CHAPTER ONE 1.0 INTRODUCTION 1.1 BACKGROUND OF STUDY Diarrhoea is a gastrointestinal tract (GIT) disorder which accounts for global deaths of nearly 1.5 million children under five years of age. It is the second most common cause of child death worldwide after pneumonia. Over half of the deaths are estimated to occur in five countries: India (386,600); Nigeria (194,000); Democratic Republic of Congo (89,900); Afghanistan (82,100); and Ethiopia (73,700) (Unicef, 2015). The most common cause of diarrhoea is an infection of the intestines due to a bacterium, virus, parasite or fungus, a condition known as gastroenteritis (Ciccarelli et al., 2013). These infections are often acquired from food or water that had been contaminated directly from another person who was infected with enteropathogens (WHO, 2013). Gastroenteritis is usually an acute and life threatening disease, resulting from loss of fluid and electrolytes particularly sodium ion (Na +) and water from the body, which requires specific anti-infective therapy (Nwachukwu et al., 2013). Enteric bacteria comprised of Salmonella species, Shigella species, Proteus species, Klebsiella species, E. coli , Pseudomonas species, Vibrio cholerae and S. aureus which are major aetiologic agents of enteric infections (Ballal and Shivananda, 2002). Enteric infections are caused by two major bacterial mechanisms including mucosal invasion and secretion of toxins (Omojate et al., 2014), are associated with Symptoms such as anorexia, nausea, vomiting, diarrhea, and abdominal discomfort and sometimes lactose intolerance (Lin et al., 1998). Treatment and prevention of infectious gastroenteritis are not limited to antibiotics therapy since indiscriminate use fosters the emergence of drug-resistant organisms. Consequently, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition and the European Society of Paediatric Infectious Diseases guidelines made a strong recommendation for the use of probiotics for the management of acute gastroenteritis (Ciccarelli et al., 2013). Probiotics are microbial food supplements that prevent pathogens from proliferation in the intestinal tract on the surface structures, aid maximum feed utilization, improve water quality and enhance the immune system of the host (Nwogu et al., 2011). Probiotics are adjuncts in health promoting food for humans and also used as therapeutic, prophylactic and growth supplements in animal production and human health (Isolauri, 2001; Amund, 2016). More importantly, the emergence of antibiotic resistance in enteric pathogenic bacteria has facilitated the prophylactic and therapeutic uses of probiotics. Consequently, probiotics should be considered as alternatives to antibiotics. 1.2 STATEMENT OF THE PROBLEM Enteropathogenic bacteria, which have been implicated in gastrointestinal diseases, are of global public health concern because of the significant morbidity and mortality especially in low and middle income countries, like Nigeria, where they place tremendous burdens on fragile healthcare systems. The major concern is the emergence of antibiotic resistant strains of enteric pathogens due to the widespread use of antibiotics. Consequently, alternative preventive and treatment measures for gastro intestinal disorders are required. Probiotics have been proven to be more effective in the management of acute gastroenteritis with no reported side effects. Probiotics are strain specific and not readily available in low income countries like Nigeria. Dependence on foreign probiotics is a challenge hence the need for isolation of indigenous strains for use. 1.3 AIM AND OBJECTIVES The overall aim of the study is to isolate potential probiotic lactic acid bacteria from fermented sorghum ( Sorghum bicolor ) gruel and beverage. 2 1.4 SPECIFIC OBJECTIVES OF STUDY The specific objectives of this research are to: Isolate and identify putative beneficial bacteria from fermented sorghum product; Examine the isolates for anti-enteropathogenic probiotic properties; Characterize and genetically identify selected isolate(s); Investigate and characterize the basis of antibacterial activity of identified isolate(s). 1.5 SIGNIFICANCE OF STUDY Lactic acid fermentation is very important in food processing in the low and medium income countries, thus has the potential for use as a measure of disease control. In order to meet the increasing demand for viable food, lactic acid fermentation is expected to play an important role in preserving fresh vegetables, cereals, fruits and other food items for feeding humanity in developing countries of the world. Lactic acid bacteria (LAB) which have been reported as probiotic strains play a major role in fermentation, enhancing nutritive values of fermented products in addition to acidifying the products which prevents the growth of some spoilage and pathogenic organisms. However, probiotic properties are strain-specific . This study will provide indigenous LAB for local use as probiotics, alternative means of treatment and prevention of gastrointestinal disorders such as diarrhoea in low income countries including Nigeria. The prospective probiotic LAB isolates should be non-resstant to antibiotics since enteric LAB have been reported to demonstrate capability to transfer antibiotic resistance gene to unrelated enteric bacteria, which is factor contributing to pathogenesis. In addition, this research will reduce dependence on imported probiotics and antibiotics in the management and prevention of diarrhoea in Nigeria thereby saving cost. The study may, however, be a source of revenue from the export of this indigenous probiotics. 3 1.6 OPERATIONAL DEFINITION OF TERMS Antibacterial: substance which inhibits growth of bacteria. Antibiotics: commercially available chemical substance derived from a mold or bacterium that can inhibit growth of microorganisms. Beverage : is a liquid food made from sorghum, boiled in water that may be more often drunk. Bran: broken coat of seeds of sorghum. Curing: to induce loss of plamid DNA. Disorder: a physical condition in which there is imbalance of normal functioning. Diarrhoea: is the condition of having at least three loose or liquid bowel movements each day which often lasts for a few days and can result in dehydration due to fluid loss. Enteric: relating to the inside of the intestines. Enzyme: a protein produced by a living organism that acts as catalyst in specific biochemical reactions. Fermentation : is a metabolic process that converts sugar to acids, gases or alcohol Filtrate: liquid substance that passes through a filter. Gene: a segment of DNA that is involved in producing polypeptide chain. Germ: a small simple structure from which a new tissue can develop into a whole organism. Gruel : is a thinner version of food porridge made from sorghum. Hull: a dry outer covering of sorghum seed. Kiln: a heating mantle for drying grains Mash: a mixture of bran and filtrate of sorghum. Peptide : is a biologically occurring short chain of amino acids linked by peptide (amide) bonds. Pomace : a crushed or ground, pulpy substance from wet-milled sorghum grains . 4 Probiotics : are microorganisms that provide health benefits when consumed by humans and animals. Proteinaceous: possessing the physiochemical properties of proteins. Surfactant: chemical substance capable of reducing surface tension of phospholipid bilayer component of microbial cell walls. 1.7 LIST OF ABBREVIATIONS AFLP – Amplified Fragment Length Polymorphism AIDS – Acquired Immune Deficiency Syndrome AOAC – Association of Official Analytical Chemists API – Analytical Profile Index ARDRA - Amplified Ribosomal DNA Restriction Analysis BLAST – Basic Local Alignment Search Tool CDV – Cardiovascular Diseases CoA – Coenzyme A DNA – Deoxyribonucleic Acid DP – Degree of Polymerisation EcN – Escherichia coli Nissle EDTA – Ethylene Diamine Tetraacetic Acid EMP – Emden-Meyerhof Panas ERIC – Enterobacterial Repetitive Intergenic Consensus FAO - Food and Agriculture Organization of the United Nations FOS – Fructo Oligosaccharides GFN - Glucose, Fructose, number GRAS - Generally Recognized As Safe 5 IBDs - Inflammatory Bowel Diseases IPTG - Isopropyl β-D-1-thiogalactopyranoside LAB – Lactic Acid Bacteria LDC – Lowe Density Lipoprotein MEGA – Molecular Evolutionary Genetics Analysis MIC - Minimum inhibitory concentration MRS – de Man Rogosa and Sharpe MUB – Mucus-Binding Proteins NAD – Nicotinamide Adenine Dinucleotide NDOs – Non-Digestible Oligosaccharides OB – Ogi baba ORT – Oral Rehydration Therapy PB – Pito Beverage PCR – Polymerase Chain Reaction PFGE – Pulse- Field Gel Electrophoresis RAPD – Randomly Amplified Polymorphism DNA rep-PCR – Repetitive Extragenic Palindromic PCR RFLP – Restriction Fragment Length Polymorphism RPM – Revolutions per minute rRNA – ribosomal Ribonucleic Acid SCFA – Short-Chain Fatty Acid TAG - Triacylglycerol UNICEF - United Nations Children’s Fund WHO – World Health Organization X-GAL - 5 Bromo, 4 Chloro,3- Indolyl-D-Galactopyranoside 6 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 PROBIOTICS The human gut flora consists of about 500 to 1,000 bacterial species (Sears, 2005; Sommer and Bäckhed, 2013), about 90% of which are obligate anaerobes (Kayser et al. , 2005). Probiotics have been suggested to protect against infectious diseases by several strain-dependent mechanisms (Foligne et al., 2010) including secretion of antipathogen substances, competitive exclusion of pathogens, maintenance of mucosal integrity, and stimulation of systemic or mucosal immune responses (Howarth and Wang,