i ANTIMICROBIAL ACTIVITY PROFILES OF TRADITIONAL FERMENTED MILK STARTER CULTURES FROM NORTH-EASTERN NAMIBIA A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (AGRICULTURE) OF THE UNIVERSITY OF NAMIBIA BY LUSIA HEITA (200627139) February 2014 Main Supervisor: Dr. Ahmad Cheikhyoussef Co- Supervisor: Dr. Martha Shikongo-Nambabi ii ABSTRACT The aim of this study was to identify and examine the antimicrobial properties of Lactic Acid Bacteria (LAB) isolated from fermented milk collected from Ohangwena, Omusati, Oshana, Oshikoto, Zambezi and Kavango regions. Traditional fermented milk in Namibia are produced by spontaneous fermentation using traditional utensils. In this study, thirty homesteads from six regions that produce and process fermented milk were selected and interviewed using semi- structured questionnaires. Omashikwa and Mashini ghakushika have similar processing method whereby fermentation is achieved by accumulation of milk; mean while Mabisi is produced by allowing the milk to ferment naturally. The pH decreased logarithmically, nonlinearly over the fermentation period from 6.5 ± 0002 from first day of fermentation to 3.92±0.001 over 4 days.There was no significant difference (p<0.05) in the pH values between the three types of fermented milk preparations. Cell free supernatants (CFS) of 180 LAB isolated from traditional fermented milk were evaluated for antimicrobial activities against selected food borne pathogens; Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Candida albicans ATCC 14053, Bacillus cereus ATTC 10876 Geotrichum klebahnii (IKST F. Lab. isolate) Escherichia coli ATCC 25922 using the well diffusion method. Twenty LAB isolates that shown the highest inhibitory effects were selected for biochemical identification using API 50 CHL were identified as; Lactobacillus plantarum (53%), Lactobacillus rhamnosus (29%), Pediococcus pentosaceus (6 %), Lactobacillus paracasei ssp. paracasei (6%) and Lactococcus lactic ssp. lactis (6%), of which Pediococus pentosaceus showed the most inhibitory effect on all the indicator strains and they have potential to produce bacteriocin Pediocin and the most inhibited indicator strain belonged to yeast Candida famata. In addition to antimicrobial activities, the major organic acids in all three types of milk were found to be lactic acid iii 0.265±0.056 mg/L. However, the levels of volatile organic compounds in the naturally fermented samples varied from one sample to another but, butyric, acetic and propionic acid were found in trace amounts. Some of the volatile flavor compounds found in Omashikwa, Mabisi and Mashini ghakushika were acetic acid, 2,3 Butanediol and Lactic acid. Further genetic confirmation of species was carried out using the 16S rDNA sequences. The research outcome addressed the establishment of the newly identified starter cultures and contributes positively to the advancement of national LAB’s profiling. The study is also the first attempt that indicates probiotic potential and exceptional preservation properties of LAB from traditional fermented milk produced in North- Eastern Namibia. iv Table of Contents ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS .......................................................................................................... vii DECLARATION ......................................................................................................................... viii DEDICATION ............................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................... xiii CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 African Traditional Fermented Milk ..................................................................................... 2 1.2. Orientation of the study ........................................................................................................ 3 1.3 Statement of Problem ............................................................................................................ 4 1.4 Objectives of the study .......................................................................................................... 4 1.5 Significance of the study ....................................................................................................... 5 1.6 Limitation of the study .......................................................................................................... 6 CHAPTER 2 LITERATURE REVIEW ......................................................................................... 7 2.1. The Origin of Fermented Milk ............................................................................................. 7 2.2 Traditional Fermented Milk .................................................................................................. 8 2.3 African traditional fermented milk ........................................................................................ 8 2.4 Fermentation Process ............................................................................................................ 9 2.5 Lactic Acid Bacteria (LAB) ................................................................................................ 12 2.5.4. Lactic Acid Bacteria in Milk Fermentation (Starter Cultures) .................................... 14 2.5.5. Types of starter cultures used in dairy industry ........................................................... 14 2.6 Microbial Compositions of Fermented Milks ..................................................................... 16 2.7. Antimicrobial Substances Produced by LAB..................................................................... 16 2.7.1. Organic Acids .............................................................................................................. 17 2.7.2. Hydrogen Peroxide ...................................................................................................... 19 2.7.3. Bacteriocins ................................................................................................................. 19 2.7.4. Aroma Compounds (Diacetyl) ..................................................................................... 21 CHAPTER 3 MATERIALS AND METHODS ........................................................................... 22 3.1. The study areas ................................................................................................................... 22 3.2. Data collection.................................................................................................................... 23 3.3. Samples Collection ......................................................................................................... 24 v 3.4. Physicochemical Analysis .................................................................................................. 24 3.4.1 pH ................................................................................................................................. 24 3.4.3. Titrable acidity of milk ................................................................................................ 24 3.5. Microbiological Analysis ................................................................................................... 25 3.5.1 Total Plate Count .............................................................................................................. 25 3.5.2 Isolation of LAB............................................................................................................... 26 3.5.3 Phenotypic Characterization of LAB ............................................................................... 26 3.6. Antimicrobial Assay of LAB ............................................................................................. 28 3.6.3 Hydrogen peroxide production ..................................................................................... 29 3.6.3. Analysis of Volatile Compounds (Diacetyl) ............................................................... 30 3.6.5. Analysis of Organic acids ............................................................................................ 31 3.7. Biochemical Identification of LAB .................................................................................... 32 3.8. Genetic Identification of LAB............................................................................................ 32 3.8.1 Genomic DNA Isolation ............................................................................................... 32 3.8.2 Amplification of 16S rDNA Region by Polymerase Chain Reaction (PCR ................ 34 3.8.3 Separation of amplified Fragments............................................................................... 35 3.8.4. Sequencing................................................................................................................... 35 3.9. Statistical analysis .............................................................................................................. 35 CHAPTER 4: RESULTS .............................................................................................................. 37 4.1 Milk Handling and Storage ................................................................................................