widespread consumption of several indigenous foods. Food, a requirement for all living things, not only gives us energy and nourishment, but also represents our attitudes and the way we think about ourselves (Roberts, 2001). It is necessary for basic level for our survival. In fact, it is a basic requirement for human survival (Baker et al., 2011). Ghana's native food come in a variety of forms, including liquid, solid, and semi-solid. The majority of Ghana's basic foods are indigenous, and the country's culture and history are strongly reflected in how they are prepared (Sefa-Dedeh, 1993). The same meal preparations in this modern day employ the same concepts and techniques as current food technology. However, the scale and application differ because they are essentially artisanal in nature. Despite their basic nature, these skills are utilized to produce a varied variety of processed and semi-processed traditional staples to fulfil the needs of consumers from diverse socio-economic classes. Cereals as the main source of energy for humans have become increasingly popular over the world (FAO, 2020), with millet ranking among the most significant cereal grains. Eleusine coracana (L.), often known as millet, is a major food crop in northern Ghana and a minor cereal University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 2 crop in Ethiopia, Nigeria, Burkina Faso, and Niger is currently underutilized. Typically, the crop is grown in India, Asia, and other African nations like Ghana, Nigeria, etc. According to SRID- MoFA (2011), the majority of Ghana's millet is grown in the Northern Region. For small-scale farmers it is a significant staple food crop (Rooney & Serna-Saldivar, 2000) grown in areas that are not prone to floods or on soils with little capacity to retain water. Millet, a typical dryland crop may also tolerate harsh weather conditions (FAO, 2001). Millet can therefore be cultivated in a variety of environments, including semi-arid to sub-humid agro-ecosystems that are prone to drought (Chandra et al., 2016). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 3 In African communities, traditional meals, especially drinks, play a significant role as they serve as a source of income for processors in both rural and urban locations (Oduro-Yeboah, 2015). Zoom-koom is one of Ghana’s non-fermented beverages that is usually consumed in the Northern part of the country and also some parts of the capital city (Accra) especially Nima and in Koforidua in the Eastern Region. The beverage which is usually consumed in a liquid form is also native to some West African countries such as Nigeria and Burkina Faso with their different ways of producing it based on the country of origin. The beverage is commonly made from pearl millet. It is gluten-free, non-acid-forming, and most of all easy to digest with low glycemic index (Muthamilarasan et al., 2016; Manjula & Visvanathan, 2014). It also helps to control blood sugar levels since it is believed to have a low glycemic index, making it an appropriate choice for people with diabetes and celiac disease (a condition brought on by eating gluten-containing cereal proteins) (Jideani & Jideani, 2011). Sorting, washing and steeping the millet grains in water that is twice as much as their mass (2:1, w/w) are the steps in the preparation of zoom-koom. According to observations made at micro-workshops, the typical steeping period is 12 hours. Before wet milling, flavouring and aromatizing spices are combined with the soaked millet grains (at a rate of 3 g/100 g for mint and 6 g/100 g for ginger). A muslin fabric (0.5 mm) is then used to filter the suspension after adding three times the mass of the wet dough in water to give a very fine wort. Finally, a sugar solution is poured to the filtrate to produce a fresh zoom-koom (Soma et al.,2019). Traditional beverages continue to be an indispensable aspect of Ghanaian culture, but disposition towards branded beverages is progressively rising (Veitch, 2021). There is therefore the need to upgrade the quality of traditional beverages through improved processing techniques. It has been suggested that there is a correlation between a product’s characteristics and its performance in terms of meeting consumers’ needs and expectations (Peri, 2006). Currently, there is limited University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 4 documentation on the production methods and product characteristics of zoom-koom and many of such traditional products. Also, various issues including processing operations and safety are of concern and need to be addressed (Amoa-Awua et al., 2007). A thorough and systematic analysis of zoom-koom and its production process can help produce an optimized process that will assure the production of a product with consistent quality. With the present trends in urbanization, and the increasing acceptance of traditional beverages such as zoom-koom among consumers, it has become necessary to address concerns with and industrially scale up zoom-koom production in order to achieve consistent and predictable quality. Based on these considerations, this study explored the optimization of some key processing parameters of zoom-Koom so as to produce zoom-koom that would meet both local and international consumer demand and acceptability. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 5 1.2 Rational for the Study Zoom-koom production, like many other traditional food processes, is considered as small-scale operations with little or no documentation on the product. Production of the beverage depends on the knowledge of the producer from handling the product over a period (Addo et al., 2016). As a result, there is always a difference in the quality of zoom-koom produced between different processors and even between batches from the same processor. Zoom-Koom have become a highly consumed beverages in some parts of West Africa including Ghana where it is becoming popular (especially in the Northern Regions and in the Accra, the capital city of Ghana) due its associated health benefits (Kannan et al., 2013). However, processing of zoom-koom is spontaneous, uncontrolled and usually made with varied steeping time, temperature and spices (Soma et al.,2017). A recent study of zoom-Koom production using the traditional method resulted in an increase in enterobacteria counts making the final product unsafe for consumption (Soma et al.,2017). This menace further reduces the possible large-scale commercialization and quality standardization of the beverage, which has been a main source of income for its producers and subsequently boost the country’s economy. Therefore, optimization of the process of the production will provide a way of standardizing the product and this can also potentially improve the nutrient profile, organoleptic and sanitary quality of the zoom-Koom. Furthermore, bioactivity of the zoom-Koom beverage may be extended because of the polyphenols and terpenoids in the spices since these are known to possess antimicrobial and bioactive properties (Ojewole et al., 2004). This study seeks to clearly explain how the zoom-koom beverage is produced in Ghana and how the production process influences the quality of the final product. Essentially, this work tackles the optimization of some key processing parameters of zoom-koom production such that the final product will satisfy local and international consumer demand and acceptability. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 6 1.3 Main objective To produce and optimize the production process for zoom-koom using Response Surface Methodology. 1.4 Specific Objectives 1. Evaluate the physicochemical, bioactive and microbial quality of zoom-koom obtained from traditional processors. 2. Optimize the zoom-koom production process using response surface methodology. 3. Determine the composition of the optimized zoom-koom. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Introduction Traditional foods are prepared from fresh materials native to a specific setting, area or locality They are prepared utilizing outmoded methods and techniques that have been handed down through the generations. According to Hamm and Bellows (2002), the right to provide one's own labour, agriculture, fisheries, food supply as well as land policies that are environmentally, socially and that is practicably suitable to one's own conditions constitutes the individual right to exercise food authority hence the widespread eating of indigenous foods leads to food sovereignty. According to Marshall and Mejia-Lorio (2012), food sovereignty refers to people's innate capabilities for obtaining and generating food, which implies that everyone has a right to foods that are both safe and healthy as well as socially and culturally acceptable. According to Shobana et al. (2013), nutrition and wellbeing are justifiable forces that maximize and improve health development of human potential energy. Comparatively, traditional foods cost less than exotic ones and this may be partly due to the fact that they are made using local food products and raw processing techniques. Sorghum, maize, and millet are indigenous crops well-known to be extremely significant and valuable essential foods in some African nations like Nigeria, Ghana, and Sudan (Taofeek et al., 2014). Depending on the region and culture, they are used to make a range of meals, including "tuo-zafi" (a dish from Ghana), porridge, various baked products and drinks (both alcoholic and non-alcoholic). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 8 2.2 ‘Zoom-Koom’ (millet beverage) Zoom-koom is a non-alcoholic drink, made from millet and rarely from sorghum, and much valued by consumers in Burkina Faso, Ghana and Nigeria. In Ghana, zoom-koom is thought to have originated from the Northern Region. Due to the traditional nature of its processing, the beverage is not produced on large scale, it is not pre-packaged and only few processors are into its commercial production. Traditional processors commonly produce the beverage on a small scale and sell it in plastic containers mixed with ice cubes to keep the beverage chilled. The beverage is usually served upon purchase into packaging materials such as clear polyethene bags and Polyethylene terephthalate bottles. Flavourings such as vanilla flavour, are commonly added to the beverage upon request. The beverage is consumed at any time of the day but usually consumed in the afternoon. It also usually served to farmers in the Northern region before lunch time. The zoom- koom beverage is sold at affordable prices to consumers considering the value of the end-product. Traditional beverages like zoom-koom have socio-cultural relevance in Ghana. In recent times there has been a rapid increase in the patronage of common traditional beverages such as zoom- koom, aliha, sobolo, palm wine at ceremonies and occasions such as weddings, engagement, naming ceremonies, funerals and parties. Classically, at occasions, the zoom-koom beverage is served in dispensers. For the traditional processing of zoom-koom, millet (pearl) is first sorted, cleaned and then steeped in water. The steeped grains are finely ground upon addition of spices such as ginger, gloves and black pepper, then water is added, it is then mashed and filtered to obtain the wort. An approximately amount of sugar is added to it to obtain fresh zoom-koom. In Ghana, there are other common traditional millet beverages similar to zoom-koom such as Frofro and Fura. Frofro and Fura are thought to have originated from the Northern Region of University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 9 Ghana. A distinguished difference between Frofro and Fura is the nature of their end product after processing. Fura needs to be mixed with water after processing whereas the latter is always in a liquid form even after processing. The unit operations involved in the processing of this traditional millet beverage include steeping, wet milling, wort extraction after which sugar is added to the wort (Soma et al.,2014). The existing documented knowledge on this traditional millet beverages in Nigeria and Burkina have only focused on their physico-chemical and microbiological quality. Soma et al., (2020) conducted research focused on evaluating the microbial quality of zoom-koom sold in some selected schools, health centres and markets in Ouagadougou in Burkina Faso. Results showed that most of the commercial zoom-koom samples collected from schools and health centres in twelve (12) different districts were found not have met the microbiological quality criteria for enterobacteria and yeasts and moulds. Soma et al. (2020) studied the microbes associated with spoilage in zoom-koom during storage. The study showed that, Lactobacillus plantarum subsp. plantarum Pediococcus pentosaceus, and Lactobacillus fermentum were linked to zoom-koom. Until now limited research has focused on characterizing and optimizing the process of producing zoom-koom as well as determining its nutrient profile. 2.2.1 Processing of Zoom-koom 2.2.1.1 Steeping/Soaking Zoom-koom is produced from the Pearl type of millet or the Finger type of millet. Steeping or soaking of the millet grains is the first stage in the process of making zoom-koom. Depending on the processor, the step's duration and conditions might vary greatly, which has an impact on the value of the final product. According to the processor, the grains are traditionally soaked in an University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 10 amount of water for a few hours until an acceptable moisture level is obtained. This, in the opinion of Woonton et al. (2005), helps in the elimination of specific colours, bacteria, and chemicals that can result in bitterness in the chosen grains. To get rid of any dirt in the soaked millet grains, it is subsequently sieved. Traditional zoom-koom preparation involves physically sorting and lightly cleaning the grains before steeping them in large bowls, buckets, or pots that may or may not be covered. As a result, there is a good chance that foreign objects including stones, sand, hair, mice, insects, and other debris will be found in the grains during steeping. 2.2.1.2 Wort extraction Following a number of operation units, wet milling is done followed by the preparation of the wort and the extraction. A combination of the steeped millet grain, spices (such as ginger, cloves, and black pepper), and water is added in a ratio according to the processor. The finished combination is given some time to stand. The mixture's insoluble components settle to the bottom and is subsequently filtered. Most people refer to this as "mashing" (Glover, 2007). Different varieties of zoom-koom exist in Ghana due to variations in the millet used, the mashing process, and the additives being used. 2.2.1.3 Addition of sweeteners and storage Addition of sweetener is the final step in zoom-koom processing. After the wort has been extracted, a quantity of sugar is added to the wort based on a ratio of 10:1. Zoom-koom processing is a spontaneous, and unfermented process as reported by Soma et al. (2019). Certain biochemical changes occur during its storage causing the beverage to undergo some type of fermentation. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 11 According to Singh and Raghuvanshi (2012), these modifications can include an increase in amino nitrogen, the breakdown of proteins, and the elimination of any inhibitors that could be present. The bacteria of the genus Lactobacillus and Weisella species, for instance, have been revealed as considerable contributors to the acidity of the zoom-koom beverage during its first souring process. (Soma et al., 2019). The beverage is put into jugs and bottles for storage and sale once the sugar has been added. It is standard practice for the majority of zoom-koom producers to offer the beverage chilled. This enables the substance to remain stable without going through fermentation. 2.3 Other Traditional millet-based beverages 2.3.1 Sur It is a fermented beverage made primarily from finger millet (Eleucine coracana) that is made in the Kullu district's Lug valley, the Kangra district's Bhangal and Luharti valleys, the Mandi district's Balh and Barot valleys, and the Sirmour region of India (Joshi et al., 2015; Kumar, 2013). Fermentation is carried out using a mixture (inocula) of roasted barley and regional herbs known as "dhaeli." The millet flour is combined with water to produce a dough, which is then allowed to naturally ferment for 7-8 days in a container. The fermented flour is cut into rotis, baked for half the time, then chilled. Then, the roti pieces, powdered dhaeli, and enough water and jaggery are then added to earthen pots that have been treated with smoke and allowed to ferment for 10 days under cover. After completion of ffermentation, the product is filtered and stored in special earthen pots that are airtight. 5–10% alcohol has reportedly been found in the product (Kumar, 2013). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 12 2.3.2 Madua In Arunachal Pradesh, India, one of the most well-liked finger-millet-based drinks is called madua. The millet is first roasted for 30 minutes, then cooled and cooked till tender. The starting culture is added to the softened grains, which are then left to ferment for 4–7 days in a perforated basket covered in ekam leaves. Hot water is poured from the top and collected in a container after the fermentation process is finished. The liquid is referred to as madua. A high-quality madua has a golden hue, a sweet flavour, and outstanding alcohol compatibility. Other finger millet-based alcoholic beverages manufactured and consumed in Arunachal Pradesh, India, including temsing, rakshi, mingri, and lohpani (Shrivastava et al., 2012). 2.3.3 Kunnu-zaki Kunnu-zaki, is another traditional non-alcoholic fermented beverage commonly consumed in Nigeria (Obadina et al., 2008; Adeleke & Abiodun, 2010; Agarry et al., 2010; Nwachukwu et al., 2010; Sekwati-Monang, 2011). Due to its stimulating property, it is accepted in other parts of the country (Amusa & Ashaye, 2009). It is produced from either millet (Pennisetum typoidum), sorghum (Sorghum bicolor), or maize (Zea mays) (Akoma et al., 2006). Like other traditional fermented non-alcoholic beverages, kunnu-zaki is consumed anytime of the day by children and adults, served to entertain visitors and at community gatherings (Amusa & Ashaye, 2009; Ndulaka et al., 2014). Sugar or honey with some number of sweet potatoes and spices (such as ginger, black pepper or cloves) added for taste and flavour (Elmahmood & Doughari, 2007). The method of preparing kunnu-zaki differs among different cultures and still produced on a small scale (Ndulaka et al., 2014). Typically, kunnu zaki is processed by soaking sorghum, millet or maize, wet milling, sieving and partial gelatinization of the slurry (Ndulaka et al., 2014). It takes about five (5) days University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 13 for the whole process to be completed and can be stored for up to three (3) days under refrigeration condition (Ndulaka et al., 2014). 2.3.4 Oshikundu Oshikundu is a traditional sour-sweet beverage from Namibia that is made from grain. Both an alcoholic and non-alcoholic version of it is produced. It is brewed with water, local sorghum (Sorghum bicolor), bran, and pearl millet (Pennisetum glaucum) meal. Oshikundu is brewed at home by rural women for daily consumption as well as for sale in several cities in northern Namibia's open markets. Boiling water is added to the mahangu meal during production, and the mixture is then allowed to cool to room temperature while being stirred occasionally. The mixture is then supplemented with bran and malted sorghum meal. Depending on the availability and preference of utilizing bran in brewing, the bran adding phase is optional. Some already fermented oshikundu is added after the mixture has been prepared. The resulting mixture is then diluted with water according to the amount of starting material used and the desired volume of the finished product. Oshikundu is then prepared by allowing the mixture to ferment for an average of one and a half hours at room temperature. Malt sorghum is fermented by yeast, which results in the production of alcohol. It is a perishable beverage that must be consumed the same day because its shelf life is less than 6 hours (Werner et al., 2012). 2.3.5 Koozh In Tamil Nadu, India, ethnic people primarily eat koozh, another fermented beverage prepared with millet flour and rice (Ilango and Antony, 2014). Although pearl millet has been mentioned in other places, finger millet (Eleucine corcana) is the primary ingredient used in its preparation. Two University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 14 fermentation phases are included in the koozh preparation steps. The millet is first ground into flour, combined with water, and then left to ferment for an entire night. The overnight fermented millet slurry is combined with broken rice (20% by weight of millet) and cooked the next day to create noyee, a thick porridge. This porridge ferments for 24 hours, producing kali, a semi-solid porridge to which the necessary amount of drinkable water is added (1:6 w/v), and salt is manually mixed in to produced koozh (Ndulaka et al., 2014). 2.4 Millet and its nutritional value In Sub-Saharan Africa, millet is a dominant crop for ensuring food security. According to Girish et al. (2014), millet has an exceptional capacity to grow and survive harsh environmental factors such as low soil fertility, insufficient rainfall, and land topography. As a result, growing millet in the Northern region of Ghana is not as difficult. India, Nigeria, Niger, China, Burkina Faso, Mali and Sudan are some of the countries with millet as a major import crop (Singh & Raghuvanshi, 2012). In Ghana, it is a chief cereal crop and essential food cultivated in the Northern part of the country. It is the second cereal crop after sorghum in terms of production area, with about 1.2 million hectares (FAO STAT, 2016). Since ancient times, Millet has been grown in Africa and the Indian subcontinent. It is thought that millet originated in Africa and was later introduced in India. According to the earliest archaeological evidence, millet was likely domesticated in Africa before moving to India around 2000 BC (Malik et al., 2002). Singh & Raghuvanshi (2012) emphasizes on the the four most common millet varieties namely; Pearl millet (Pennisetum glaucum), Foxtail millet (Setaria italica), Proso millet or white millet (Panicum miliaceum), and finger millet (Eleusine coracana). Pearl millet accounts for 40% of the world's production. The millet kernel's high dietary fiber content is mostly due to the seed University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 15 coat, embryo (germ), and endosperm (FAO, 1995), which also has a hypoglycaemic impact when ingested. Complex carbohydrate in high fiber diets in the product is also gradually processed and absorbed, leading to a decrease postprandial glucose level (Singh & Raghuvanshi, 2012) when consume. Although there are variants available such as yellow, white, tan, red, brown, or violet in colour, but only the red types are typically grown around the world (Shobana et al., 2013). According to Ajiboye et al. (2014), addition of ancient based cereals such as sorghum and millet in our daily meals can decrease the risk of long-lasting disease, making them vital crops in Ghana. Aside from pito, millet is used in Ghana to make a wide range of dishes, including hausa koko, weaning foods, tuo zafi, and numerous other baked goods. According to Singh and Raghuvanshi (2012), millet contains a total carbohydrate content of roughly 72% to 79.5% and a range of 5.6% to 12.7% in protein. According to Mbithi et al. (2000), dark seeded varieties have higher protein levels than white seeded varieties. They also claimed that the necessary amino acids in a protein determine its quality. Due to the high levels of lysine, threonine, and valine contents in finger millet, in contrast, the essential amino acid balance is significantly better (Ravindran, 1992). Ravindran (1992) found an inverse relationship between the amounts of the amino acid’s lysine and methionine in the finger millet grain and its protein content. With a ratio of 2 between leucine and isoleucine content, finger millet almost has the same amount of isoleucine as rice and wheat (Ravindran, 1992). Grain cereals supply essential dietary elements to people all over the world, making plant nutrients extremely important in the food sector (Shobana et al., 2013). Proteins can be altered to modify their structure, and presumably its physicochemical and functional properties by employing physical, chemical, and biological approaches like fermentation or enzymatic treatment (Amadou et al., 2013). Magnesium and phosphorus are present in millet in reasonable proportions (Girish et al., 2014). Magnesium can lessen the impact of heart-related problems and University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 16 migraines. On the other hand, phosphorus is an essential part of a precursor to adenosine triphosphate (ATP), which is necessary for the body to produce energy and function normally. A spontaneously fermented millet-based food (koko) was organically utilized as a probiotic therapy for diarrhoea in young children in an innovative intervention (Lei et al. 2006). According to Shobana et al. (2013), the millet grain contains full of phytochemicals, including phytic acid, which is known to lower cholesterol levels. Also, phytate is believed to be effective in cancer risk reduction. The variety of potential chemo-preventive molecules known as phytochemicals, which include antioxidants present in very high amounts in foods like millet, are responsible for these health advantages (Izadi et al., 2012). 2.4.1 Structure and Chemical Composition of Millet The kernel structure of millet is comparable to that of sorghum. The pearl millet is caryopsis, where the pericarp is completely linked to the endosperm, and it is made up of the pericarp, germ, and endosperm. However, the endosperm and the pericarp-like sack are only weakly attached at one point in the finger millet. These millet kernels are referred to as utricles because their pericarp can easily separate from the testa, which serves as a shield for the endosperm (McDonough et al., 2000). Figure 1: Typical Structure of Millet Seed (Singh & Raghuvanshi, 2012) University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 17 The relative distribution of the three components of the kernel are 8.4% of the pericarp, 75% of the endosperm, and 16.5% of the germ. As a result, endosperm to germ is about 4.5:1 in pearl millet, but 8.4:1 in sorghum in proportion. Due to the finger millet's small germ, the endosperm to germ ratio is smaller than that of sorghum and pearl millet, ranging from 11:1 to 12:1. The 1000 kernel weight for finger millet is relatively modest, and there are differences between the visual colours of pearl and finger millets and hence the texture of the millets should be taken into consideration when preparing them (Abdelrahman, 1984). Dry milling corneous kernel types yields more grain than soft floury kernel types do. When producing thick porridge, cultivars with higher levels of corneous endosperm are desirable, also, in the baking process either fermented or unfermented bread, the flour obtained from soft endosperm are much preferred (Rooney, 1986). Figure 2: Cross section diagram of millet grain (Singh & Raghuvanshi, 2012). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 18 2.4.2 Millet Production and consumption in Ghana In Northern Ghana, millet farming has existed around 1459 BC (D'Andrea et al., 2001). According to SRID-MoFA (2011), millet is mostly grown in Ghana's Northern, Upper East, and Upper West regions, accounting for 29% of the country's total land area. In Northern Ghana, millet grows better than other crops because it grains yields in hot, dry weather and on soils with little water retention capacity (CGIAR, 1996). The value of millet as a commercial crop is secondary to its importance as a food crop. It is a customary crop that is cultivated by the majority of homes for food and is only ever sold to make money as a last option. Millet is also known to be the first crop to be harvested following a prolonged dry season, which is why it is considered a hunger-buster grain (Kudadjie et al., 2004). According to USDA (2022), Ghanaians consume 223 pounds of millet domestically, with a growth rate of -3.43%. Millet is turned into a number of products in Ghana, including koko, fura, and maasa (Lei & Jakobsen, 2004). According to the (MoFA 2018 Annual Report), millet has also generated revenue source for individuals and Ghana as a whole by contributing its fair share to the country's Gross Domestic Product. The annual production of millet in Ghana from 2006 to 2020 is shown in Table 1. Some of the high yielding varieties of millet cultivated in Upper East and Upper West Regions of Ghana are Kaanati, Akad-kom, Naad- kohblug, Afribeh-naara and Waapp-naara (MoFA 2018). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 19 Table 1: Production of Millet in Ghana from 2006 to 2020 Year Volume (1000 metric tonnes) 2006 165 2007 113.04 2008 193.84 2009 245.55 2010 218.95 2011 183.92 2012 179.68 2013 155.93 2014 155.00 2015 157.37 2016 159.02 2017 163.48 2018 181.56 2019 190.00 2020 170 Source: FAO, 2020 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 20 2.5 Nutrient Profile of millet – based beverages 2.5.1 Phenolic compounds and Antioxidant activity The extremely diverse class of chemicals known as phenolic compounds include the phenol functional group as a primary constituent. These are conveniently divided into phenolic acids, flavonoids, and tannins. The subcategories of phenolic acids include hydroxybenzoic, hydroxycinnamic, hydroxyphenylacetic, and hydroxyphenylpropanoic acids. By using HPLC- DAD-ESI-MSn (Dykes &Rooney, 2006; Chandrasekara and Shahidi 2011) identified and categorize the free, hydrolyzed (esterifed and etherifed), and bound phenolic chemicals in millets. The soluble fraction of finger millet has the highest concentrations of flavonoids (1896 g/g) and hydroxybenzoic acid derivatives (62.2 g/g). Little millet (173 g/g) and foxtail millet (171 g/g) had the highest quantities of hydroxycinnamic acid and its derivative in soluble form. The insoluble bound phenolics that are affixed to the cell wall make up the largest portion of the overall phenol content. Free form flavonoids are more common. According to Dykes and Rooney (2006), millet's phenols exhibit antioxidant, anti- mutagenic, anti-oestrogenic, anti-inflammatory, antiviral, and platelet aggregation inhibitory properties. Total antioxidant capacity of finger millet, little, foxtail and proso millets is high due to their high total carotenoid and tocopherol content which varied from 78 to 366 and 1.3 to 4.0 mg/100 g respectively, in different millet varieties (Dykes &Rooney, 2006). The chemical characteristics of millet are suppressed by phenolics during the enzymatic breakdown of complex carbohydrates, delaying the absorption of glucose and ultimately controlling the postprandial blood glucose level. These chemical qualities include amylase and -glucosidase activities. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 21 Table 2: Phenolic compound content (μg/g defatted meal) in different types of millets Phenolic compound Pearl Finger Proso Foxtail Kodo Methyl vanillate 19.8 _ _ _ _ Protocatechuic acid 11.8a 23.1a, 48.2 69.7 10.2 39.7 p-Hydroxybenzoic acid 22a 8.9a, 1.7 55.4 14.6a, 5.63 10.5 Vanillic 16.3a, 7.08 15.2a, 85.8 87.1a, 22.1 40.1 Syringic 17.3a 7.7a _ 93.6a _ Gentisic acid 96.3a 61.5a _ 21.5a _ Cafeic acid 21.3a 16.6a, 11 _ 10.6a, 34 276 p-Coumaric acid 268.9a, 53.5 36 1188 2133.7a, 848 767 Trans-ferulic acid 637 331 332 631 1844 Cis-ferulic acid 81.5 65.3 18.6 101 100 8,8′-Aryl ferulic acid _ _ _ 19.6 94.8 5,5′-Di ferulic acid 57 11.8 5.44 62.2 173 Flavonoidsb 7.1 1896 1.9 169 179 Kumar et al., 2018. (Adapted from Chandrasekara and Shahidi (2011) (content of phenolic compounds in bound form). values are taken from Dykes and Rooney (2006) (expressed as μg phenolic acid/mg samples) b Content of phenolic compounds in soluble fraction of millet grains c Data not available University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 22 The antioxidant activity of millets is attributed to the presence of high polyphenol and tannin content (Rao et al., 2017, Rathore et al., 2019). Plants with antioxidant qualities scavenge free radicals which are the root cause of some deadly illnesses and disorders like cancer, diabetes, liver disease, renal failure, and degenerative diseases. Free radicals are organic substances that interfere with the regular functioning of body metabolism, and millet varieties are known sources of antioxidants that prevent their build up in the body (Sies, 1993; Oboth and Rocha, 2007). 2.5 Physicochemical and Microbial Quality of “Zoom-koom” 2.5.1 Physicochemical Quality of “Zoom-koom” and millet-based beverage Physicochemical properties are the intrinsic physical and chemical characteristics of a food substance, this includes pH, Acidity, Total Soluble solids and colour. According to Tapsoba et al. (2017), who investigated the physicochemical properties of zoom-koom and the impact of fermentation on the microbial community during processing in Burkina Faso, except for the millet zoom-koom ranged from 4.2 to 4.1 for the fermented zoom-koom without and with sugar, respectively, the pH of the unfermented zoom-koom did not significantly decrease. The study also showed a non-significant variation in the pH between the unfermented zoom-koom and the sugar- and tamarind-sweetened zoom-koom, ranging from 5.2 to 3.5 respectively. With the exception of the millet zoom-koom, the titratable acidity of the unfermented zoom- koom likewise showed a non-significant decline, ranging from 0.25 to 0.39% for the fermented zoom-koom without and with sugar, respectively. The study also reveals non-significant differences of the acidity with respect to the unfermented zoom-koom ranging from 0.19 to 0.24% for the zoom-koom without sugar, with sugar and Tamarind respectively. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 23 Additionally, Soma et al. (2019) investigated into how adding a starting culture of lactobacillus to liquid zoom-koom and instant flour zoom-koom can enhance their nutritional, hygienic, and sensory qualities. The liquid zoom-koom samples had titratable acidities of between 1.21 and 1.89 g of lactic acid per 100 g of product, with a pH range of 5.02 to 5.13. Both forms of zoom-koom are acidic drinks (pH 6), according to the findings. Food acidification was a method used to stabilize the products. The "Kounou" samples were kept at room temperature and in the refrigerator for 4 days before the TSS values were read, according to Bayoi et al. (2022), who studied the Physicochemical Changes of Commercial "Kounou"- a millet beverage During Short Term Storage at Room and Refrigerated Temperatures. The TSS fell considerably (p≤ 0.05) in the ambiently stored samples, but not statistically significantly (p ≥ 0.05) in the samples kept in the refrigerator. Between the first and fourth days, the TSS changed from 7.26 °Brix to 4.98 °Brix and 8.78 °Brix to 8.13 °Brix. A natural lacto-alcoholic fermentation process, which is the primary characteristic of the indigenous African beverages consumed while they are still in an active state of fermentation, was indicated by the reduction of TSS after storage (Tusekwa et al., 2000). This process gradually degrades soluble solids like sugars. Additionally, research showed that samples kept at ambient temperature had lower total soluble solids values than samples kept in a cold environment. A similar fermented beverage made from cereal that was produced in Nigeria and kept at the same temperature was found to exhibit the same pattern (Noah et al., 2013). Abiodun et al. (2017) studied the physicochemical, microbial and sensory properties of kunuzaki beverage sweetened with black velvet tamarind (Dialium guineense) and observed that the lightness, L* value ranged from 38.23 for the control to 33.32 for the treatment with the highest amount of velvet tamarind, indicating a reduced lightness with increment in the incorporation with the velvet University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 24 tamarind. They also noted that the redness, a* and yellowness, b* values increased with the addition of the tamarind. 2.5.2 Microbial Quality of “Zoom-koom” and millet-based beverage Soma et al. (2019), investigated into how adding a starter culture of lactobacillus to liquid zoom- koom and instant flour zoom-koom can enhance their nutritional, sanitary, and organoleptic qualities. According to the microbiological quality findings, the amount of plate count (aerobic) in the liquid zoom-koom samples ranged from 1.0 10 6 cfu/ml to 1.0 108 cfu/ml. The concentration of lactic acid bacteria varied from 9.5 105 to 1.5 108 cfu/ml. Total coliforms ranged from 1.5 ×102 to 4.9104 cfu/ml. The concentration of the thermotolerant coliforms ranged from 9.0 101 to 4.9 104 cfu/ml. Yeast and mould concentrations ranged from 4.0 101 to 7.6 10 4 cfu/ml. The zoom- koom made and sold in Ouagadougou city contained high levels of coliforms, according to earlier studies (Barro et al., 2003; Bsadjo-Tchamba et al., 2014) and those on beverages with a similar composition, such as the traditional millet-based drink kunun-zaki from Nigeria (Gaffa et al., 2002; Elmahmood and Doughari 2007). Additionally, there is no pasteurization stage in the zoom-koom processing in Burkina Faso to guarantee the products' safety (Soma et al., 2017; Tapsoba et al., 2017). According to Tapsoba et al. (2017), starters could greatly enhance the hygiene of the zoom- koom. According to the recommendations made by CECMA (2009), the quantities of aerobic mesophilic bacteria and total coliforms in unfermented liquid zoom-koom and instant flour zoom- koom on the one hand, and in fermented liquid zoom-koom and fermented instant flour zoom-koom on the other hand, were determined to be non-conforming. However, it was discovered that the levels of yeast and moulds in all of the zoom-koom samples complied with CECMA's (2009) requirements. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 25 2.6. Microbial Safety of Beverages Along the food value chain, risk factors for poor quality and safety include inadequate food safety knowledge, the use of contaminated raw materials, polluted water, unhygienic practices and environments, unstandardized production processes, mixed-culture processing, poor packaging, and inadequate preservation techniques (Adekoya et al., 2019). 2.6.1 Enterobacteriaceae Enterobacteriaceae are a group of gram-negative aerobic or facultatively anaerobic, asporogenous, rod-shaped bacteria (Lister & Barrow, 2008). Some genera of Enterobacteriaceae are resident intestinal bacteria and are found in fecal matter (Lister & Barrow, 2008). The presence and dominance of Enterobacteriaceae raises concern because pathogenic microorganisms such as Shigella, Salmonella and Escherichia coli are members of this family (Gabaza et al., 2019; Blandino et al., 2003). They are usually linked with poor sanitation and their presence could mean a possible health risk (Nyambane et al., 2014). According to Dinardo et al. (2019), natural cereal fermentations occur in microbial successions of Enterobacteriaceae, Enterococcus, Leuconostoc, Pediococcus and/or Weissella species before acid-tolerant lactobacilli takes over. In cereal beverages, the risk of food-borne illnesses may occur when Enterobacteriaceae persists (Todorov & Holzapfel, 2015). According to the Ghana Standards Authority (GSA) microbiological standards for ready to eat foods, microbial counts less than 3 log cfu/ml for Enterobacteriaceae is considered satisfactory. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 26 2.6.2 E. coli E. coli is a fecal coliform that is naturally found in the intestines of humans and vertebrates. According to Pachepsky et al. (2016), E. coli like other fecal contamination indicators such as the thermotolerant coliforms are used by monitoring bodies to check for the presence of fecal and pathogenic infection. It has been shown that E. coli pathovars cause enteric diseases such as bloody and non-bloody diarrhoea and chronic sequelae such as hemolytic uremic syndrome (Buchholz et al., 2011; Gault et al., 2011). In Doza et al. (2017)’s study, it was found that pathogenic E. coli genes were found in 14% of E. coli-positive food and 2% of E. coli positive flies after a multiplex PCR test of a subset of E. coli positive food and fly samples. 2.6.3 Staphylococcus aureus Staphylococcus aureus is a naturally occurring pathogen found in the environment and on human skin and mucous membranes of most healthy humans (Rasigade & Vandenesch, 2014). Humans are significant hoarders of this microorganism (Boucher & Corey, 2008; Chambers, 2005). It does not usually cause infections on healthy skin but if it enters the blood stream it could cause a variety of infections (Rasigade & Vandenesch, 2014). They are commonly associated with community acquired infections and hospital-acquired infections in which treatment is still a challenge due to the emergence of Methicillin-Resistant-S. aureus (USCDC, 2003; Boucher & Corey, 2008). Hundred thousand (100,000) cfu/ml of S. aureus in food is needed to produce 1 µg of toxin and the temperature range for Staphylococcus aureus to form toxin is between 10 to 45°C and optimal at around 35 to 40°C. Standard refrigeration temperature can therefore limit the development of Staphylococcal enterotoxin (Centre for Food Safety, Food and Environmental Hygiene Department, Hong Kong, 2014). According to Kalita et al. (2017), S. aureus can cause health University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 27 complications like septic shock, multiple organ failure, microvascular abnormalities development, disseminated intravascular coagulation and necropsy. 2.6.4 Yeast and Mould The uses of fungi (yeast and mould) are quite enormous; it can be a pathogen and act as causative agent of diseases and infections (Prescott, 2002). In most cases, food spoilage happens or occurs through moulds (Mariott and Gravani, 2006). Fungi can serve as a source of antibiotic; it can serve as a fruiting agent as in the case of mushrooms. There are fungi diseases, which adversely affects both humans and animals. There will be an adverse consequence on food supplies, health, the economy etc. if fungi degenerate. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 28 CHAPTER THREE 3.0 Materials and Methods 3.1.1 Sampling site and procedure Samples of Zoom-koom (20) were obtained from 5 processors conveniently selected from the Ashaiman-Tulaku, Amansaman, Nima-main market (Greater Accra Region) and Koforidua Zongo (Eastern Region). Each commercial processor was visited to study the production process for zoom-koom and samples of about 100ml was collected from each processor for analysis. Samples were transported under chilled conditions to the laboratory in the Department of Nutrition and Food Science at the University of Ghana for microbiological, bioactivity and physicochemical analysis. The control sample was also produced in the laboratory based on how the local processors produced their zoom-koom. It was produced in the laboratory because of safety and hygiene reasons. Addition of water Dripping of water Addition of spices Addition of water and filtration Addition of sugar Sampling point Figure 3: Process for commercial production of zoom-koom. (Soma et al., 2015) Sorting Milling Steeping Milled sample Millet Zoom-koom University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 29 Table 3: Analysis of zoom-koom from commercial processors Commercial processors Physicochemical and chemical analysis Microbiological analysis Nima (5) Koforidua (5) Ashaiman (5) Amansaman (5) Colour, pH, Titratatable Acidity, brix, Total phenolic compound, Antioxidant activity Total plate counts, yeasts and moulds, Total coliform, E. coli, Staphylococcus aureus. 3.1.2 Microbiological analysis 3.1.2.1 Sample preparation Ten grams of the samples was measured and homogenized in a sterile stomacher bag for one minute using a stomacher blender (Steward Stomacher blender 400 circulator) in 90ml of 0.1 % peptone water to suspend the microorganisms. The stock preparation was serially diluted from 10- 1 to 10-9 and the appropriate dilution used. 3.1.2.2 Media preparation All microbiological media used: Potato Dextrose agar for yeasts and moulds, MacConkey agar for E. coli, Mannitol Salt Agar for Staphylococcus aureus and Violet Red Bile Glucose Agar for Enterobacteriaceae (E. coli) were prepared following the instructions specified by the manufacturer. The media were sterilized in an autoclave for 15 min at 121℃ and tempered at 50°C. The plates were checked for sterility by incubating uninoculated agar plates to check for growth. Peptone water (0.1%) used as a diluent was also prepared following manufacturer’s instructions and incubated at 121℃ for 15 min (Benulah et al.,2022). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 30 3.1.2.3 Enumeration of Total bacteria, yeasts and moulds, total coliform, Escherichia coli and Staphylococcus aureus Aliquot (0.1ml) of appropriate dilutions were pipetted unto duplicate sterile agar plates using the spread plate technique. The MacConkey agar, Mannitol Salt Agar and Violet Red Bile Glucose Agar plates were incubated in an inverted position at 37℃ for 24h while Potato Dextrose agar plates were incubated at 25℃ for 3-5 days. The agar plates of De Man Rogosa Sharpe agar were incubated anaerobically in an anaerobic jar at 37℃ for 24h. After incubation, the bacterial colonies were observed and counted using a colony counter. The number of microorganisms counted were multiplied by the reciprocal of the dilution factor to give the count per gram. The method used was a modification from Morello et al. (2003). 3.2 Physicochemical Analysis 3.2.1 pH The pH of the samples was evaluated using an Orion 2-star pH meter in accordance with AACC (2000) procedure. The pH of 60ml of sample was measured and recorded using the probe of the pH meter already calibrated according to manufacturer instructions. 3.2.2 Titratable Acidity The titratable acidity, given as percent lactic acid, was calculated using the AACC (2000) method, which involved titrating 10 ml of sample against 0.1 N NaOH with 1% phenolphthalein (2 - 3 drops) as an indicator until a light pink color appeared (endpoint). Triplicate determination per sample were made. The following formula was used to compute the titratable acidity: University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 31 Titratable Acidity(% Lactic acid) = (𝑉 × 𝑁 × 9.08)/(𝑊 × 10) Where V = titre value N = normality of the titrant W = sample weight 9.08 = Equivalent weight of predominant acid (lactic acid) 3.2.3 Brix Brix was determined using a digital refractometer (Hanna digital refractometer model HI 96801) at room temperature. 3.2.4 Colour The colour of zoom-koom samples was determined using a colourimeter (Digital handheld colorimeter-FRU® 10QC160226). Samples of zoom-koom were poured into a plastic petri dish. The measuring head of the colourimeter was cautiously positioned on top of the petri dish and the colour measurement taken. Three different colour readings were done on each sample and the mean calculated by the CIE L* a* & b* colour value index. Colour change (∆E) was calculated using the formular: ∆E = √(𝐿ₒ∗ − 𝐿∗ ) + (𝑎ₒ∗ − 𝑎∗ ) + (𝑏ₒ∗ − 𝑏∗ ) The control zoom-koom sample was the reference point, with colour parameters L* a* and b*denoted by Lₒ, aₒ &bₒ, where L is lightness, a is the positive value of red and negative value of green; b is the positive value of yellow and the negative value of blue (Sumnu et al., 2005). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 32 3.3 Chemical Analysis 3.3.1 Determination of total phenol compounds 3.3.1.1 Sample preparation Using the Folin-Ciocalteu reagent and a slightly modified Adusei et al. (2019) method, the total phenol content of zoom-koom drink was measured, the calibration curve was plotted using gallic acid as a reference standard. 1 ml aliquot of 10, 20, 40, 80, and 100 mg/ml gallic acid solutions was mixed with 2 ml Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) and neutralized with 4 ml sodium carbonate solution (7.5 percent w/v). For colour development, the reaction mixture was incubated at room temperature for 30 minutes with intermittent shaking. A single beam UV-VIS spectrophotometer (Ultraspec Pro) was used to quantify the absorbance of the ensuing blue colour at a wavelength of 765 nm. For sample determination, 1 ml of the test sample was mixed with 80% methanol (v/v). The sample mixture was then vortexed using a (Standard mini Vortexer 009060) for 2 minutes, after which the sample was centrifuged using an (Eppendorf centrifuge 5417C) for 5 mins at a speed of 10000rpm. The supernatant was then discarded into a test and 2ml of the Folin-Ciocalteu and sodium carbonate reagents were then added to the test sample. The mixture was then incubated for 30mins with intermittent shaking and absorbance values read at wavelength of 765nm.All measurements were performed in triplicate for each analysis. The total phenols content was determined from the linear equation of a standard gallic acid curve and the result expressed as mg/ml gallic acid equivalent (GAE). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 33 3.3.2 Antioxidant Activity Radical Scavenging Activity of DPPH.) The DPPH assay was carried out according to Pandey et al., (2018). The sample (1 mL) was combined with 3 mL of DPPH solution (4 mL of stock DPPH solution in 96 mL of 80 percent methanol) and maintained at room temperature for 30 minutes. A UV-Vis spectrophotometer was used to measure the mixture's absorbance at 520 nm (Ultraspec Pro). As a blank, a mixture of 1 mL methanol and 3 mL DPPH solution was utilized. The percent inhibition of the extract's antioxidant activity was calculated using the following equation: Inbition = (A control − A sample)/(A control) × 100 where A control = is the blank's absorbance value and A sample = the absorbance of extract and DPPH solution 3.4 Optimization of process for zoom-koom using Response Surface Methodology 3.4.1 Materials The local variety of millet (pearl), ginger, black pepper and gloves were obtained from the local market in Koforidua, Ghana. 3.4.2 Design of optimization The Box - Behnken design was used to identify the combinations of process variables that provided the best quality characteristics for sensory acceptability, physicochemical properties, chemical properties, and microbial quality of zoom-koom (Box and Behnken, 1960; Montgomery, 2001). These factors include the blend ratio of steeped millet and spices, steeping time, and steeping temperature. The variables utilized in the experiment are listed in Table 4 along with their values. University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 34 Table 4: Variables and their levels used in the Box-Behnken design Variables Symbols Levels Coded -1 0 1 Blend ratio (g) of steeped millet and spices X1 700:50 700:100 700:150 Steeping time (hrs) X2 2 6 12 Steeping temperature (℃) X3 25 35 45 The number of experimental runs (N) in a Box-Behnken design was calculated using the formula 𝑁 = 𝐾2 + 𝐾 + 𝐶, where (k) is the number of components and Co is the number of replications at the center point (Aslan and Cebeci, 2006). For Box-Behnken designs, 𝑁 = 2𝑘(𝑘1) + 𝐶𝑜, was used to determine N, however for central composite designs, 𝑁 = 2𝑘 + 2𝑘 + +𝐶𝑜 was used (Ferreira et al., 2007), where k is the number of components, and Co is the number of centre points. So, for the three component Box-Behnken design in this work, a total of 15 experimental runs was used (Table 5). In order to construct the design matrix (factor combinations per experimental unit) for the Box-Behnken design, the level of one of the components was set at the centre point while mixtures of all levels of the other factors were used (Myers and Montgomery 2002). According to Table 4, all levels of factors X1 and X2 were merged after determining the level of component X3 (the steeping temperature), and the same methods were then applied for the factors X2 and X1, respectively. The final rows of the design matrix contained the three replicate center positions (Table 5). The data from the trials were fixed into the second order polynomial model (Montgomery 2001): University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 35 𝑌 =b0 + b1x1 + b2x2 + b3x3 + b11x1 2 + b22x2 2 + b33x3 2 + b12x1x2 + b13x1x3 + ……… The independent variables in this equation are X1, X2, and X3. The linear coefficients are b1, b2, and b3. The interaction coefficients are b12, b13, and b23. The quadratic coefficients are b11, b22, and b33 (Montgomery, 2001). Y is the response and bo is the model constant. Table 5: Box-Behnken Design matrix of variables (k=3) for optimization of the zoom-koom Variants X1 X2 X3 Blend ratio (g) Steeping time (hrs) Steeping temperature (OC) 1 -1 -1 0 700:50 2 35 2 1 -1 0 700:150 2 35 3 -1 1 0 700:50 12 35 4 1 1 0 700:150 12 35 5 -1 0 -1 700:50 7 25 6 1 0 -1 700:150 7 25 7 -1 0 1 700:50 7 45 8 1 0 1 700:150 7 45 9 0 -1 -1 700:100 2 25 10 0 1 -1 700:100 12 25 11 0 -1 1 700:100 2 45 12 0 1 1 700:100 12 45 13 0 0 0 700:125 7 35 14 0 0 0 700:125 7 35 15 0 0 0 700:125 7 35 University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 36 3.4.3 Preparation of zoom-koom One kilogram (1 kg) each of millet grains and the spices (ginger, gloves & black pepper) were washed and steeped in water with respect to time and temperature according to the experimental design (Table 4 and 5). Five (5%) sodium benzoate was added to the steep grain was added to the beverage to control the microorganism that may be present in the steeped sample (Terna and Ayo, 2002). The steeped grains were washed again and milled according to the blend ratio of the steeped millet and spices based on the experimental design using a multifunction blender robot (SHB- 3088). The slurry was prepared by adding sterilized water in a ratio of 10:1 to the fresh extract and filtered using muslin cloth to obtain the wort. About 0.05kg/L of sugar was added to produce the final zoom-koom. Addition of water Addition of sterilized water & Filtration Addition of Sugar (0.05kg/L) Figure 4: Process flow showing Laboratory Processing of zoom-koom Millet Sorting Steeping time(2,6 12hrs) & Steeping temp.(25, 35 & 45) Blend ratio of millet & spice (700:50, 700 :100 & 70:150) Wort Zoom-koom Sodium sorbate(5%) University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 37 Table 6: Analysis of zoom-koom samples Physicochemical Microbiological Analysis Bioactive properties Colour, pH, TTA, oBrix Total plate counts, yeasts and moulds, E. coli, Staphylococcus aureus, Enterobacteriaceae Antioxidant Activity, Total phenolics. 3.4.4 Consumer sensory acceptance of zoom-koom The sensory acceptability of the 15 zoom-koom samples excluding the control obtained as detailed in the preceding section was examined. The study used the balanced incomplete block design described by Cochran and Cox (1957) and Montgomery (2001) with 15 treatments (samples), k = 5 (samples per judge), r = 7 (replicates), b = 21 (number of blocks/judge), = 2, and N=105. Each panellist was given the opportunity to examine five samples at a time because it would be increasingly challenging for a single consumer to evaluate all 15 zoom-koom samples in one session without using up valuable time. Panellists’ (n=21, k= 5 samples per judge) from the University of Ghana were chosen at random amounting to a total number of 105 consumers. Measures for recruiting included ensuring that panellists were frequent zoom-koom consumers. The panellists were given the zoom-koom in disposable cups that were coded with randomly generated three-digit codes. panellists were asked to evaluate the samples based on the taste, aftertaste, colour, aroma, and overall acceptability using a 9-point hedonic scale (9 = like greatly, 5 = neither dislike nor like, and 1 = dislike significantly) (Prinyawiwatkul et al. 1997; Peryam and Pilgrim 1957). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 38 3.5 Proximate composition of optimized zoom-koom 3.5.1 Moisture content determination An amount of 3 g zoom-koom was weighed using an analytical balance (SARTORIUS B120S, GERMANY). The weight of the petri dish and each sample was determined and recorded. The petri dish and its content were placed in a drying oven (FISHER Isotemp® Oven, SENIOR MODEL) at a temperature of 105oC for 12 h. It was then removed and placed in a desiccator and then weighed. The procedure was repeated for each sample in triplicates (AOAC, 2000). The moisture content was determined as follows: Moisture (%) = (𝑊1−𝑊2 )𝑋 100 𝑊2 Where: W1= weight (g) of sample and crucible before drying and W2= weight (g) of sample and crucible after drying. 3.5.2 Crude fat determination by goldfish apparatus method An amount of 3 g zoom-koom with a known a moisture content was used. The beakers were weighed using an analytical balance (SARTORIUS B120S, GERMANY). The samples were placed into a thimble and placed in the holding chamber of the Goldfish apparatus. An amount of petroleum ether (25ml) was poured into each of the beaker. The beaker containing the solvents was also connected to the gaskets. The tap was then opened to allow free flow of water through the apparatus to facilitate the condensing of the solvent extracted within 4h. The beakers were and its content dried in an oven (FISHER Isotemp® Oven, SENIOR MODEL) for 30 min cooled in a desiccator for 30 min and weighed using an analytical balance (SARTORIUS B120S, GERMANY) to determine the difference in weight of the flask. The procedure was repeated for University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 39 each sample in triplicates (AOAC, 2000). The fat content was calculated using the formula: Crude Fat (%) = 𝑊1 𝑋 100 𝑊2 Where: W1=Fat weight and W2 = Sample weight 3.5.3 Ash content determination An amount of 3 g zoom-koom was weighed using an analytical balance (SARTORIUS B120S, GERMANY). The weight of the crucible and each sample was determined and recorded. The crucible and its content were placed in a muffle furnace (THERMO SCIENTIFIC) at a temperature of 600oC for 2 h. The crucibles were removed and allowed to cool in a desiccator after which was weighed. The procedure was repeated for each sample in triplicates (AOAC, 2000). The formula used to calculate ash content: Ash (%) = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ 𝑠𝑎𝑚𝑝𝑙𝑒−𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 sample weight × 100 3.5.4 Crude fiber content determination The sample utilized for the fat analysis was also used for the study of the raw fiber. The defatted sample was put into a 500 ml Erlenmeyer flask, to which 0.5 g of asbestos and 200 ml of 1.25% boiling H2SO4 were added. The condenser was then attached, and the flask was placed on a heated plate. It was then be filtered and washed with boiling water till filtrate was no longer basic and 15ml alcohol was used to do a final washing and residues transferred into silica crucibles and dried in an electric oven (FISHER Isotemp® Oven, SENIOR MODEL) for one hour at 100oC. The weight loss was calculated after the crucibles and their contents have been heated for 30 minutes in a muffle furnace, cooled in a desiccator, and weighed. Each sample was done in triplicate (AOAC, 1990). The crude fibre content was calculated using the formula: University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 40 Crude fibre(%) = 𝐶1−𝐶2 ) 𝑋 100 𝐶3 , where: C1= Weight of dried sample, C2 = Weight of ashed sample, and C3 = Weight of defatted sample 3.5.5 Crude protein content determination Using an analytical balance (SARTORIUS B120S, Germany), 3 g of zoom-koom were weighed and put into a digestion flask. Kjeldahal catalysts and concentrated H2SO4 amount to 25 milliliters. The digested sample was next filtered into a 100 ml volumetric flask, filled to the appropriate level with 60 ml of distilled water, and thoroughly mixed. The Kjeldhal apparatus was heated while containing 10 ml of sample and 17 ml of NaOH for the ammonia distillation process. Twenty-five millilitres of 4% boric acid were measured into the conical flask to receive the liberated ammonia gas. The nitrogen content was estimated by titrating the ammonium borate formed with standard 0.096N HCl using mixed indicator and titre values recorded. The procedure was repeated for each sample in triplicates (AOAC, 2000). Protein content was calculated using the formula: Crude protein(%) = (𝐴−𝐵) 𝑥 14.007 𝑥 6.25 𝑊 Where: A= volume (ml) of 0.2 N HCL used sample titration, B= volume (ml) of 0.2 N HCL used sample titration, N= Normality of HCL, W= weight (g) of sample, 14.007= atomic weight of nitrogen, and 6.25= the protein – nitrogen conversion factor. 3.5.6 Carbohydrate /nitrogen free extract The estimated amount of total carbohydrates was calculated by deducting the total of moisture, ash, protein, fat, and crude fiber from 100 and expressing the result as a percentage. (AOAC, 2000). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 41 % 𝐂𝐚𝐫𝐛𝐨𝐡𝐲𝐝𝐫𝐚𝐭𝐞 = 𝟏𝟎𝟎 − (% 𝐦𝐨𝐢𝐬𝐭𝐮𝐫𝐞 + % 𝐀𝐬𝐡 + %𝐂𝐫𝐮𝐝𝐞 𝐟𝐢𝐛𝐫𝐞 + %𝐂𝐫𝐮𝐝𝐞 𝐩𝐫𝐨𝐭𝐞𝐢𝐧 + %𝐂𝐫𝐮𝐝𝐞 𝐟𝐚𝐭) 3.5.7 Energy The energy content of zoom-koom was determined by multiplying a factor of four (4) to the protein, a factor of nine (9) to the fat and a factor of four (4) to the carbohydrate and the results summed up (AOAC, 1990). The energy content of bread was calculated using the formula: Energy (kcal) = (4 x protein content) + (9 x fat content) + (4 x carbohydrate content) 3.6 Data and Statistical analysis Mean ± SD was used to summarize physicochemical, bioactive properties and microbiological values. ANOVA was used to check the significance of differences at an alpha level of 0.05 between the microbial counts and physicochemical values of the commercially processed samples. Differences were assessed using a post hoc Tukey test using Minitab software version 20.0. For the optimization, response surface regression techniques were used to analyse the experimental data, and the F-test was used to determine the significance of the regression coefficients, the collected data were fitted to polynomial models, and the models' fitness were assessed in terms of R2 values, the absence of fit errors, and the prediction error sum of squares (PRESS). University of Ghana http://ugspace.ug.edu.ghUniversity of Ghana http://ugspace.ug.edu.gh 42 CHAPTER FOUR 4.0 Results and Discussion 4.1 Physicochemical properties of commercially processed zoom-koom The physicochemical properties of zoom-koom samples along the commercial processing chain sourced from four commercial processors in the Greater Accra Region, one commercial processor in Koforidua in the Eastern Region of Ghana and the control sample treatment is presented in Table 7. Generally, there was a reduction in pH and a rise in TTA. Th