PROXIMATE AND FREE FATTY ACID COMPOSITION OF SELECTED ANIMAL PRODUCTS FROM TWO MARKETS IN THE ASHIEDU KETEKE SUB METRO IN ACCRA BY MADUFORO, ALOYSIUS NWABUGO (10509385) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MSC DIETETICS JULY, 2016 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION This is to certify that this thesis is the result of research undertaken by Maduforo, Aloysius Nwabugo towards the award of the Master of Science Degree in Dietetics in the Department of Nutrition and Dietetics, School of Biomedical and Allied Health Sciences, College of Health Sciences, University of Ghana, Legon …………………………………………… MADUFORO, ALOYSIUS NWABUGO (STUDENT) ………………………………... DR. MATILDA ASANTE (SUPERVISOR) ………………………………... MRS. ANNA AMOAKO - MENSAH (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh iii DEDICATION This research work is dedicated to Almighty God who saw me through this University and who showed me an everlasting love by giving his only begotten SON for my redemption. He gave me the FAITH to embark on this journey even in the mist of financial difficulties. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENT My most profound gratitude goes to God Almighty for sustenance throughout my stay in this University while working on this research. I thank my supervisors Dr. Matilda Asante, and Mrs. Anna Amoako - Mensah whose thorough supervision made this work realisable. I also extend my gratitude to Mrs Mary Avagah, a good friend, who took me in the first day I relocated to Ghana and had me settled. My success story is not complete without my wonderful sisters: Mrs Mmezi Chinaka, Ms. Lilian Maduforo, Mrs Onuh Kelechi and Mrs Uzowuru Ada Zion who financially, morally and spiritually motivated me to complete this work. I also thank my brother whom I have taken to be my earthly father, Mr. Maduforo Anayo, your support and encouragement will be rewarded. I am also grateful to all my siblings for their support and prayers. I have siblings in the faith whom God used in manifold ways to bless my life, my friend and brother, Dr. Hayford T. Ayerteh, and my brethren: Samual Hanyor, Abigail Amorin, Charles Kafando, Benjamin Kwame, Ruth Ukwelenwa, Mary Nafaye, Appronti Deborah, Lydia, Georgina, Derrick and many others, thank you very much. I appreciate the effort of my pastors and their wives in Deeper Life Campus Fellowship in Ghana, Pastor, Francis Fosu, Frank Nartey (Fiffi), Degraft Boateng and Emmanuel Buabeng. Also my lecturers in University of Ghana: Prof. Steiner Matilda Asiedu, Prof. Christiana Nti, Dr. Gladys Peprah Boateng, Dr. Charles Brown, Dr. Benjamin Arko-Boham, Mrs Freda Intiful, Mr Frank Hayford, Mrs Lauren Boateng and many others. I am also grateful to all the clinical preceptors at 37 Military Hospital, PML children Hospital, and Trust specialist hospital who University of Ghana http://ugspace.ug.edu.gh v mentored me at different occasions during the study. All the laboratory scientist and technicians at CSIR Food Research Institute Ghana especially Mr Nelson, I am grateful for your assistance. I also thank my friends and classmates Mr Tamimu Yakubu and Mr Rauf Issah for their encouragement. The best mother in the world is my mother Mrs. Maduforo M. who encouraged, supported and prayed for me, mummy thank you. I also appreciate Mr Adams Shehu and Mr Bilal Obeng Musah for their help during the statistical analysis of the study, thank you all. University of Ghana http://ugspace.ug.edu.gh vi ABSTRACT Background: Knowledge of the nutritional composition of foods is absolutely indispensable for proper diet therapy and dietetic counselling. Cow hide (wele), cow intestines, cow tripe, and cow foot serve as special delicacies in Ghana, but the nutritional composition of these animal products are either missing in the Ghanaian food composition tables or have not been updated for over 40 years. Furthermore, the Ghana food composition tables do not have any information on the fatty acid composition of foods in the database. This affects the accuracy and reliability of dietary data analysis. Aim: The aim of the research was to determine proximate (protein, fat, carbohydrate, moisture and ash) and free fatty acid composition of four animal products from cow (cow hide, intestine, tripe and cow foot). Methods: Four selected samples; cow hide (wele), cow offals (intestine and tripe) and cow foot were purchased from Makola and Agbogbloshie markets in Asiedu Keteke Sub Metro Area of Accra. Samples were analysed chemically using standard analytical methods at the Food Research Council for Scientific and Industrial Research Food Research Institute of Ghana. Nutrient composition analysis was performed on raw and cooked samples. Results were summarized as means, and standard deviation. Percentage changes in the nutrient composition after cooking of the samples were calculated. Data from the raw and cooked samples were compared using the independent sample t – test. Results: Moisture composition of the fresh (uncooked) samples ranged from 66.23±0.21 g/100 g to 82.44±0.01 g/100 g. There was a significant decrease (P<0.05) in the moisture content of the cooked samples: ranging from 61.63±0.06 g/100 g to 70.47±0.02 g/100 g. Ash and fat University of Ghana http://ugspace.ug.edu.gh vii content of the raw samples differed significantly (P<0.05) from the cooked samples and ranged from 0.29±0.01 g/100 g to 0.87±0.09 g/100 g. The fat content of the raw and cooked samples ranged from 0.24±0.01 g/100 g to 9.16±0.01 g/100 g and 0.37±0.01 g/100 g to 18.16±0.02 g/100 g respectively. Protein increased in all the samples after cooking. The protein composition in the uncooked samples ranged from 9.44±0.04 g/100 g to 28.42±0.07 g/100 g. The highest percentage increase (169%) as well as the highest moisture loss was observed in the intestine. The least increase in protein after cooking was seen in cow hide which increased by 16.64%. The protein composition of the cooked samples ranged from 25.44±0.19 g/100 g to 39.78±0.15 g/100 g. The free fatty acid (FFA) as oleic in the uncooked samples ranged from 0.49 g/100 g of fat to 13.71 g/100 g of fat. There was an increase in all the samples after cooking, however this was significant in (P<0.05) in two of the samples (cow hide and cow foot). Conclusion: The study has provided data on the proximate and free fatty acid composition of some animal products. Cooking of the samples had a significant effect on nutrients composition. All the analysed products contained protein though the quality of protein in cow hide and cow feet was not ascertained in this study. Free fatty acid (FFA) as oleic increased in all the sample after cooking and was highest in cow hide, confirming that extensive heat treatment increases the level of FFA. Results from the study can be used as a guide in counselling clients and educating the general public to make wise choices when they are cooking with these animal products. University of Ghana http://ugspace.ug.edu.gh viii TABLE OF CONTENTS DECLARATION ............................................................................................................... ii DEDICATION ................................................................................................................. iii ACKNOWLEDGEMENT ................................................................................................ iv ABSTRACT ..................................................................................................................... vi LIST OF TABLES .......................................................................................................... xii LIST OF FIGURES ........................................................................................................ xiii CHAPTER ONE............................................................................................................... 1 1.0 INTRODUCTION .................................................................................................. 1 1.1 Background ............................................................................................................. 1 1.2 Problem Statement .................................................................................................. 4 1.3 Significance of the study ......................................................................................... 5 1.4 Aim of the study ..................................................................................................... 5 1.5 Specific objectives .................................................................................................. 5 CHAPTER TWO ............................................................................................................... 6 2.0 LITERATURE REVIEW ........................................................................................ 6 2.1 Meat and animal products ....................................................................................... 6 2.2 Importance of animal products ................................................................................ 6 2.3 Food habits and animal products ........................................................................... 10 2.3 Importance of meat in West Africa ........................................................................ 11 2.4 Cow hide, tripe, intestine and cow foot consumption in Africa .............................. 12 2.5 Nutrient composition of animal products ............................................................... 14 2.6 Proximate and fatty acid composition of meat ....................................................... 17 2.6.1 Protein composition of meat .............................................................................. 17 2.6.2 Ash composition of meat ....................................................................................... 19 University of Ghana http://ugspace.ug.edu.gh ix 2.6.3 Moisture composition of meat ............................................................................... 22 2.6.4 Carbohydrate composition of meat ........................................................................ 23 2.6.5 Fat composition of meat ........................................................................................ 23 2.6.5.1Fatty acid composition of meat.............................................................................. 24 2.6.5.2Free fatty acids (FFA) as oleic .............................................................................. 28 2.7 Cooking of Meat ................................................................................................... 29 2.7.1 Effect of cooking on nutritional quality of meat .................................................... 30 2.7.2 Effect of cooking on moisture composition of meat ............................................... 30 2.7.3 Effect of cooking on fat and fatty acid composition of meat .................................. 31 2.7.4 Effect of cooking on protein composition of meat ................................................. 32 2.7.5 Effect of cooking on micronutrient composition of meat ....................................... 34 CHAPTER THREE ......................................................................................................... 36 3.0 METHODS ........................................................................................................... 36 3.1 Study design ......................................................................................................... 36 3.2 Study site and Laboratory...................................................................................... 36 3.3 Procedure for data collection ................................................................................. 36 3.4 Sample preparation ............................................................................................... 38 3.5 Methods for Nutrients analyses ............................................................................. 39 3.5.1 Moisture content determination ............................................................................. 39 3.5.1.1 Principle.............................................................................................................. 39 3.5.1.2 Procedure ............................................................................................................ 39 3.5.2 Protein content determination ................................................................................ 40 3.5.2.1 Principle.............................................................................................................. 40 3.5.2.2 Procedure ............................................................................................................ 41 3.5.3 Fat content determination ...................................................................................... 42 University of Ghana http://ugspace.ug.edu.gh x 3.5.3.1 Principle.............................................................................................................. 42 3.5.3.1 Procedure ............................................................................................................ 42 3.5.4 Ash content determination ..................................................................................... 43 3.5.4.1 Principle.............................................................................................................. 43 3.5.4.2 Procedure ............................................................................................................ 43 3.5.5 Carbohydrate content determination ...................................................................... 44 3.5.6 Energy content determination ................................................................................ 44 3.5.7 Free fatty acid as oleic content determination ........................................................ 45 3.5.7.1 Principle.............................................................................................................. 45 3.5.7.2 Procedure ............................................................................................................ 45 3.6 Data Analysis ........................................................................................................ 46 3.7 Ethics ....................................................................................................................... 46 CHAPTER 4.................................................................................................................... 47 4.0 RESULTS ............................................................................................................. 47 4.1 Proximate and free fatty acid (Oleic) composition of cow hide (wele)....................... 47 4.2 Proximate and free fatty acid (oleic) composition of cow foot ................................... 48 4.3 Proximate and free fatty acid (oleic) composition of cow intestines .......................... 49 4.4 Proximate and free fatty acid (oleic) composition of cow tripe .................................. 50 4.5 Proximate and free fatty acid (as oleic) composition of all the raw samples .............. 51 4.6. The proximate and free fatty acid (as oleic) composition of all the cooked samples .. 52 CHAPTER 5.................................................................................................................... 53 5.0 DISCUSSION AND CONCLUSION .................................................................... 53 5.1 Discussion............................................................................................................. 53 5.2 Limitations of Study ................................................................................................. 60 5.3 Conclusion ............................................................................................................ 60 University of Ghana http://ugspace.ug.edu.gh xi 5.4 Recommendations ................................................................................................. 60 References ...................................................................................................................... 62 Appendix 1: ..................................................................................................................... 72 Pictures of the samples at the various stages of the study ................................................. 72 Appendix 2: ..................................................................................................................... 77 Official raw data result from CSIR Food Research Institute, Accra, Ghana ...................... 77 Appendix 3 ..................................................................................................................... 79 Ethical and Protocol Committee Approval Letter ............................................................. 79 University of Ghana http://ugspace.ug.edu.gh xii LIST OF TABLES Table 2.1: Contribution of various food groups to world food supplies………………………...8 Table 2.2: Meat and milk consumption in selected countries (kg/head/year) ………………….9 Table 2.3: Contribution of animal products to human diets…………………………………...10 Table 4.1: Proximate and free fatty acid (oleic) composition of cow hide (wele) …………..47 Table 4.2: Proximate and free fatty acid (oleic) composition of cow foot……………..……..48 Table 4.3: Proximate and free fatty acid (oleic) composition of cow intestines………………49 Table 4.4: Proximate and free fatty acid (oleic) composition of cow tripe……………………50 Table 4.5: Proximate and fatty acid (oleic) composition of all the raw samples….………….51 Table 4.6: Proximate and fatty acid (oleic) composition of all the cooked samples…………52 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF FIGURES Figure 3.1: Animal products as displayed in Makola market in Accra, Ghana………………37 Figure 3.2: Flow chart for sample purchase, transport and preparation…………………….....38 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Meat and other animal products such as fish, milk, milk products and eggs are used primarily as food for man. However, some serve as components of feed for other animals (McDonald, et al., 2010). Animal by-products especially those from meat and fish are used for different purposes. They may be used to feed pets, such as cats and dogs and may also be integrated in the diets of farm animals such as meat, fish and bone meal. Although animal products are used principally as sources of food, they can also serve other beneficial purposes. They may be used as skins, wool and hair that are employed in the production of fabrics such as carpets and clothing (McDonald, et al., 2010). Slaughtered animals such as cows provide many edible products other than the carcass meat which humans normally consume. Such products include offals (examples: intestine, tripe, liver, kidney and tongue), cowhide, cow head and cow foot. Cow hide popularly known in Ghana as “wele” and "ponmo" or "canda" in Nigeria is one of the highly patronized meat products not only in Ghana and Nigeria but also in other parts of Africa as well (Essumang, Dodoo, & Adokoh, 2007; Essumang, Dodoo, & Hadzi, 2011; Okiei, Ogunlesi, Alabi, & Osiughwu, 2009). In addition, intestine, tripe and cow foot are available in different markets in Ghana and patronized by many people who cook and eat them as a special delicacy at home. Consumers have different misconceptions about the nutritional quality of these animal products. For instance, some Ghanaians believe that products like cow hide ‘has nothing inside’, that is, lacks any nutrient at all. Thus many a dietitian has at one time or the other dealt with hyperlipidemic and patients who frequently indulge in eating these animal products. These University of Ghana http://ugspace.ug.edu.gh 2 products are also offered on the menu of local eateries popularly refered to as ‘chop bars’, restaurants and hotels. According to Gerber (2007), animal products especially meat, suffer from an ugly image as a major source of cholesterol in relation to dietetic value by health professionals. However, it is important to recognize that meat substantially adds to the supply of valuable and essential nutrients. Also, the contribution of less required compounds, like saturated fatty acids, is often overrated because of inaccurate data in some food composition tables and because losses that occur during cooking, as well as trimming before eating, are not usually considered (Gerber, 2007). The performance of an accurate nutrient analysis is critical for proper and accurate documentation of a food composition data base (United States Department of Agriculture, 2014). Knowledge of the nutritional composition of foods is very essential for proper diet therapy, dietetic counselling and nutrition practice. All food products thus require analysis to determine the nutrient composition of the particular food (Nielson, 2010). In every food nutrient analysis, the proximate especially the macronutrient composition is always the initial focus due to its importance to supply energy, build body and also produce chemicals like hormones and enzymes for major body processes (Mahan, Escott-Stump, & Raymond, 2012). According to Nielson (2010) macronutrients (lipids, proteins, and carbohydrates) constitute the main structural components of foods. An accurate and precise quantitative and qualitative analysis of lipids in foods therefore is important for accurate nutritional labelling, determination of whether the food meets the standard of identity for meat products, and to ensure that the product meets manufacturing specifications. University of Ghana http://ugspace.ug.edu.gh 3 Proximate composition of foods refers to the major components of food which include; moisture, ash (total minerals), lipids, protein, and carbohydrates (Nielson, 2010). Macronutrients are nutrients that are needed in large amounts by the body of humans to provide energy, support growth, maintain body processes and to ensure good health status. Overnutrition (in the case of overweight and obesity) and undernutrition (for example kwashiorkor, marasmus, stunting, wasting) are mostly due to excessive intake and inadequate intake of macronutrients, respectively (Mahan, Escott-Stump, & Raymond, 2012; Rolfes, Pinna, & Whitney, 2011). Ash and vitamins in foods are micronutrients, which regulate body processes (Mahan, Escott-Stump, & Raymond, 2012). There is enough nutrition research for the diet-heart (lipid) hypothesis, supporting the idea that an imbalance of dietary cholesterol and fats are the primary cause of atherosclerosis and cardiovascular disease (CVD) (Griel & Kris-Etherton, 2006). Hence, the recommendation of health professionals world-wide is a reduction in the overall consumption of saturated fatty acids (SFAs), trans-fatty acids (TAs) and cholesterol, while emphasizing the need to increase intake of essential fatty acids like omega - 3 (n-3) polyunsaturated fatty acid (Kris-Etherton & Innis, 2007; Griel & Kris-Etherton, 2006). These recommendations on fat consumption are mainly based on findings from epidemiologic studies reporting strong positive correlations between intake of SFA and the incidence of CVD, a condition believed to emanate from the concomitant rise in serum low-density-lipoprotein (LDL) cholesterol as intake of SFA increases (Center for Disease Control and Prevention, 2008). Research findings have revealed that for every 1% increase in energy from SFA, LDL cholesterol levels increases by 1.3 to 1.7 mg/dl (Mensink, Zock, Kester, & Katan, 2003). It is important to recall that previously, the Dietary Guidelines for Americans recommended that daily cholesterol intake be limited to no more than 300 mg/day, however, in a recent guideline on dietary cholesterol consumption, the University of Ghana http://ugspace.ug.edu.gh 4 2015 Dietary Guidelines Advisory Committee (DGAC) declined to bring forward this recommendation because available evidence did not reflect appreciable relationship between consumption of dietary cholesterol and serum cholesterol, therefore, it was stated that “cholesterol is not a nutrient of concern for overconsumption” (USDA, 2015). 1.2 Problem Statement An accurate knowledge of the chemical composition of foods is fundamental and essential in the dietary treatment and management of diseases or in any quantitative study of human nutrition (McCance & Widdowson`s, 2002). In the recent food composition table published by Food and Agriculture Organization (FAO) of the United Nations in 2012, it was reported that some of the data were a collection of research conducted in Africa and outside Africa. However, in West Africa, few food composition tables are published and many of them contain information copied from other food composition tables compiled between 1960 – 1980’s (Food and Agriculture Organization of The United Nations, 2012). Information in the food composition table for Ghana does not include meat offals (intestines and tripe) and cow foot. Although it does include cow hide (wele), this information like the rest of the foods in the database needs to be updated as it was published 40 years ago (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). In addition, the Ghana food composition table has no information on the fatty acid composition of foods presented in the database. This can have a negative impact on dietetic practice since proper dietary counselling requires an evidence-based information (Mahan, Escott-Stump, & Raymond, 2012). This therefore highlights the need for the current research to analyse the nutrient composition of commonly consumed foods including meat and meat products. University of Ghana http://ugspace.ug.edu.gh 5 1.3 Significance of the study Analysis of the proximate and fatty acid composition of foods is very important in dietetic practice, in the food industry for proper and accurate food labelling and nutritional information and for proper food selection by the general public. This research will provide an evidence based report of the macronutrient composition of cow hide (wele), meat offal (tripe and intestine), and cow foot. This will be a handy tool in diet counselling for the dietitian and nutritionist. Findings of the study will also provide relevant information which will assist consumers of these products to make informed choice with regards to consumption of the products. Data from this research will increase and add to the food composition database for Ghana and also be a guide for further studies. 1.4 Aim of the study To determine the proximate and free fatty acid composition of selected animal products from two markets in the Ashiedu Keteke Sub Metro in Accra raw and cooked samples of cow hide, cow foot, cow intestine and tripe. 1.5 Specific objectives The specific objectives of the study were as follows: 1. To determine the nutrients composition by measuring the protein, fats, carbohydrate, ash and fatty acid (as oleic) of uncooked samples of cow hide, cow foot and meat offals (intestines and tripe). 2. To determine the nutritional composition by measuring the protein, fats, carbohydrate, ash and fatty acid (as oleic) of cooked samples of cow hide, cow foot and meat offals (intestines and tripe). 3. To compare the nutritional composition of the raw and cooked samples. University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Meat and animal products There is an increased demand for animal products globally, principally driven by a combination of population growth, urbanization and rising income (Givens, 2005). Meat is the flesh and organ of animals that are eaten as food. The definition of meat differs from one country to another and from one culture to another. What some cultures consider as a "taboo" due to differences in belief and culture, or undesirable is acceptable for another (Heinz & Hautzinger, 2007). In Ghana, ‘wele’, meat offals and cow foot are regarded as meat. However, they are regarded as by-products of meat since they are not part of the main carcass. These meat parts therefore conform with the definition by the Food Standards in Australia New Zealand (FSANZ) who defined meat as ‘the whole or part of the carcass of any buffalo, camel, cow, deer, goat, hare, pig, poultry, rabbit or sheep, slaughtered other than in a wild state, but does not include eggs, or foetuses’ in their Food Standard Code (Food Standards Australia New Zealand, 2002). People commonly use ‘meat’ to refer to only flesh (skeletal muscle plus any attached connective tissue or fat), but the FSANZ definition also includes offal (i.e., meat other than meat flesh, including brain, heart, kidney, liver, pancreas, spleen, thymus, tongue and tripe) although it excludes bone and bone marrow (Food Standards Australia New Zealand, 2002). 2.2 Importance of animal products The importance of animal products in world food supply and nutrition cannot be overemphasized. Animal products supply about one-sixth of energy supplies and one-third of protein supplies globally (FAO, 2008; McDonald, et al., 2010). Meat was found to be the major contributor, followed by milk and milk products (FAO, 2008). However, individual countries vary in their figures. Typical examples are North America and most European University of Ghana http://ugspace.ug.edu.gh 7 countries, where meat consumption is 30–40 times higher than in the countries of Africa and the Indian subcontinent, though for India the difference for milk consumption is not as high (FAO, 2008; McDonald, et al., 2010). The global averages of animal products contribution are about 2.0 MJ of energy and 29 g of protein per person per day. While in the developed countries of Europe and North America, and also in pastoral countries Australia and Argentina, animal products contribute about 4 MJ of energy per day, or about one-third of total energy intake. Their contribution of protein is about 70 g per day, which is higher than total requirements for man (FAO, 2008). However, variations exist between countries in the proportions of the three categories of animal products. For instance, the USA, with their highly developed dairy industry, have reduced consumption of animal fats (i.e. butter) (FAO, 2008). Also in Spain, there is low consumption of butter, and this is attributed to their use of olive oil. An exception is China, where energy contribution from meat is relatively high, but protein intake is relatively low because the meat eaten is mainly high fatty pork (FAO, 2008; McDonald, et al., 2010). Contrary to this, Japan has a low energy contribution from animal products, but protein intake is high because they consume more fish (FAO, 2008; McDonald, et al., 2010). The contribution of animal products in India, is only 0.64 MJ and 10 g of protein per day (or, respectively, 6 and 15 per cent of total intakes), while in the USA, the equivalent figures are 26 and 64 per cent (FAO, 2008). In Nigeria and in many of the countries of West and Central Africa, including Ghana, the contributions of animal products are very small. Kenya, has a higher contribution due to their pastoral agriculture (FAO, 2008; McDonald, et al., 2010). The tables 2.1 to 2.3 below show the contribution of animal products in the global and country food supply. University of Ghana http://ugspace.ug.edu.gh 8 Table 2.1: Contribution of various food groups to world food supplies Food group Energy (%) Protein (%) Cereals Roots, tubers and pulses Nuts, oils, vegetable fats Sugar and sugar products Vegetables and fruits 47 7 10 8 6 43 10 4 2 7 All plant products 78 66 Meat Eggs Fish Milk Other Animal fats 8 1 1 5 1 2 15 2 5 11 1 0 All animal products 18 34 Other foods 5 0 Source: Food and Agriculture Organisation of the United Nations (2008). University of Ghana http://ugspace.ug.edu.gh 9 Table 2.2: Meat and milk consumption in selected countries (kg/head/year) Country or region Meat Milk USA 125.6 257.6 Argentina 86.1 164.2 France 93.4 290.2 UK 84.3 243.5 Burundi 3.7 3.6 Bangladesh 3.3 15.0 India 5.7 63.5 Sri Lanka 6.9 32.8 Source: Food and Agriculture Organisation of the United Nations (2008). University of Ghana http://ugspace.ug.edu.gh 10 Table 2.3: Contribution of animal products to human diets Energy (MJ/day) Protein (g/day) Meat and Offal Milk and Eggs Animal Fats Total Animal Total Animal (including Fish) World 0.96 0.60 0.25 1.81 29 France 2.07 1.69 1.12 4.88 74 Spain 1.89 1.27 0.30 3.46 70 UK 1.96 1.52 0.64 4.12 59 USA 1.92 1.71 0.46 4.09 74 Australia 2.12 1.15 0.96 4.32 70 Argentina 2.12 1.01 0.30 3.43 57 China 1.96 0.48 0.19 2.63 37 Japan 0.74 0.72 0.15 1.61 51 India 0.09 0.31 0.24 0.64 10 Kenya 0.33 0.60 0.03 0.96 15 Nigeria 0.15 0.08 0.02 0.25 8 Source: Food and Agriculture Organisation of the United Nations (2008). 2.3 Food habits and animal products The major factor that determines the intake of animal products is the economic status of people (McDonald, et al., 2010). However, this factor is controlled by many other factors, such as the availability of substitute sources of animal based foods, religious beliefs, food taboos and social customs of consumers (McDonald, et al., 2010; Maduforo, 2010; Maduforo, Nwosu, Ndiokwelu, & Obiakor-Okeke, 2013). Inhabitants of some regions of the world like arctic and desert areas, where cultivation of crop is not possible depend mainly on animals for their University of Ghana http://ugspace.ug.edu.gh 11 protein supply (FAO, 2008; McDonald, et al., 2010). Eskimos in arctic areas, eat fish and other animals that also feed on fish. In desert areas, nomadic people depend on animal products for their survival (McDonald, et al., 2010). Many major religious groups in the world such as Muslims, Jews and Hindus prohibit the consumption of meat from pig (pork) because, they are regarded as ‘unclean’ animals. Hindus who are most often vegetarians and many Muslims do not eat beef. The intake of milk, milk products and eggs is restricted by fewer religious and social groups, although the extreme vegetarians known as vegans exclude these from their diets and this is mostly common among Hindus, Seventh Day Adventist and Eckist groups. In some parts of West Africa, different types of animals that are worshipped or regarded as sacred are not consumed. Also, in some parts of Nigeria, pregnant women, lactating mothers and children are prohibited from eating eggs and some types of meat because of different food taboos associated with them in those areas (FAO, 2008; McDonald, et al., 2010; Maduforo, 2010; Maduforo, et al., 2013). Diets are often considered as bland and unexciting when they are wholly vegetable and plant source by many people in this region. However, meat and other animal products are used to add variety to make it more palatable and acceptable by people. Modern methods of preservation of animal products, such as refrigeration, heat processing, canning and vacuum sealing, have made it easier for people to enjoy an uninterrupted supply of these products especially in the fresh form (McDonald, et al., 2010). 2.3 Importance of meat in West Africa Animal products especially, meat, egg and milk, can make an invaluable contribution to the diets in developing countries like Ghana. Meat has less nutritional importance in many developed countries where a wide variety of foods of all kinds is available (Bender , 1992). University of Ghana http://ugspace.ug.edu.gh 12 Several foods consumed in West Africa and other developing countries are cereals or root crops based and are relatively bulky, especially where fats are in short supply, and this can limit the total energy intake (FAO, 2008). This is typical of complementary foods introduced to children during infancy and foods given to young children in most homes in Ghana. Meat is important in the diet because it is a concentrated source of protein which is not only of high biological value but its amino acid composition complements that of cereal and other vegetable proteins. It is also a good source of iron and zinc and several B vitamins, and liver is a very rich source of vitamin A (Bender 1992; Cunningham & Lupien, 1992; Whitney & Rolfes, 2008). Offal, on the other hand, is the major ingredient for making sausage (Edwards, 2013). It is made from the small intestine of a goat, cow or sheep, stuffed with chilli and small chunks of meat, fatty meat, and blood (however, blood can be excluded which some people prefer) (Edwards, 2013). 2.4 Cow hide, tripe, intestine and cow foot consumption in Africa In Ghana, Nigeria and many other parts of Africa and the world, cow hide (wele) and different types of meat offal is a special delicacy that is consumed generally and during special occasions (Edwards, 2013). Cow foot is called “kotodwe” in Akan language (personal communication) and “ukpa efi” in Igbo language. Cow foot contains hides in it with the addition of bone. Cow foot consumed more commonly in Accra are sourced from Northern Ghana or from neighbouring West Africa countries such as Burkina Faso and Togo (personal communication). Tripe is a type of edible offal from the stomachs of various farm animals (Helou, 2011). Cow tripe is another delicacy that is commonly consumed around Africa as well as in other parts of University of Ghana http://ugspace.ug.edu.gh 13 the world. Beef tripe is produced from the first stomach or rumen (blanket/flat/smooth tripe), also known as the paunch or plain tripe, and second stomach the reticulum (also known as the honeycomb or pocket tripe) of the cow (Scottish Government, 2009). The third stomach or omasum (bible/book/leaf tripe) is also processed for human consumption (being of most value for producers to be made into sausage, stew or soup) (Houlihan, 2011). However, it is difficult to clean, as it needs a lot of water and agitation to clean between the flaps. It also deteriorates if not processed, chilled and packed quickly. There is a very small market for the fourth stomach or abomasum (the reed) and as a result this is only processed in factories if the processing factory has an established market (Houlihan, 2011; Scottish Government, 2009; Bender, 1992). Offal is used extensively in Ghana to prepare different dishes, especially the small intestine. It is used to prepare different type of soups and stews such as light soup, okro soup and tomato stew. Nigerians also eat offals in different forms. It can be added to different types of stews and soups or cooked alone as pepper soup. In South Africa meat offal is relished by South Africans of diverse social class and strata. Hence, the popularity of this dish made it one of the few cultural foods consumed by both white and black South Africans (Edwards, 2013). Reports have revealed that offal dishes in South Africa are mostly limited to stomach, hide (skin), sheep's head, shin and very rarely brains (Helou, 2011; Edwards, 2013). One of the popular methods to cook offals in South Africa is to cook it with small potatoes in a curry sauce served on rice (Edwards, 2013). Also, in Zimbabwe, offal is enjoyed by people of all cultures. Offal dishes made from cow are the most common and they include: stomach, hooves, shin, intestines, liver, head, tongue and University of Ghana http://ugspace.ug.edu.gh 14 very rarely in certain communities, testicles. Chicken offal dishes is however, consumed which include: foot, liver, intestines and gizzards. Another meat offal popularly enjoyed is made from goat or sheep offals (Helou, 2011; Edwards, 2013). Meat offal is also a relished delicacy in Kenya and is associated with traditional beliefs. In some areas, the Kikuyu tradition, grilled kidneys are a delicacy usually reserved for young ladies, however, anybody can eat it nowadays (Edwards, 2013). Equally, only young men eat the tongue and testicles, while the ears were to be consumed by little girls (Edwards, 2013). Liver is consumed by all. The heads, lungs and hooves of animals are boiled to make soup and sometimes mixed with herbs for medicinal purposes (Edwards, 2013). 2.5 Nutrient composition of animal products Foods derived from animals are an important source of nutrients in the diet, they are especially known to supply the body with high biological value nutrients. For instance, red meat contains protein of high biological value and important micronutrients that are needed for good health throughout life. It is also known to contain a range of fats, including essential omega-3 polyunsaturated fats (Givens, 2005; Williams, Droulez , Levy , & Stobaus, 2006; Williams, 2007). Compared to plant foods, food from animal sources can meet micronutrient needs even in smaller portions than plant source foods. Also, animal food sources provide many vitamins and minerals simultaneously, which may be important in diets that are marginally lacking in more than one nutrient (Allen, 2002; Murphy & Allen , 2003). University of Ghana http://ugspace.ug.edu.gh 15 The existence of vegetarians especially, vegans, establishes the non-essentiality of animal products for man; hence, all the essential nutrients for man can be met by foods of plant origin. However, there are, numerous major nutritional benefits in meeting man’s nutritional need from both animal and plant sourced foods instead of from only plant sources (McDonald, et al., 2010). The first is that animal products supply nutrients of high biological value and in proportions closer to those required by man (Whitney & Rolfes, 2008; McDonald, et al., 2010). This is best illustrated by the essential amino acids: a growing child needs 2 g of lysine and 40 g of total protein a day, a ratio of 5 g lysine per 100 g protein (McDonald, et al., 2010). In cereals like rice and wheat proteins, the lysine/protein ratio is much lower (2.8 g and 3.1 g, respectively), and hence these cereals need to be complemented in the diet by a lysine-rich protein source (Whitney & Rolfes, 2008; McDonald, et al., 2010). A good-quality plant protein such as that of the soya bean has a lysine/protein ratio of 6.4, but animal proteins in milk and beef have even more favourable ratios of 8.2 g and 9.1 g, respectively (McDonald, et al., 2010). Therefore, animal proteins are important for complementing the proteins of staple foods such as cereals by supplying lysine and other essential amino acids, and this is particularly important for growing children, for whom amino acid requirements are most critical (McDonald, et al., 2010). If lysine requirements have to be met with cereal proteins, then protein intake has to be high and much of it is wasted (McDonald, et al., 2010). Children however, might not be able to consume the quantity of food that will supply all the needed lysine. Legumes can as well be used to complement for lysine but legumes are also having methionine as a limiting amino acid (Rolfes, Pinna, & Whitney, 2011). University of Ghana http://ugspace.ug.edu.gh 16 One essential nutrient, vitamin B12 (cyanocobalamin), synthesised by microorganisms and present in animal products is virtually absent from plant derived foods (McDonald, et al., 2010; Brown, et al., 2011). Vegans are especially counselled so that they have a supply of this nutrient from a supplementary source such as yeast (Brown, et al., 2011). Animal products are also good sources of other vitamins, especially vitamin A, thiamine, riboflavin and niacin (McDonald, et al., 2010). Animal sourced food contain nutrients that are of high biological value and hence, are more accessible for digestion than those of plant-derived foods (McDonald, et al., 2010; Sizer & Whitney, 2014). Plant cell walls delay digestion in the stomach and small intestine, however, they may be digested in the large intestine, but, the subsequent release of nutrients may be too late to permit efficient absorption (McDonald, et al., 2010). Some minerals in plant tissues are bound in compounds that resist digestion called antinutrients. Good examples are phosphorus in phytates and calcium in oxalates (Wardlaw, Hample, & DiSilvestro, 2004). Animal products are good sources of the minerals iron, calcium and zinc (Gerber, 2007). Meat is regularly related with a negative health image owing to its “high” fat content and in the case of red meat is perceived as a cancer-stimulating food (Gerber, 2007). There is a recommendation of a low meat intake, particularly red meat to avoid the associated health risk such as cancer, obesity and metabolic syndrome (Biesalski, 2005). It was also reported that higher intake of red and processed meats was identified as detrimental compared to lower intake (USDA, 2015). However, this argument did not consider the fact, that meat is an essential source of high biological value protein and some essential micronutrients such as trace elements and vitamins, which are either not present in plant derived food or have a poor bioavailability (Gerber, 2007). Meat is low in carbohydrate and hence, contributes to a low University of Ghana http://ugspace.ug.edu.gh 17 glycemic index, which is attributed to be advantageous for people with obesity, diabetes development and cancer (essential component of ketogenic diet) (Biesalski, 2005). Generally, meat contains many essential and important nutrients, which are beneficial for human health and development. As a crucial part of a diversified diet, meat ensures adequate intake of essential micronutrients and amino acids and is involved in regulatory processes of energy metabolism (Gerber, 2007). 2.6 Proximate and fatty acid composition of meat The proximate composition which includes, protein, ash, moisture, fat and carbohydrate as well as fatty acid composition will be discussed. 2.6.1 Protein composition of meat Crude protein is total nitrogen multiplied by protein factor which is 6.25 for meat and meat products. It is usually expressed in g per 100 g sample. Total nitrogen content includes nitrogen primarily from proteins and to a lesser extent from all organic nitrogen containing non-protein substances. However, for practical purposes, non-protein nitrogen is assumed to be of little significance (ASEAN Network of Food Data Systems , 2011). Studies have shown that a typical meat muscle consists of about 75% moisture, 20% protein, 3% fat and 2% soluble non-protein substances (Gerber, 2007). However, it is reported that proteins are the major component of the dry matter of lean meat (Briggs & Schweigert, 1990). The food composition table of Ghana shows that cow hide (wele) contains 77.7% moisture, 21.7%, protein 0.7% fat and 0.2% ash (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). There are nine essential amino acids in proteins (Sizer & Whitney, 2014). The human body is unable to synthesize essential amino acids hence, they must be consumed from our diet (Rolfes, Pinna, & Whitney, 2011). This therefore, necessitates the consumption of dietary protein to meet the requirement for the dietary essential amino acids, and also to meet the need University of Ghana http://ugspace.ug.edu.gh 18 for non-specific nitrogen required for the synthesis of the dietary non-essential amino acids and other physiologically important nitrogen containing compounds such as nucleic acids, creatine, porphyrins (Pellett & Young, 1990). The digestion of proteins into their constituent amino acids and their absorption are essential for the biosynthesis of endogenous proteins. These processes are important for the various physiological functions of the human body as well as for the growth and repairing of the tissues, for the proper functions of the antibodies and for the regulation of enzymes and hormones (Gerber, 2007). Various foods differ in their protein and amino acid composition. Proteins in different foods have their own unique amino acid composition (Mahan, Escott-Stump, & Raymond, 2012). The essential amino acids present in proteins in foods are used to determine the nutritional quality of that protein (Gerber, 2007). High quality dietary proteins are found to contain the necessary quantities of essential amino acid to meet the needs of the human body. While low quality dietary proteins display an imbalanced ratio of essential amino acids. The essential amino acid that is most lacking in a protein is referred to as limiting amino acid (Gerber , 2007). Studies have shown that the amino acids present in meat protein is comparatively the same regardless of the cut or organ from which the meat is obtained (Pellett & Young, 1984; Bender, 1992). However, a distinguished exception is for meats containing large amount of connective tissue, because of the distinctly different amino acid composition of collagen and elastin (Pellett & Young, 1984). Cow hide and cow foot which are mostly made up of collagen and elastin therefore will have a different amino acid profile even though they might be having protein present in them as reported (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, University of Ghana http://ugspace.ug.edu.gh 19 1975). Studies have reported that collagen varies from most other proteins by the amount of the amino acids, hydroxylysine and hydroxyproline and no cysteine or tryptophan (Pellett & Young, 1984; Bender, 1992). Elastin, also present in connective tissue, has less hydroxylysine and hydroxyproline. Therefore, meat cuts that are richer in connective tissue have lower protein quality (Bender, 1992). The high composition of connective tissues in cow hide and cow foot makes them tough. These cuts of meat are sold at a lower prices compared to lean meat and that is probably the reason most people including the poor in the society patronize these meat parts in Ghana (Bender, 1992). Generally, meats are known to contain high levels of the main essential amino acids, lysine, total sulphur amino acids, threonine and tryptophan (Gerber, 2007). The amino acid pattern of egg protein is considered optimal, therefore, it is usually used as a criterion to define protein quality (Gropper, Smith, & Groff, 2009). Studies have shown that animal proteins, such as meat, milk and cheese have higher protein quality than plant proteins (Mahan, Escott-Stump, & Raymond, 2012). Animal proteins are more bioavailable because they have better digestibility compared to plant proteins (Gerber, 2007). Scientist have tried to explain this to some extent with the fact that plant proteins are mostly embedded into polysaccharide matrices (cell walls) where they cannot be reached by the proteolytic enzymes (Gerber, 2007). However, a healthy nutrition demands a balanced combination of different food proteins. Consumption of plant and animal foods ensures a high nutritional quality of the food due to complementary effect of the diet to meet amino acid need for protein synthesis. 2.6.2 Ash composition of meat Ash refers to the inorganic residue remaining after either ignition or complete oxidation of organic matter in a foodstuff in this case, the meat sample (Marshall, 2010). Ash content University of Ghana http://ugspace.ug.edu.gh 20 represents the total mineral content in foods (Marshall, 2010). Minerals and vitamins are called micronutrients in food. Micronutrients are nutrients needed in small specific quantities in the body. Most of them are not generated in the body but are derived from food intake, making them essential (Whitney & Rolfes, 2008; Rolfes, Pinna & Whitney, 2011, Mahan, Escott- Stump, & Raymond, 2012). Examples of these micronutrients include vitamins A and B12, iron, folates, iodine, and zinc. Prolonged inadequate intake of foods rich in these micronutrients result in their deficiencies (Sizer & Whitney, 2014). Most developing countries mostly in Asia and sub-Saharan Africa are plagued with hunger, poverty and high rate of unemployment. This results to food insecurity in most of the households (Uchendu, 2011). Ash and vitamins are involved in essential metabolic processes. They are crucial part of the diet because they cannot be synthesized by the body at all or in sufficient amount (Gerber, 2007). Minerals such as phosphorus, zinc and iron are required for metabolism of energy giving food nutrients such as fats, carbohydrates and proteins (Sizer & Whitney, 2014). Minerals are present in an adequate amount in a balanced diet however, when they are deficient, many metabolic processes are disrupted in the body and other vital roles such as blood formation, immunity and body building in the body are affected (Mahan, Escott-Stump, & Raymond, 2012). Protein rich animal sourced foods are rich sources of micronutrients. Report have indicated that lack of consumption of foods that are adequate in protein and energy is one of the immediate causes of micronutrient deficiency (National Planning Commission, 2014). Micronutrients of public health importance in sub-Saharan Africa including Ghana are vitamin A, iodine, iron, and zinc and folates. It is reported that globally, almost 63% of women are anaemic and 31% University of Ghana http://ugspace.ug.edu.gh 21 are iodine deficient, while close to 30% of under-fives are vitamin A deficient (VAD) and 20% are zinc deficient (World Health Organization, 2016; Micronutrient Initiative, 2013). Subject to the composition of the individual diet, the bioavailability of iron can vary 5- to 10- fold (Gerber, 2007). The best available haem iron are sourced from meat, fish, poultry and offal while the non-haem iron are sourced from plant foods in our diet (Rolfes, Pinna, & Whitney, 2011). It is important to note as well that meat does not contain iron absorption inhibitory factors like phytate, tannins, oxalate and fibers (Gerber, 2007). About 1 – 10 % of the non- haem iron is absorbed from the diet compared to 20 – 25 % absorption from haem iron (Whitney & Rolfes, 2008). Studies have shown that meat and offals contain a wide variety of mineral salts (Bender, 1992). The composition of these mineral salts such as iron, zinc and copper differ significantly in different species, liver being considered as the richest source of these minerals compared with muscle tissue (Bender, 1992). The feed of animals containing high levels of minerals do not necessarily increase the level of that mineral in the flesh and there is a complex inter-relation between minerals (Byerly, 1975; Bender, 1992). For instance, the molybdenum content of mutton (sheep meat) increases with dietary molybdenum on the condition when dietary sulphate levels are low. Dietary molybdenum prevents the accumulation of copper which is partly counterbalanced by increased manganese (Bender, 1992). Copper present in the liver decreases and molybdenum increases as the amount of molybdenum. There are other interrelationships between mineral salts such as calcium and zinc (Byerly, 1975; Gropper, Smith, & Groff, 2009). However, it is reported that when pasture/feed is deficient in minerals, particularly phosphorus and cobalt, the amount in the muscle are reduced (Bender, 1992). University of Ghana http://ugspace.ug.edu.gh 22 There is dearth of published data on the ash composition of the samples analysed in this study. However, a study in Ghana showed that the ash composition of cow hide (wele) was 0.2% (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). 2.6.3 Moisture composition of meat Moisture in this report refers to the amount of free water and volatile substances that are lost by drying the food under controlled temperature in an air oven (ASEAN Network of Food Data Systems , 2011). It is expressed in g per 100 g sample. Every food product contains moisture, but the composition of moisture varies from one food product to another. Moisture content is required to express the nutrient content per dry weight basis. Research has reported that a typical meat muscle consists of about 75 % moisture (Briggs & Schweigert, 1990). In previous study, the moisture composition of cow hide was 77.7 g/100 g moisture (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). Microbial growth in foods is supported by high moisture content, hence, micro-organisms cannot grow unless there is enough moisture available to them (Bender, 1992). Drying of meat reduces moisture content and the water activity (Aw) thus preventing microbial growth. "Water activity" is defined as the ratio of water vapour pressure measured in the product to the pressure of a saturated water vapour atmosphere at the same temperature (Bender, 1992). Therefore, the free water in a food product, i.e. excluding the water bound to proteins, is called the water activity (aw). Free water is that part that can be removed as water vapour (it is different from the total moisture content) (Bender, 1992). When meats are kept without proper storage, it is predisposed to deterioration by microbes. It is reported that drying meat under conditions of natural temperatures and humidity with circulation of air and the assistance of sunshine is the oldest method of preservation (FAO, 1990). University of Ghana http://ugspace.ug.edu.gh 23 There is variation in the minimum moisture content necessary for bacterial growth depending on the type of organism. Normal bacteria have the minimum water activity value of 0.91; normal yeasts minimum water activity value is 0.88; normal moulds lowest water activity value is 0.80; and for salt-tolerant (halophilic) bacteria the lowest water activity value is 0.77. Therefore, water activity must be reduced below these levels to ensure preservation of food and meat products (Bender, 1992). “Muscle meat of almost any kind can be dried but it is necessary to use lean meat since fat becomes rancid during the drying process. Drying involves the removal of moisture from the outer layers and the migration of moisture from the inside to the outside, so the pieces of food must be thin. The meat is cut into long thin strips or flat thin pieces and preferably salted, either dry or by dipping into salt solution, to inhibit bacterial growth and to protect from insects” (Bender, 1992). 2.6.4 Carbohydrate composition of meat Meat and meat products are not good sources of carbohydrate (Gerber, 2007). However, studies have shown that freshly slaughtered meat might contain carbohydrate in the muscle and the liver where the glycogen are mostly stored. Hence, immediately after rigor mortis, the carcass contains almost 2.5% carbohydrate, made up of lactic acid, glucose and their derivatives (Bender, 1992). Because, meat is low in carbohydrate, it has a low glycemic index, which is attributed to be beneficial with respect to obesity, diabetes development and cancer (Biesalski, 2005). After cooking or long storage of meat, the carbohydrate present will be lost. 2.6.5 Fat composition of meat Fat includes fatty acids, triglycerides, esters, long chain alcohols, hydrocarbons, other glycol esters and sterols. It is usually expressed as g fat per 100g sample (ASEAN Network of Food Data Systems , 2011). Fat is a concentrated dietary source of energy, containing 9 calories per University of Ghana http://ugspace.ug.edu.gh 24 kilogram body weight as against protein and carbohydrate that has 4 calories each (Whitney & Rolfes, 2008). Fats supplies important nutrients such as essential fatty acids as well as precursors of compounds that regulate a number of physiological functions (e.g. prostaglandins) and helps to absorb fat-soluble vitamins (A, D, E and K) (Gerber, 2007). Fats are found at three major sites in the body of animals (Bender, 1992): first, the storage deposits under the skin and around the organs. This makes up the obvious, visible fat in a piece of meat and can be as much as 40 – 50% of the total weight in fatty meat. Second, the small streaks of fat are observable between the bundles of muscle fibres, intermuscular fat, i.e. in the lean part of the meat; this is known as "marbling" and can amount to 4-8% of the weight of lean meat. Third, the small amount of fat within the structure of the muscle - intra muscular or structural fats - in amounts varying with the tissue. This can be 1-3% of the wet weight of muscle and 5-7% of the weight of the liver (Bender, 1992). The quantity of intermuscular and depot fat present in a meat cut differs, and is subject to the fat excretion of the animal and how the cut has been trimmed (Seuss, Honikel, & Scholz, 1988). According to Gerber (2007) structural fats are basically phospholipids and include long chain fatty acids. 2.6.5.1 Fatty acid composition of meat A fatty acid refers to an organic acid-a chain of carbon atoms with hydrogen attached that has an acid group (COOH) at one end and a methyl group (CH3) at the other end (Whitney & Rolfes, 2008). Fatty acids are the simplest of the lipids, they are components of the more complex lipids. Fatty acids definition has included fatty acids derived from triacylglycerols, partial glycerides, phospholipids, glycolipids, sterol esters, or free fatty acids (FFAs). However, in food analysis, the amount is expressed as grams of triacylglycerols (Nielson, 2010). University of Ghana http://ugspace.ug.edu.gh 25 There are three types of fatty acids which include: saturated fatty acids (SFA), mono- unsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) (Gerber, 2007). Fatty acids are classified as follows: saturated (SFA) – when there is no double bond occurring between the carbon-carbon atoms, monounsaturated (MUFA) – possessing one carbon-carbon double bond or polyunsaturated (PUFA) – having two or more carbon-carbon double bonds (Gropper, Smith, & Groff, 2009). According to Gerber (2007) about half of the fatty acids in meats are unsaturated contrary to the widespread belief that animal fat is mainly composed of SFA. According to Romans, et al. (1994) meat lipids usually contain less than 50 % SFA, and up to 70 % (beef 50-52 %, pork 55-57 %, lamb 50-52 % and chicken 70 %) unsaturated fatty acids. Oleic acid is said to be the most common MUFA and it is present in substantial amounts in both animal and plant sources (Food and Agriculture Organization, 2010). Typical sources include all fats and oils, especially olive oil, canola oil, high-oleic sunflower and safflower oil (Food and Agriculture Organization, 2010). Reports have shown that the most abundant fatty acids in meat are oleic (C18:1), palmitic (C16:0), and stearic (C18:0) acid (Bender, 1992; Gerber, 2007). Linoleic acid (C18:2n-6) is the predominant PUFA (0.5 – 7 %), followed by alpha-linolenic acid (C18:3n-3) (Gerber, 2007). Trans-fatty acids contain below 0.5 % of total fatty acids across all types of meat from monogastric animals; in ruminant meats they represent around 2 – 4 % (Pfalzgraf, Timm, & Steinhart, 1994 in Geber, 2007; Valsta, Tapanainen, & Männistö, 2005). However, whether fatty acids, exist in free form or as component of complex lipids, they have various functions in metabolism. These functions include: providing most of the calories from dietary fat, serving as an important constituent of all membranes, and playing a role in gene University of Ghana http://ugspace.ug.edu.gh 26 regulation (Rustan & Drevon, 2005). Polyunsaturated fatty acids which are sourced from dietary lipids serve as precursors of powerful locally acting metabolites i.e. the eicosanoids (Rustan & Drevon, 2005). It is relevant to state that acetic acid is the shortest fatty acid, with a chain of two carbon atoms in length (Gropper, Smith, & Groff, 2009). However, majority of fatty acids occurring naturally are made up of even numbers of carbons in their chains-up to 24 carbons in length (Whitney & Rolfes, 2008). Different types of fatty acids have been attributed to cause different chronic diseases especially saturated fatty acids when consumed in excess of recommended dietary intake (RDI) (Kris-Etherton & Innis, 2007). Fatty acids could also be regarded as essential when they must be supplied through dietary sources and a deficiency symptoms manifest when they are lacking or non-essential. Non – essential fatty acids are those the body can make from other fatty acids (Whitney & Rolfes, 2008; Gropper et al., 2009). Epidemiological studies have shown a strong positive correlation between intake of SFA and the incidence of CVDs, a condition believed to result from the concomitant rise in serum low-density-lipoprotein (LDL) cholesterol as SFA intake increases (Posner, et al., 1991; Hu, et al., 1997; Center for Disease Control, 2008). Several factors have been attributed to influence the fatty acids composition of meat. Among such factors are genetic factors, level of fatness of the animal and dietary factors (Bender, 1992; Gerber, 2007). However, genetic factors are reported to influence fatty acid composition of meat to lower extent than dietary factors (Gerber, 2007). Saturated and monounsaturated (MUFA) fatty acids contents increase with higher fat content of meat than does the polyunsaturated fatty acids (PUFA) content (Gerber, 2007). Muscle lipids are composed of polar lipids, mostly phospholipids found in the cell membranes, and neutral lipids consisting largely of triacylglycerols in the adipocytes that are found along the muscle fibers and in the University of Ghana http://ugspace.ug.edu.gh 27 interfascicular area. A lesser amount of triacylglycerols is also present as cytosolic droplets in the muscle fibres (Gandemer, 1999). According to Gerber (2007) the value of phospholipids in the muscle is comparatively independent of the total fat content and varies between 0.2 and 1 % of muscle weight. Conversely, the triacylglycerols composition of muscle is strongly related to the total fat content and varies from 0.2 % to more than 5 % (Sinclair & O’Dea, 1990; Fernandez, Monin, Talmant, Mourot, & Lebret, 1999; Gandemer, 1999). Studies have shown that phospholipids are particularly rich in PUFA, while triacylglycerols contain lower amounts of PUFA (Bender, 1992; Gerber, 2007). The proportion of SFA to unsaturated fatty acids in phospholipid is strictly controlled in order to maintain membrane properties, because phospholipids are membrane components (Gerber, 2007). Research findings have shown that the PUFA content of triacylglyerols can be influenced by dietary factors, especially in monogastric animals, it is diluted by de novo fatty acid synthesis consisting of SFA and MUFA, hence resulting in a decrease in the PUFA/SFA ratio with increasing fat deposition (Bender, 1992; Gerber, 2007). The majority of saturated, monounsaturated and polyunsaturated fatty acids are sourced from the diet (Gerber, 2007). However, it can also be synthesized in the body with exception of the omega – 3 (n-3) and omega – 6 (n-6) fatty acids (Bender, 1992; Gerber, 2007). Therefore, these two groups of PUFAs are essential and have to be supplied by the food (Mahan, Escott-Stump, & Raymond, 2012). Eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), which are physiologically important long-chain polyunsaturated n-3 fatty acids (LC n- University of Ghana http://ugspace.ug.edu.gh 28 3 PUFA) have been shown to contribute various beneficial health effects, and meat is a good source of this LC n-3 PUFA (Simopoulos, Leaf, & Salem, 1999). There is very limited in vivo synthesis of EPA and DHA from dietary alpha-linolenic acid in adult humans, EPA and DHA are primarily incorporated into muscle tissue phospholipids. Meats therefore can be regarded as a transformer of alpha-linolenic acid to EPA and DHA through their synthesis and following storage in their muscles. It is consequently, recommended that lean meat be taken into consideration when dietary LC n-3 PUFA intakes are being determined (Burdge, Finnegan, Minihane, Williams, & Wootton, 2003, Lopez-Garcia, et al., 2004). It is also important to note that fatty fish and seafood have been attributed to be a rich source of the LC n-3 PUFA, hence, the public health recommendations for regular consumption of fatty fish (Kris-Etherton, Harris, & Appel, 2003). However, research findings have shown that fatty fish may contain toxic compounds which have negative effect on health. Such toxic compounds include: fat soluble methyl mercury, dioxins and polychlorinated biphenyls found in fatty fish (Hooper, et al., 2006). 2.6.5.2 Free fatty acids (FFA) as oleic The acid value of meat is expressed as per cent of FFA calculated as oleic acid (Food Safety and Standards Authority of India, 2012). The acid value is used in the determination of FFA as oleic in meat fats. Acid value is defined as the number of milligrams of potassium hydroxide (KOH) required to neutralize the FFA present in one gram of fat. It is a relative measure of rancidity as FFA are normally formed during decomposition of oil/fat glycerides (Food Safety and Standards Authority of India, 2012). The analytical importance of acid value is that the value is a measure of the amount of fatty acids which have been liberated by hydrolysis from the glycerides due to the action of moisture, University of Ghana http://ugspace.ug.edu.gh 29 temperature, and/or lipolytic enzyme, lipase (Food Safety and Standards Authority of India, 2012). 2.7 Cooking of Meat Meat purchased from the market, may contain bone, visible fat, gristle and tendons which may be removed by some people to some varying degree before cooking, in order to have what they want to see "on the plate". Hence, the size and nutrient composition can vary significantly when compared with raw meat (Bender, 1992). Meat and meat products are considered cooked when the centre of the product is maintained at a temperature of 65-70°C for 10 minutes since the proteins will then be coagulated and the meat tenderised by partial hydrolysis of the collagen. The vegetative form of bacteria, but not spores, will have been destroyed (thermoresistant spores can survive heating above 100°C) (Bender, 1992). The meat cooking process is completed when there is a change of colour from red to brown (red to pink in cured products) and flavours are developed (Bender, 1992). However, it is reported that meat from older animals are higher in connective tissue thereby demands longer cooking at 50 - 60°C. This temperature is also used for collagen hydrolysis. Cow hide and cow foot are normally cooked for longer cooking durations. Due to their low collagen composition, the intestines and tripes are normally not cooked for longer durations as the cow hide and cow foot (Gerber, 2007). The denaturation of protein occurs in the red myoglobin and is converted to brown myohaemochromogen (Lawrie, 1991). This process starts at the temperature of 40°C and almost complete at 80-85°C (Lawrie, 1991). It is also reported that cooked flavour emanates University of Ghana http://ugspace.ug.edu.gh 30 from series of reactions including changes in lipids, carbohydrate and protein, resulting to heat breakdown of peptides and amino acids and reactions between proteins and carbohydrates (Bender, 1992). It is also reported that if meat from older animals is heated for long periods at temperatures above 80°C, their amino acids begin to decompose with the production of unpleasant flavours hence, the hydrolysis of collagen is rapid during the canning process when high temperatures are employed for only a short time (Bender, 1992). As earlier stated, offals are the major ingredient used in the production of meat products, such as sausages. It is reported that in sausage production, the particles of meat become bound together during cooking through coagulation of extracted proteins (Bender, 1992; Edwards, 2013). 2.7.1 Effect of cooking on nutritional quality of meat Meat is regarded as a complex food that is made up of a highly structured nutritional composition (Gerber , Scheeder, & Wenk, 2009). The aim of cooking meat is to make it palatable and safe for consumption (Tornberg, 2005). Studies have demonstrated that heat treatment may result in undesirable changes in meat, such as the loss of the nutritional value of food, mainly due to vitamin and mineral losses, and changes in the fat and fatty acid composition due to leaching into water used for cooking and lipid oxidation (Rodriguez- Estrada, Penazzi, Caboni, Bertacco, & Lercker, 1997). 2.7.2 Effect of cooking on moisture composition of meat Several studies have shown that moisture is lost during cooking. However, the amount of moisture lost during cooking depends on some factors such as duration of cooking, temperature, method of cooking, size of sample and heat penetration (Bender, 1992; Cunningham & Lupien, 1992; Gerber, 2007; Nielson, 2010). University of Ghana http://ugspace.ug.edu.gh 31 Reduction in the moisture composition of meat results in an increase in amount of the fat and protein per 100g. However, changes in fat composition depends on the method of cooking. Also, water-soluble vitamins, minerals (ash) and protein are lost in the juices. Some studies have reported significant reduction or increase while some have reported insignificant reduction or increase of the nutrients after cooking. However, in most cooking procedures the juices are usually consumed with the meat (Bender, 1992; Cunningham & Lupien, 1992; McCance & Widdowson`s, 2002; Gerber, 2007; Nielson, 2010). Hence, with so many factors effecting nutrient changes during cooking, research findings are rarely comparable - except when the study has been carried out in the same laboratory - and cannot be expected to do more than indicate the general effects (Bender, 1992). The reduction in moisture composition during cooking invariably affects the nutrient composition of the meat by increasing the amount per unit weight of some nutrients. 2.7.3 Effect of cooking on fat and fatty acid composition of meat Lipids (fats and oils) are essential structural and functional components of food which have a crucial effect on food quality even when the amount of lipid is low (Frankel, 1998; Gerber, 2007). The effect of cooking on the fatty acid composition of meat has been documented in various literatures with fluctuating results making it impossible to draw a general conclusion (Janicki & Appledorf, 1974; Ono, Berry, & Paroczay, 1985; Slover, Lanza, Thompson, Davis, & Merola, 1987; Smith, Savell, Smith, & Cross, 1989; Heymann, Hendrick, Karrasch, Eggeman, & Ellersieck, 1990). A previous study by Janicki and Appledorf (1974) it was reported that in raw ground beef patties with 18.4% initial fat had PUFA/SFA ratio of 0.052, whereas cooked portion had ratios ranging from 0.061 – 0,063. In a similar study, Ono et al. (1985) reported in similar study ground beef patties with 21.5% initial fat, had PUFA/SFA ratios of 0.060 – 0.061 for the raw and cooked respectively. Later research on these studies University of Ghana http://ugspace.ug.edu.gh 32 found that subject to the type of product, fatness or the extent to which it is cooked, the meat may or may not be higher in the percentage of unsaturated fatty acids (Rhee, 2000). A more recent study found that cooking reduced total fat content by about 17.9 to 44.4 % and hence, concurrently influenced the content of different fatty acids as well (Gerber, 2007). Oxidation of fatty acids occurs more in the presence of heat (Wood, et al., 2003; Alfaia, et al., 2010). Studies have shown that cooking increases the level of free fatty acids in meat (Bender, 1992; Wood, et al., 2003; Gerber, 2007). In Ghana, cow hide and cow foot normally pass through extensive heat treatment, such as singeing with car tyre or fire wood (Essumang, Dodoo, & Adokoh, 2007; Essumang, Dodoo, & Hadzi, 2011). This will result in more oxidation of unsaturated fatty acids, hence producing more free fatty acids (Wood, et al., 2003; Alfaia, et al., 2010). Further cooking of the meat will increase the level of free fatty acids in meat (Alfaia, et al., 2010). Studies have shown that the ability of unsaturated fatty acids, particularly those containing more than two double bonds, to rapidly oxidise, is important in regulating the shelf life of meat (Bender, 1992; Wood, et al., 2003; Gerber , Scheeder, & Wenk, 2009; Alfaia, et al., 2010) 2.7.4 Effect of cooking on protein composition of meat Subjecting meat to a high temperature has been reported to have effect on damaging proteins especially, when part of an essential amino acid is rendered unavailable. This has been attributed to first lysine at temperatures around 100°C; then cystine and methionine at temperatures around 120°C, and other amino acids after prolonged heating (Bender, 1978). When meats are cooked at a low temperature, there is little loss of available lysine and no loss of methionine and cystine (Bender, 1992). Very old studies revealed that there was change in protein quality after roasting in an open pan at 163°C when the internal temperature did not University of Ghana http://ugspace.ug.edu.gh 33 rise above 80°C; or when the meat was browned in an oven for 30 minutes, and later sterilised in a can (Mayfield & Hedrick, 1949; Rice, 1978). Exposure of meat to high temperatures especially during roasting makes the external brown due to Maillard or browning reaction which is a reaction between the lysine and reducing substances present and hence produces the desired roast flavour. The roasted part is only a small proportion of the total piece of meat and the internal temperature does not exceed about 80°C, however, there is no measurable change in the quality of the protein as a whole (Bender, 1992). In another report, apart from the inherent quality of the various proteins a decrease in quality is assumed if there is damage to amino acids when the food is cooked. It is important to note that when proteins are coagulated at a temperature not below 100°C, there is no change in nutritional quality (Bender, 1992). The nutritional quality of the proteins of meat rich in connective tissue such as cow hide and cow foot used in this study is low since collagen and elastin are poor in the sulphur amino acids (Pellett & Young, 1990; Gerber, 2007). Studies have shown that - there is only 0.8 g of each (collagen and elastin) per 100 g of total protein as against the values of 2.6 and 1.3 of each respectively in "good meat (Bender, 1992). Meat that has high level of collagen and elastin is tough to eat and such meat is mostly used for canning since the relatively high temperature involved in the sterilisation process partly hydrolyses the collagen so making the product more palatable. However, the outcome is still a product with net protein utilization (NPU) that is as low as 0.5 as against a value of 0.75 - 0.8 for good quality meat (Lang, 1970; Bender, 1992). The total damage to protein caused by cooking is of little practical significance and hence, meat is mostly consumed with the diet which contain protein from other food ingredients, therefore, University of Ghana http://ugspace.ug.edu.gh 34 the shortfall in meat quality can be compensated with other protein sources in the diet (Bender, 1992). 2.7.5 Effect of cooking on micronutrient composition of meat Meat is known as a rich source of micronutrients (Sizer & Whitney, 2014). The B vitamins and trace elements present in meat, greatly contributes to the daily intake of these micronutrients in the diet (Lombardi-Boccia, Lanzi, & Aguzzi, 2005). It is important to note that data presented in most food composition tables are based on nutritional composition of raw meat, hence, losses occurring during cooking are only stated in general comments and generally little or no details are given (Gerber , Scheeder, & Wenk, 2009). Some vitamins like thiamine and riboflavin are water-soluble and heat-labile vitamins (Whitney & Rolfes, 2008). Thiamine however, is more susceptible to thermal degradation when compared with riboflavin (Gerber , Scheeder, & Wenk, 2009). Studies have shown that as much as almost 100 % of thiamine can be lost by different processing methods. Riboflavin on the other hand, also showed retention ranging from 58 % to 20 % (Chan, Brown, Lee, & Buss, 1995; Lombardi-Boccia, Lanzi, & Aguzzi, 2005). Cooking time, temperature, and cooking method have been classified as factors that contribute to losses of micronutrients. Therefore, it is crucial to be acquainted with the extent of losses occurring during cooking (Gerber , Scheeder, & Wenk, 2009). The availability and quantity of minerals in meat have been shown not to be significantly affected by commonly used cooking and processing methods (Jansuittivechakul, Mahoney, Cornforth, Hendricks, & Kangsadalampai, 1985). However, variable amounts do leach into the broth, hence, the nutritional consequences of these are challenging to evaluate because the drippings from meat may or may not be consumed with the diet (Zenoble & Bowers, 1977). Therefore the degree of mineral loss is determined by the cooking medium and utilization of the dripping (Gerber , Scheeder, & Wenk, 2009) University of Ghana http://ugspace.ug.edu.gh 35 Meat has an iron-absorption enhancing factor which makes it a unique source of iron. There is absence of absorption inhibitors, and the presence of heme iron in meat (Lee & Shimaoka, 1984). Many studies have demonstrated that the iron composition of meat cooked in iron cookware increased and was available as natural food iron (Mistry, Brittin, & Stoecker, 1988; Cheng & Brittin, 1991; Kumar, Srivastava, & Srivastava, 1994). This can invariably increase the ash content of meat. A more recent study revealed that mineral salts including calcium, sodium, potassium, magnesium and phosphorus were affected by cooking processes by decreasing during the aforementioned minerals after cooking processes (Gerber , Scheeder, & Wenk, 2009). This can have effect in generally reducing the ash composition of meat. Contrary to the other minerals that decreased after cooking, iron and zinc were found to increase in beef after cooking processes which can as well have an effect in increasing the ash composition of the meat. However, in the same study, all the vitamins measured decreased after cooking with thiamine showing the highest losses, from 73 up to 100 % (Gerber , Scheeder, & Wenk, 2009). Other factors identified by several studies that can affect the nutrient composition of meat include: continuous innovations and changes of the breeds as well as the rearing practices, feed composition, slaughtering methods and age of the animal. (Bender, 1992; Lombardi-Boccia, Lanzi, & Aguzzi, 2005; Gerber, 2007; Gerber , Scheeder, & Wenk, 2009). Also, the method used in processing cow hide in Ghana has been found to increase the heavy metal composition which includes iron, mercury, cadmium and zinc are all included and this invariably increases the ash composition of meat (Essumang, Dodoo, & Adokoh, 2007; Essumang, Dodoo, & Hadzi, 2011). University of Ghana http://ugspace.ug.edu.gh 36 CHAPTER THREE 3.0 METHODS 3.1 Study design An experimental laboratory study design was employed for this research. 3.2 Study site and Laboratory The meat samples were obtained from two markets namely Makola and Agbogbloshie within Ashiedu Keteke Sub Metro Area of Accra. These markets were purposively selected because they are the major markets that sell the selected animal products in large quantities in that area. (Accra Municipal Assembly, 2008). The samples were processed and analysed in the chemistry laboratory of the Council for Scientific and Industrial Research (CSIR) – Food Research Institute Ghana (FRI). The CSIR – FRI is adjacent to the Ghana Standards Authority, on the Adamafio Crescent, near the Tetteh Quarshie Interchange, Accra. The CSIR – FRI provides technical, analytical services, contract research and consultancy services to governmental agencies, micro-medium and multinational agro-food processing industries and international development agencies. The chemistry laboratory is accredited by South African National Accreditation System (SANAS). 3.3 Procedure for data collection The samples of each of the animal products (cow hide, cow foot, cow tripe and cow intestines) were purchased from four (4) different points of sale in each of the markets (Makola and Agbogbloshie) by purposive sampling. Sampling from the two major central markets served as a good representative sample as the other markets were smaller and many of them did not sell all the animal products used in this study. Figure 3.1 below shows the different cow meat parts used in this study as displayed in the market. University of Ghana http://ugspace.ug.edu.gh 37 Figure 3.1: Animal products as displayed in Makola market in Accra, Ghana KEY: A = Cow hide (wele) B = Cow foot C = Cow tripe D = Cow intestine The purposive sampling method was adopted to purchase the samples because they were not sold at a particular cluster in the markets, rather their points of sale were located at different points throughout the market. About one pound (lb)/ 0.5 kg each of cow foot, cow tripe and cow intestines was purchased from each seller. In the case of cow hide however, 3.5 lb (1.6 kg) was purchased from each seller to facilitate extracting adequate fat from the sample for FFA analysis. A B C D University of Ghana http://ugspace.ug.edu.gh 38 Thus, eight (8) samples of each of the four products were purchased from the two markets (that is, 4 samples of each product from each of the two markets). Overall, a total of 32 samples were purchased (8x4). The samples were carried in a polythene bag and transported to the laboratory immediately. The samples were kept in the freezer at less ≤-18 0C (see figure 3.1). Figure 3.2: Flow chart for sample purchase, transport and preparation 3.4 Sample preparation The samples from both markets were divided into two equal parts: one to be analysed as a raw sample and the other to be cooked before analysis (see figure 3.1). The meat products were cooked by boiling separately inside an aluminium sauce pan on a Gerhadt electric burner (Gerhadt Bonn, App No, SN: 01191159). The raw products were cooked without the addition of any ingredients except water. In the cow tripe 1000 ml of water was added and it was allowed to boil for 40 minutes, 1000 ml of water was added to cow hide and it was allowed to boil for University of Ghana http://ugspace.ug.edu.gh 39 30 minutes, 500 ml of water was added to cow foot and it was allowed to boil for 40 minutes and 500 ml of water was added to cow intestine and it was allowed to boil for 30 minutes. Cooked and uncooked samples were ground and homogenized separately with an industrial food blender (Panasonic, MX-AC300, Mixer Grinder) to ensure uniform mixture of the different samples. Laboratory samples were collected from the homogenized mixture, vacuum packed and frozen at ≤ -18 0 C until analysis. The homogenized samples were subjected to chemical analysis in duplicates using the standard assay. Each analysis was carried out in triplicate, however, the two most accurate were used to determine the mean. 3.5 Methods for Nutrients analyses Standard laboratory analytical procedures were applied to analyse the various animal products. Proximate (moisture, ash, protein, fat and carbohydrates) and free fatty acid (oleic) were determined. 3.5.1 Moisture content determination The moisture content of the homogenized samples was determined using the Association of Official Analytical Chemists (AOAC) procedure for air oven method of moisture determination (AOAC, 1990). 3.5.1.1 Principle This method is based on the drying of a food sample under controlled pressure and temperature until constant weight is obtained. Moisture content is required to express the nutrient content per dry weight basis (ASEAN Network of Food Data Systems , 2011). 3.5.1.2 Procedure A drying container was placed in the drying oven at 100±5 0 C for 1 -2 hours until constant weight was obtained. It was then cooled in a desiccator for about 30 min and weighed. Two (2) grams of each sample was weighed into the dried weighed crucible. The samples were placed University of Ghana http://ugspace.ug.edu.gh 40 in the air oven pre-heated to 100±5 0 C for 2 – 3 hours. The dried samples were transferred into a desiccator, allowed to cool for 30 min and reweighed. The heating procedure was repeated until constant weight was obtained. The difference in weight between two consecutive weighing did not exceed 5 mg. The difference in weight was calculated as a percentage of the original sample as follows: Moisture (g/100 g) = 𝑾𝟐−𝑾𝟑 𝑾𝟐−𝑾𝟏 𝒙 𝟏𝟎𝟎 𝟏 Where W1 = weight of container or empty dish (g) W2 = weight of container + sample before drying (g) W2 - W1 = weight of sample (g) W3 = weight of container + sample after drying (g) W2 – W3 = loss of weight (g) Total solid (%) = 100 - % moisture (w/w) Test results were reported in g per 100 g sample to two decimal places. 3.5.2 Protein content determination Protein content was determined using the automated Micro-Kjeldahl method (AOAC, 1990). 3.5.2.1 Principle The method is based on the digestion of proteins and other organic food components in the sample with sulphuric acid in the presence of a catalyst e.g. sodium or potassium sulphate to release nitrogen from protein and retain it as ammonium salt. Ammonia gas is liberated upon addition of excess alkali (concentrated sodium hydroxide) and distilled into a boric acid solution to form ammonium-borate complex. The ammonia liberated from the complex is titrated with standardized hydrochloric acid. The amount of nitrogen in the sample is determined from the milligram equivalent of the acid used. Crude protein is determined by University of Ghana http://ugspace.ug.edu.gh 41 multiplying the nitrogen content with a conversion factor (6.25) which is specific to the food matrix (meat) (ASEAN Network of Food Data Systems , 2011). 3.5.2.2 Procedure Digestion: About 2 g of each sample was weighed into a 100 ml micro-Kjeldahl digestion flask. About 2 g of copper sulphate and 10 g sodium sulphate were added to the flask, thoroughly shaken and placed on the digestion rack in an inclined position. The sample in the flask was digested by heating in a flame chamber until frothing ceased. The temperature was increased, allowed to boil for about one hour until the colour changed to bluish green. The clear digested sample was allowed to cool. Distillation: Distilled water was added to the digested sample with a wash bottle to 100 ml in a 100 ml volume metric flask. Ten (10) ml of the digest was pipetted and transferred into a micro-Kjeldahl distillation flask followed by the addition of 60 ml of 60% sodium hydroxide (NaOH) solution. The flask was immediately fixed to the splash head of the distillation apparatus. Four per cent (4%) boric acid was added into a 100 ml receiving conical flask, 2 drops of methyl red indicator was added, in such a way that the outlet of the adapter of the delivery tube was extended under the surface of the boric acid solution. The mixture was heated to liberate ammonia into the receiving conical flask containing 100 ml boric acid and the indicator until yellowish green colour distillate was obtained. Titration: The distillate was titrated with 0.1N hydrochloric acid (HCl) until the end point of pink colouration was obtained. Percentage (%) protein was then calculated as shown in the next section. Calculation Nitrogen content was calculated and multiplied with 6.25 to obtain the crude protein content. Percentage Nitrogen = (𝟏𝟎𝟎 𝐱 𝟎.𝟎𝟎𝟏𝟒 𝐱 𝟔.𝟐𝟓 )𝐓 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 Where University of Ghana http://ugspace.ug.edu.gh 42 Where T = titre value of the sample 0.0014 = correction factor of the acid Test results were reported in g per 100 g sample to two decimal places. 3.5.3 Fat content determination Fat content of the samples was determined using Soxhlet Extraction method (AOAC, 2000). 3.5.3.1 Principle In this method, the sample is hydrolyzed by hydrochloric acid at 70-80oC. Protein, if any, can be dissolved in the acid, crude fat is then manually extracted by diethyl and petroleum ether. The solvent is removed by evaporation and the oil residue is dried and weighed (ASEAN Network of Food Data Systems , 2011). 3.5.3.1 Procedure The homogenized sample that was kept in screw-cap bottle in a freezer at ≤-18 0 C until analysis was used. The sample was thawed to room temperature and mixed thoroughly using a high speed mixer. Two (2) g of the homogeneous sample were weighed into a container (W1). An antioxidant namely pyrogallic acid was added to the sample for fatty acid determination. Hundred (100) mL of 4 N HCl was added to the sample in the digestion flask. Some glass beads were added, then the flask was connected to an air condenser and refluxed with gentle boiling for 30 min - 1 h. The digestion mixture was filtered and residue was washed with warm water until the filtrate was free from acid (indicated by pH paper). The filter paper containing the residue was dried in an oven at 50-60 0 C overnight. It was then transferred into an extraction thimble. The thimble was placed in the reservoir part of the soxhlet apparatus. The extraction cup was dried in an oven at 100 0 C for 1 h. It was cooled in a desiccator and weighed (W2). Fifty (50) mL petroleum ether was added into the pre-weighed extraction cup. The extraction cup was placed into the fat extraction system. The sample was extracted in the thimble by immersing it in warmed solvent for 30 min. The solvent was evaporated in each extraction cup University of Ghana http://ugspace.ug.edu.gh 43 on a water bath in a fume hood. The extraction cup was dried in an oven at 100 ±5 0 C for 30 min and cool in a desiccator. It was re-heated and weighed again every 30 min until constant weight was obtained (W3). However, in the case of the fat that was used for fatty acids determination, the heating temperature was decreased to 50-60 0 C. Calculation Total Fat (g/100 g) = (𝐖𝟑−𝐖𝟐 ) 𝐗 𝟏𝟎𝟎 𝐖𝟏 Where: W1 = Weight of sample W2 = Weight of dried extraction cup before fat extraction W3 = Weight of dried extraction cup after fat extraction Test results were reported in g per 100 g sample to two decimal places. 3.5.4 Ash content determination Ash was determined according to AOAC dry ashing methods (AOAC, 2000). 3.5.4.1 Principle This method involves the separation of minerals from the food matrix by destruction of the organic matter of the sample through dry ashing (ASEAN Network of Food Data Systems , 2011). 3.5.4.2 Procedure Marked crucibles were heated in a furnace at 500 – 550 ºC for 2 - 3 h. The furnace temperature was lowered to 180 ºC and the crucibles were transferred into a desiccator, cooled for 30 min and weighed (W1). About 2 g of dried sample was weighed in duplicate into the pre-weighed crucible dish (W2). The dried samples, were charred over a hotplate, initially at low temperature to avoid spattering. The temperature was gradually increased until smoking ceased. The charred samples were incinerated in a furnace at 500-550ºC until the residue University of Ghana http://ugspace.ug.edu.gh 44 became uniformly white. The temperature of the furnace was decreased to 180ºC and crucibles were transferred into a desiccator, cooled for 30 min and weighed (W3). Calculation Ash (g/100 g) = (𝐖𝟑−𝐖𝟏 ) 𝐗 𝟏𝟎𝟎 𝐖𝟐−𝐰𝟏 Where: W1 = weight of crucible W2 = weight of crucible + sample W3 = weight of crucible + ash Test results were reported in g per 100 g sample to two decimal places. 3.5.5 Carbohydrate content determination This was determined by difference method. The summation of all the proximate values was subtracted from 100% (FAO, 2003). 3.5.5.1 Principle This is based on the fact that once all the other proximate parameters are determined, the balance of the residue is regarded as carbohydrates. Calculation Carbohydrate was calculated by difference (100 – % Moisture + Ash + Protein + Fat). 3.5.6 Energy content determination Energy was determined by the “Atwater factor”. The energy value of the samples was calculated by multiplying the values for fat, carbohydrate and protein with 4:9:4 the “Atwater factors” respectively (Nielson, 2010). Where: protein = 4 Kcal /g fat = 9 Kcal /g carbohydrate = 4 Kcal /g University of Ghana http://ugspace.ug.edu.gh 45 Test results were reported in kcal per 100 g sample to two decimal places. 3.5.7 Free fatty acid as oleic content determination Free fatty acid as oleic was determined by the ISO 660 (1996-05-05) method which involves the determination of acid value of the fat (Food Safety and Standards Authority of India, 2012). 3.5.7.1 Principle The acid value is determined by directly titrating the oil/fat in an alcoholic medium against standard potassium hydroxide (KOH) or sodium hydroxide (NaOH) solution (Food Safety and Standards Authority of India, 2012). 3.5.7.2 Procedure Fat of each sample was melted separately and the oil was mixed to ensue homogeneity before it was weighed. Ten (10) gram of each cooled oil sample was weighed in a 250 ml conical flask. A 100 ml of freshly neutralized hot ethyl alcohol was added and about one ml of phenolphthalein indicator solution was also added. The mixture was boiled for about five (5) minutes and titrated while it was hot against standard potassium hydroxide (alkali) solution shaking vigorously during the titration. The weight of the fat that was taken for the estimation and the strength of the alkali used for titration were such that the volume of alkali required for the titration did not exceed 10 ml. Free fatty acids as oleic (g/100 g fat) = (𝟐𝟖.𝟐 𝐱 𝐕 𝐱 𝐍 ) 𝐗 𝟏𝟎𝟎 𝐖 Where: V = Volume of ml of standard potassium hydroxide used N = Normality of the potassium hydroxide solution W = Weight in g of the sample Test results were reported in g per 100 g fat sample to two decimal places. University of Ghana http://ugspace.ug.edu.gh 46 3.6 Data Analysis Data obtained from chemical analyses was analysed using the statistical package for social sciences (SPSS) version 22 and Microsoft excel 2016 version. Results were summarized as means, and standard deviation. The percentage changes in the nutrient composition after cooking of the samples was calculated. Data from the raw and cooked samples was compared using the independent sample t – test to determine their p – values. 3.7 Ethics Approval for the study was obtained from the Ethics and Protocol Review Committee of the School of Biomedical and Allied Health Sciences, University of Ghana. Permission was also obtained from the authorities and leaders of the markets as well as the traders before the study started. Also, the traders were aware of the intent of purchasing their goods and they granted verbal permission. University of Ghana http://ugspace.ug.edu.gh 47 CHAPTER 4 4.0 RESULTS 4.1 Proximate and free fatty acid (oleic) composition of cow hide (wele) Table 4.1 shows the proximate and free fatty acid (FFA) (as oleic) composition of raw and cooked cow hide. Moisture and ash content were significantly higher (p < 0.05) in the raw cow hide than the cooked cow hide. The energy, fat, protein and the FFA (as oleic) content of cooked cow hide were significantly higher (p < 0.05) compared to raw cow hide. Table 4.1: Proximate and free fatty acid (Oleic) composition of Cow Hide (Wele) Parameter Raw Cooked P-Value %Changes After Cooking Moisture g/100g 72.57±0.010a 68.08±0.07b 0.0001 -6.19 Energy kcal/100g 115.80±0.22b 135.90±0.94a 0.0012 17.36 Ash g/100g 0.87±0.09a 0.48±0.01b 0.0268 -44.83 Fat g/100g 0.24±0.01b 0.37±0.01a 0.0029 54.17 Protein g/100g 28.42±0.07b 33.15±0.21a 0.0012 16.64 Carbohydrates g/100g 0.000±0.00a 0.000±0.00a 1.0000 0.00 FFA (as Oleic) g/100g fat 13.70±0.01b 17.87±0.01a <0.0001 30.44 Means in the same row with the different superscript (a-b) were significantly different at P < 0.05. University of Ghana http://ugspace.ug.edu.gh 48 4.2 Proximate and free fatty acid (oleic) composition of cow foot Table 4.2 shows the proximate and free fatty acid (FFA) (as oleic) composition of raw and cooked samples of cow foot. Compared to the cooked cow foot, moisture, ash and fat content of the raw cow foot were significantly higher (p < 0.05). The energy, protein and FFA (as oleic) content were significantly higher in cooked cow foot (p < 0.05) than raw cow foot. Table 4.2: Proximate and free fatty acid (oleic) composition of cow foot Parameter Raw Cooked P-Value %Changes After Cooking Moisture g/100g 66.23±0.21a 64.40±0.29b 0.0193 -2.76 Energy kcal/100g 164.30±1.49b 185.10±0.11a 0.0026 12.66 Ash g/100g 0.78±0.01a 0.29±0.01b 0.0005 -62.82 Fat g/100g 5.78±0.13a 2.89±0.08b 0.0013 -50.00 Protein g/100g 28.07±0.08b 39.78±0.15a 0.0001 41.72 Carbohydrates g/100g 0.00±0.00 0.00±0.00 0.00 FFA (As Oleic) g/100g fat 0.49±0.09b 1.38±0.09a 0.0105 181.63 Means in the same row with the different superscript (a-b) were significantly different at P < 0.05. University of Ghana http://ugspace.ug.edu.gh 49 4.3 Proximate and free fatty acid (oleic) composition of cow intestines The proximate and free fatty acid (FFA) (as oleic) composition of raw and cooked sample of cow intestines are shown in Table 4.3. Comparisons between the two samples showed that the moisture and carbohydrate content of the raw cow intestines were significantly higher (p < 0.05) while the ash content was significantly lower (p = 0.002). However, the energy, fat and protein content of cooked cow intestines were significantly higher (p < 0.05) compared to raw cow intestines. There was no significant difference in the FFA (as oleic) content of cooked cow intestines and raw cow intestines (p > 0.05). Table 4.3: Proximate and free fatty acid (oleic) composition of cow intestines Parameter Raw Cooked P-Value %Changes After Cooking Moisture g/100g 74.53±0.19a 61.63±0.06b 0.0001 -17.31 Energy kcal/100g 145.80±0.86b 265.10±0.95a 0.0001 81.82 Ash g/100g 0.46±0.01b 0.60±0.01a 0.0022 30.43 Fat g/100g 9.16±0.13b 18.16±0.02a <0.0001 98.25 Protein g/100g 9.44±0.04b 25.44±0.19a <0.0001 169.49 Carbohydrates g/100g 6.13±0.16a 0.00±0.00 b 0.0003 -100.00 FFA (As Oleic) g/100g fat 3.14±0.48ba 3.24±0.18 ab 0.8093 3.18 Means in the same row with the different superscript (a-b) were significantly different at P < 0.05. University of Ghana http://ugspace.ug.edu.gh 50 4.4 Proximate and free fatty acid (oleic) composition of cow tripe Table 4.4 shows the proximate and free fatty acid (FFA) (as oleic) composition of raw and cooked sample of cow tripes. The moisture content was significantly higher (p < 0.05) in the raw cow tripes than the cooked cow tripes. Energy, ash, fat and protein content were all significantly higher (p < 0.05) in cooked cow tripes than raw cow tripes. The carbohydrate content of raw cow tripes is not significantly higher (p > 0.05) than cooked cow tripes with a 100% change after cooking. No significant difference (p > 0.05) was found in the FFA (as oleic) content of the raw and cooked sample. Table 4.4: proximate and free fatty acid (oleic) composition of Cow Tripe Means in the same row with the different superscript (a-b) were significantly different at P < 0.05. Parameter Raw Cooked P- Value %Changes After Cooking Moisture g/100g 82.44±0.01a 70.47±0.02 b 0.0001 -14.52 Energy kcal/100g 83.82±0.32 b 141.20±0.64 a 0.0001 68.46 Ash g/100g 0.37±0.01 b 0.42±0.01 a 0.0283 13.51 Fat g/100g 2.98±0.06 b 3.63±0.01 a 0.0048 21.81 Protein g/100g 13.28±0.25 b 27.16±0.17 a 0.0002 104.52 Carbohydrates g/100g 0.95±0.32 ab 0.00±0.00 ba 0.0524 -100.00 FFA (As Oleic) g/100g fat 2.17±0.07 ba 2.24±0.32 ab 0.7168 3.23 University of Ghana http://ugspace.ug.edu.gh 51 4.5 Proximate and free fatty acid (as oleic) composition of all the raw samples Table 4.5 shows the proximate and free fatty acid (as oleic) composition of all the raw samples analysed in the study. According to the result, cow hide contained the highest ash, protein, FFA (as Oleic) and least in fat content. Cow foot contained the highest energy, cow intestine contained the highest fat and cow tripe contained the highest moisture. Table 4.5: Proximate and fatty acid (as oleic) composition of all the raw samples Parameter Cow Hide Cow Foot Cow Intestines Cow Tripe Moisture g/100g 72.57±0.010 66.23±0.21 74.53±0.19 82.44±0.01* Energy kcal/100g 115.80±0.22 164.30±1.49* 145.80±0.86 83.82±0.32 Ash g/100g 0.87±0.09* 0.78±0.01 0.46±0.01 0.37±0.01 Fat g/100g 0.24±0.01 5.78±0.13 9.16±0.13* 2.98±0.06 Protein g/100g 28.42±0.07* 28.07±0.08 9.44±0.04 13.28±0.25 Carbohydrates g/100g 0.000±0.00 0.00±0.00 6.13±0.16* 0.95±0.32 FFA (as Oleic) g/100g fat 13.70±0.01* 0.49±0.09 3.14±0.48 2.17±0.07 The animal (cow) product containing the highest mean value of the nutrients analysed is indicated with *. University of Ghana http://ugspace.ug.edu.gh 52 4.6. The proximate and free fatty acid (as oleic) composition of all the cooked samples Table 4.6 shows the proximate and free fatty acid (as oleic) composition of all the cooked samples. The table shows that cow hide contained the highest FFA (oleic) and least fat content. Cow foot contained the highest protein and least in ash content. Cow intestines contained the highest level of energy, ash and fat. Cow tripe contained the highest moisture level. Table 4.6: The proximate and fatty acid (as oleic) composition of all the cooked samples Parameter Cow Hide Cow Foot Cow Intestines Cow Tripe Moisture g/100g 68.08±0.07 64.40±0.29 61.63±0.06 70.47±0.02* Energy kcal/100g 135.90±0.94 185.10±0.11 265.10±0.95* 141.20±0.64 Ash g/100g 0.48±0.01 0.29±0.01 0.60±0.01* 0.42±0.01 Fat g/100g 0.37±0.01 2.89±0.08 18.16±0.02* 3.63±0.01 Protein g/100g 33.15±0.21 39.78±0.15* 25.44±0.19 27.16±0.17 Carbohydrates g/100g 0.000±0.00 0.00±0.00 0.00±0.00 0.00±0.00 FFA (as Oleic) g/100g fat 17.87±0.01* 1.38±0.09 3.24±0.18 2.24±0.32 The animal (cow) product containing the highest mean value of the nutrients analysed is indicated with *. University of Ghana http://ugspace.ug.edu.gh 53 CHAPTER 5 5.0 DISCUSSION AND CONCLUSION 5.1 Discussion The moisture composition of the fresh (uncooked) samples ranged from approximately 66% in cow foot to about 82% in cow tripe. This range is similar to the finding from a previous study that indicated that a typical meat muscle contained about 75 g/100 g moisture (Briggs & Schweigert, 1990). Another study on the role of meat in human nutrition for the supply with nutrients, particularly functional long-chain n-3 fatty acids, reported that a typical muscle contained 62% to 75% moisture (Gerber, 2007). In the Ghana food composition table, cow hide is reported to contain 77.7% moisture (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975), which is higher than the moisture content found in cow hide in this present study. The high moisture content of meat reduces its keeping quality (Bender, 1992; Onuoha, Oly- Alawuba, Okorie, Tsado, & Maduforo, 2015). As a result, meat often requires various preservative processes such as drying to slow deterioration. Drying meat reduces the moisture content and makes the water unavailable for microbial growth by reducing the water activity (Aw) of the meat (Bender, 1978; Alfaia, et al., 2010; Onuoha, Oly-Alawuba, Okorie, Tsado , & Maduforo, 2015). In this study, tripe contained the highest level of moisture, making it most susceptible to spoilage and cow foot contained the least moisture hence, making it the least predisposed to microbial spoilage (Bender, 1992) Moisture content of cooked samples reduced after cooking and ranged from approximately 62% in intestine to 70% in tripe. The change in moisture composition after cooking can be attributed to coagulation of protein and leaching out of the moisture (Cunningham & Lupien, 1992). Level of moisture lost during cooking is determined by various factors such as duration University of Ghana http://ugspace.ug.edu.gh 54 and method of cooking, temperature, size of sample and heat penetration (Bender, 1992; Cunningham & Lupien, 1992; Gerber, 2007; Nielson, 2010). The implication of reduced moisture content of meat is a corresponding increase in the density of fat and protein and leaching out of micronutrients especially water soluble and heat labile vitamins as well as minerals (Bender, 1992; Cunningham & Lupien, 1992; McCance & Widdowson`s, 2002; Gerber, 2007; Nielson, 2010). It was also noted in a study that, changes in fat composition was determined by the cooking method and protein is also lost in the juices alongside the micronutrients (Gerber, 2007). While research generally agrees that cooking causes changes in the nutrient density, differing results have resulted in different conclusions regarding the direction and extent of change. Consequently, some studies have reported significant reduction or increase while some have reported insignificant reduction or increase of the nutrients after cooking. Since several factors affect nutrient changes during cooking, research findings are rarely comparable unless the study has been carried out in the same laboratory and even then is not expected to do more than indicate the general effects (Bender, 1992). In any case, reduction of moisture content during cooking invariably affects the nutrient composition of meat and nutrients which are lost from the meat by leaching will not necessarily be lost to the consumer if the recipe includes consumption of the meat with the juice (Bender, 1992; Cunningham & Lupien, 1992; McCance & Widdowson`s, 2002; Gerber, 2007; Nielson, 2010). Ash content of the raw samples ranged from 0.37g/100 g in cow tripe to 0.87±0.09 g/100 g in cow hide. Apart from cow hide, information on the nutrient content of the other meat products analysed, namely tripe, intestine, and cow foot is largely absent from food composition tables used in Ghana and West Africa. University of Ghana http://ugspace.ug.edu.gh 55 The ash content of cow hide obtained in the present study is close to levels reported by a study in Zurich that found that ash formed around 1% of the composition of a typical meat muscle (Gerber, 2007). It is however, much higher than that obtained in a study conducted in Ghana about 41 years ago, which obtained an ash content of 0.2% (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). Several factors may account for this difference. Current research in Ghana shows that cow hide is processed by singeing with car tyres, plastics, petrol and/or wood (Essumang, Dodoo, & Hadzi, 2011). Another study showed that the level of heavy metals such as mercury, lead, iron, and cadmiun, increased in cow hide processed with this method, and these may have contributed to the high level of ash observed (Essumang, Dodoo, & Adokoh, 2007). It is also reported that the type of feed or pasture of animals can influence the level of specific minerals which in turn invariably affects total ash content (Bender, 1992). For instance, when pasture/feed is deficient in minerals, particularly phosphorus and cobalt, the amounts in the muscle are reduced (Bender, 1992). After cooking, significant (p<0.05) changes were observed in the ash levels of all the samples analysed. However, the percentage changes after cooking varied. There was a significant reduction (p<0.05) in cow hide (44.83%) and cow foot (62.82%) after cooking: while the ash content of intestines and tripe significantly (P<0.05) increased after cooking (30.43% and 13.51% respectively). Thus, while cooking resulted in leaching of minerals from cow hide and cow foot, it resulted in a higher loss of moisture in the intestines and tripe which led to a higher mineral density of the latter two products. Fat content of the raw samples ranged from 0.24±0.01 g/100 g in cow hide to 9 g/100 g in intestine. A report on the nutrient composition of a typical muscle of a meat shows that a typical University of Ghana http://ugspace.ug.edu.gh 56 muscle contained about 3 % fat (Gerber, 2007). Several factors may affect the level of fat in a meat sample. In the case of cow hide, one of these factors is processing method. Cow hide processing in the study area involves extensive heat treatment which can burn up the fat during the processing period. (Essumang, Dodoo, & Adokoh, 2007; Essumang, Dodoo, & Hadzi, 2011). This may be a contributory factor to the fact cow hide contained the least amount of fat among the products analysed. Cooking did not change the trend in fat content. Cow hide remained lowest in fat (0.37±0.01 g/100) and intestines the highest (18.16±0.02 g/100 g). Cooking resulted in an increase in the density of fat in all the products with the exception of cow foot which lost fat. Incidentally, cow foot lost the least amount of moisture. Several authours have reported varying results in the fat composition after heat treatment of meat, although their studies did not use exactly the same samples used in this study, hence a general statement cannot be drawn from this study (Janicki & Appledorf, 1974; Ono, Berry, & Paroczay, 1985; Slover, Lanza, Thompson, Jr. Davis, & Merola, 1987; Smith, Savell, Smith, & Cross, 1989; Gerber , Scheeder, & Wenk, 2009). The broth obtained on cooking the meat products showed that fat may have leaked during cooking. In view of the negative health implications of consuming fat above recommended intake including risk of artheriosclerosis, hypertesion, obesity, cardiovascular diseases, and dyslipidemia (Mahan, Escott-Stump, & Raymond, 2012), it will be prudent to encourage the public to discard the fatty broth rather than use it in cooking as is usually the case. Another option is to freeze the broth so that much of the fat can easily be skimmed off before the broth is used. University of Ghana http://ugspace.ug.edu.gh 57 According to research findings, a typical meat muscle consists of 20% protein (Gerber, 2007). In this study, protein content of the uncooked samples ranged from 9.44±0.04 g/100 g in intestines to 28.42±0.07 g/100 g in cow hide. Cooking increased the protein density in all the samples. Protein content of cow hide was higher than that recorded in the food composition tables commonly used in Ghana (21.7 g/100 g). (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975). Even though this study has demonstrated that cow hide and cow foot contain higher crude protein than a typical muscle and studies have shown that the amino acids present in meat protein is comparatively the same regardless of the cut or organ from which the meat is obtained (Bender, 1992). However, there is a notable exception for meats containing large amounts of connective tissue, because of the distinctly different amino acid composition of collagen and elastin (Pellett & Young, 1984). Among all the meat samples used in this study, cow hide is subjected to the highest level of heat processing, hence even though the meat is said to be deficient in essential amino acids like lysine because it is mainly made up of collagen, further high temperature treatment will further reduce the protein quality (Bender, 1978; Pellett & Young, 1984; Bender, 1992; Essumang, Dodoo, & Adokoh, 2007). Cow hide and cow foot are mostly made up of collagen and elastin, therefore, they have a different amino acid profile even though they contain higher protein as reported in this study. Research findings have revealed that collagen varies from most other proteins by the amount of the amino acids, hydroxylysine and hydroxyproline and no cysteine or tryptophan (Bender, 1992). Elastin, also present in connective tissue, has less hydroxylysine and hydroxyproline (Bender, 1992). Therefore, meat cuts of cow hide and cow foot that are richer in connective tissue have lower protein quality (Bender, 1992). Studies have also shown that there is only 0.8 g of each (collagen and elastin) per 100 g of total protein as against the values of 2.6 and 1.3 of each respectively in "good meat (Bender, 1992). Meat that has high University of Ghana http://ugspace.ug.edu.gh 58 level of collagen and elastin is tough to eat and such meat is mostly used for canning since the relatively high temperature involved in the sterilisation process partly hydrolyses the collagen thus making the product more palatable (Bender, 1992). However, the outcome is still a product with net protein utilization (NPU) that is as low as 0.5 as against a value of 0.75 - 0.8 for good quality meat (Lang, 1970; Bender, 1992). After cooking, there was an apparent increase of protein in all the samples analysed in this study. This is attributed to the decrease in moisture which concentrated the nutrient composition of the samples ( (Bender, 1992; Gerber, 2007; Gerber , Scheeder, & Wenk, 2009). The percentage increase in the value of protein was highest in intestines (about 169%), which also lost the highest amount of moisture. The protein composition of the cooked samples ranged from 25.44 g/100 g in intestine to 39.78 g/100 g in cow foot. Treatment of meat with a high temperature has been reported to have a damaging effect on proteins from the nutritional perspective, especially, when part of an essential amino acid is rendered unavailable (Mayfield & Hedrick, 1949). This has been observed in lysine at temperatures around 100°C; cystine and methionine at temperatures around 120°C, and other amino acids after prolonged heating (Bender, 1978). Among all the meat samples used for this study, cow hide was subjected to the highest level of heat processing, hence in addition to being deficient in essential amino acids like lysine because it is mainly made up of collagen, further high temperature treatment will further reduce the protein quality of cow hide (Bender, 1978; Pellett & Young, 1984; Bender, 1992; Essumang, Dodoo, & Adokoh, 2007). The total damage to protein caused by cooking is of little practical significance if as is often the case, meat is consumed with a diet which contains protein from other food University of Ghana http://ugspace.ug.edu.gh 59 ingredients, since, the shortfall in meat quality can be compensated with other protein sources in the diet (Bender, 1992). In this study, uncooked intestine and tripe were found to contain 6.31 g/100 g and 0.72 g/100 g of carbohydrates respectively. None of the other samples contained carbohydrates. Furthermore, after cooking there was 100% loss of the carbohydrates detected in the intestines and tripe. Several studies have shown that meat is deficient in carbohydrates (Eyeson, Ankrah, Sundararajan, Karinpaa, & Rudzka, 1975; Gerber, 2007; Food and Agriculture Organization of The United Nations, 2012). However, it was reported that freshly slaughtered meat might contain carbohydrate in the muscle and the liver where the glycogen is mostly stored. Hence, immediately after rigor mortis there is almost 2.5% carbohydrate present in the form of lactic acid, glucose and derivatives (Bender, 1992). However, when meat is cooked, the carbohydrate present if any is lost in the process as shown in this study. The free fatty acid (FFA) as oleic in the uncooked samples ranged from 0.49 g/100 g in cow foot to 13.71 g/100 g in cow hide. The value increased in all the samples after cooking, thus confirming a previous report which stated that cooking animal products increases their FFA as oleic (Food Safety and Standards Authority of India, 2012). However, it was statistically significant in only cow hide and cow foot. The fact that cow hide had the highest FFA as oleic may be attributed to the extensive heat treatment it undergoes during processing. Studies have shown that FFA are normally formed during decomposition of oil/fat glycerides, therefore, the level of FFA as oleic in foods is a relative measure of rancidity (Food Safety and Standards Authority of India, 2012). University of Ghana http://ugspace.ug.edu.gh 60 It is crucial to state that FFA as oleic is derived by determining the acid value of food, hence the analytical importance of acid value is that it is a measure of the amount of fatty acids which have been liberated by hydrolysis from the glycerides due to the action of moisture, temperature, and/or lipolytic enzyme, lipase (Food Safety and Standards Authority of India, 2012). Therefore, the fact that cow hide had the highest level of FFA as oleic in this study is not unexpected since it has been subjected to a more extensive heat treatment than the other samples (Janicki & Appledorf, 1974). 5.2 Limitations of Study There was no place where a comprehensive fatty acid analysis and amino acid profile could be carried out in the study region, hence these parameters were not included in the study. Also, the sample size used in the study was small and sampling was limited to just one sub-metro in Accra, therefore, these results may not be fully applicable to the whole country although it can serve as baseline data for a more comprehensive study that would collect data in other regions in Ghana. 5.3 Conclusion The study has provided data on the proximate and free fatty acid composition for raw and cooked cow hide, cow foot, cow intestines and cow tripe. All the analysed products contained protein though the quality of protein in cow hide and cow feet was not ascertained in this study. Cooking had a significant effect on the nutrients composition of all the samples. In many cases, the nutrient content of cooked samples was higher. These apparent increases were due to reduction in moisture content of the meat products on cooking. In practise, this implies that less amount of cooked meat than raw is required to obtain a given amount of nutrients. 5.4 Recommendations Further studies should be done to ascertain the amino acid profile, fatty acid profile and cholesterol levels of these products, and to determine the health risk associated with eating University of Ghana http://ugspace.ug.edu.gh 61 these products in terms of fatty acid profile and cholesterol levels. It will also be very important to replicate the study in other regions in Ghana in order to come up with a comprehensive data that can be added to the food composition table for use in Ghana. Result from the study can be used as a guide in counselling clients and educating the general public to make wise choices when they are cooking. Dietitians should advice consumers to discard the broth of products such as the intestines and the tripe which has high level of fat since fats found in animal products are mostly saturated fats and cholesterol. Furthermore, consumers should be encouraged to consume these products in moderation and diversify their diet to include other sources of proteins like meat muscle, fishes, plant proteins to obtain an adequate diet. Consumers need to be discouraged from habitual consumption of these animal products as a consistent substitute to meat muscle and other rich sources of protein. 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University of Ghana http://ugspace.ug.edu.gh 72 Appendix 1: Pictures of the samples at the various stages of the study Fig. 1: Samples displayed in Makola market in Accra, Ghana KEY: Sample A = Cow hide (wele) Sample B = Cow foot Sample C = Cow intestine Sample D = Cow tripe A B C D University of Ghana http://ugspace.ug.edu.gh 73 Fig 2: Parts of uncooked samples being weighed at Chemistry laboratory of CSIR Food Research Institute, Accra, Ghana KEY: Sample A1 = Uncooked Cow hide (wele) Sample B1 = Uncooked Cow foot Sample C1 = Uncooked Cow intestine Sample D1 = Uncooked Cow tripe A1 B1 C1 D1 University of Ghana http://ugspace.ug.edu.gh 74 Fig 3: Cooked samples at chemistry laboratory of CSIR Food Research Institute, Accra, Ghana KEY: Sample A2 = Cooked Cow hide (wele) Sample B2 = Cooked Cow foot Sample C2 = Cooked Cow intestine Sample D2 = Cooked Cow tripe A2 B2 C2 D2 University of Ghana http://ugspace.ug.edu.gh 75 Fig 4: Cooked broth (Juices) from samples at chemistry laboratory of CSIR Food Research Institute, Accra, Ghana KEY: Sample A2 = Cooked broth (Juices) from Cow hide (wele) Sample B2 = Cooked broth (Juices) from Cow foot Sample C2 = Cooked broth (Juices) from Cow intestine Sample D2 = Cooked broth (Juices) from Cow tripe A2 B2 C2 D2 University of Ghana http://ugspace.ug.edu.gh 76 Fig 4: Some of the euipment used for the study at chemistry laboratory of CSIR Food Research Institute, Accra, Ghana KEY: A = Industrial food blender (Panasonic, MX-AC300, Mixer Grinder) used for homogenization of sample B = Industrial food blender (Panasonic, MX-AC300, Mixer Grinder) used for homogenization of sample C = Gerhadt electric burner (Gerhadt Bonn, App No, SN: 01191159) D = Researcher cooking with aluminium sauce pan on Gerhadt electric burner A2 B2 C2 D2 University of Ghana http://ugspace.ug.edu.gh 77 Appendix 2: Official raw data result from CSIR Food Research Institute, Accra, Ghana University of Ghana http://ugspace.ug.edu.gh 78 University of Ghana http://ugspace.ug.edu.gh 79 Appendix 3 Ethical and Protocol Committee Approval Letter University of Ghana http://ugspace.ug.edu.gh 80 University of Ghana http://ugspace.ug.edu.gh