Molecular Characterization and Population Dynamics of Lactic Acid Bacteria During the Fermentation of Sorghum
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Molecular characterization and population dynamics of lactic acid bacteria during the fermentation of sorghum by EVELYN MADOROBA Submitted in partial fulfilment of the requirements for the degree of Philosophiae Doctor in the Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria March 2009 © University of Pretoria TABLE OF CONTENTS DECLARATION…………….………...………………………………….…….………......iii ACKNOWLEDGEMENTS…………………………………………….……..……………iv DEDICATION………………………………………………………………………………v SUMMARY………………………………………………………………………………….vi LIST OF FIGURES………………………………………………………………………...viii LIST OF TABLES…………………………………………………………………………..ix LIST OF ABBREVIATIONS………………………………………………………………x RESEARCH COMMUNICATIONS……………………………………………………....xii CHAPTER ONE…………………………………………………………………………….1 LITERATURE REVIEW CHAPTER TWO……………………………………………………………………………55 POLYPHASIC TAXONOMIC CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM SPONTANEOUS SORGHUM FERMENTATIONS USED TO PRODUCE TING, A TRADITIONAL SOUTH AFRICAN FOOD CHAPTER THREE…………………………………………………………………………70 DIVERSITY AND DYNAMICS OF BACTERIAL POPULATIONS DURING SPONATNEOUS FERMENTATIONS USED TO PRODUCE TING, A SOUTH AFRICAN FOOD CHAPTER FOUR………………………………………………………………………….103 USE OF STARTER CULTURES OF LACTIC ACID BACTERIA IN THE PRODUCTION OF TING, A SOUTH AFRICAN FERMENTED FOOD CHAPTER FIVE………………………………………………………………………..….139 GENERAL DISCUSSION AND CONCLUSIONS APPENDICES……………………………………………………………………………...143 ii DECLARATION I declare that the thesis, which I hereby submit for the degree, Philosophiae Doctor (Microbiology) at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at another University. Signed: …………………………………… Date: ………………………………... iii ACKNOWLEDGEMENTS I am greatly indebted to Prof. T. E. Cloete for awarding me an opportunity to work in his laboratory just when my academic career was at the crossroads. Prof. Cloete supplied crucial advice and inspiration throughout my studies. I am also grateful to Prof. J. Theron and Dr. E. T. Steenkamp, who have been my co- promoters, for their expert advice and academic passion in my PhD studies. Both co- promoters supplied a plethora of significant information that improved this thesis. Dr. Geert Huys, Dr. Ilse Scheircklinck and Ann Van Schorr are thanked for assistance with polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), which gave a refreshing dimension to this thesis. I sincerely wish to express special thanks to Prof. Gert Marais for assistance with identification of fungi. I also wish to express my heartiest appreciation to Dr. H. L. De Kock for expert advice regarding sensory evaluation of ting. This project was made possible by financial support from the National Research Foundation, South Africa. I am also indebted to the Third World Organisation for Women in Science for awarding me a PhD fellowship. My sincere gratitude is extended to my family and friends for their unwavering support and encouragement during times of distress. I shall never forget the patience and prayers of Chawapiwanashe Chipo and Masimba Michael Madoroba. My husband is thanked for his support during my absence from home. Above all, I would like to thank the invisible force behind the success of all my experiments, and this is none other than the Almighty God. iv DEDICATION This thesis is dedicated to my parents, the late Mr. Henry Mahembe and Mrs. Grace Mahembe for emphasizing the significance of a sound education. v SUMMARY Molecular characterization and population dynamics of lactic acid bacteria during the fermentation of sorghum by Evelyn Madoroba Promoter: Prof. T.E. Cloete Co-promoters: Prof. J. Theron and Dr E.T. Steenkamp Department of Microbiology and Plant Pathology University of Pretoria for the degree Ph.D Ting is a cooked fermented sorghum food that is popular amongst southern Africans for its sour taste and unique flavour. However, major challenges are associated with large-scale production and marketing of this spontaneously fermented food due to inconsistent microbiological and sensory quality. The use of starter cultures may circumvent these limitations. Prior to engaging starter cultures, detailed knowledge of the microbial diversity and dynamics during fermentation is important. Therefore, the aim of this study was to investigate microbial diversity and dynamics during sorghum fermentations, and to clarify the role of starter cultures regarding the microbiological safety and consumer acceptance of sensory characteristics of fermented ting. A culture-independent approach, based on the use of PCR-denaturing gradient gel electrophoresis (DGGE), revealed that Lactococcus lactis, Lactobacillus curvatus, Weissella cibaria and some Enterobacteriaceae were predominant at the end of spontaneous sorghum fermentations. Culture-dependent methods indicated that Lb. fermentum, Lb. plantarum, Lb. rhamnosus, E. faecalis, E. mundtii, W. cibaria and L. lactis were predominant at the end of fermentation. These results not only indicated the predominant bacteria during sorghum fermentation, but also indicated that a combined approach is required to reveal microbial diversity and dynamics during spontaneous sorghum fermentations. vi Based on the above results, L. lactis, Lb. fermentum, Lb. plantarum and Lb. rhamnosus were evaluated as starter cultures for production of ting. All the starter cultures were able to ferment sorghum, but the lowest pH and highest lactic acid was produced in naturally fermented sorghum inoculated with L. lactis. This fermentation showed an increase in the number of lactic acid bacteria and yeasts, whilst pathogen counts decreased. Ting from this fermented gruel, in contrast to naturally fermented sorghum, had sensory properties preferred by panelists. The results indicated that the use of L. lactis in starter cultures may result in ting with consistent and acceptable attributes. vii LIST OF FIGURES Fig. 1.1 Generalized scheme for the formation of important metabolic products from pyruvate by lactic acid bacteria…………………………………………………………………….…...8 Fig. 1.2 Neighbor-joining phylogenetic tree, based on 16S rRNA gene sequence analysis, showing the relationships among type species of the representative genera from the families of Lactobacillales……………………………………………………………………………17 Fig. 1.3 Main steps involved in DNA-based methods for characterization of LAB…………………23 Fig. 1.4 Main steps involved in DNA-based methods for studying microbial population dynamics of LAB……………………………………………………………………………………….28 Fig. 2.1 Maximum likelihood (ML) phylogeny for the Lb. plantarum, Lb. casei and Lb. reuteri Groups based on 16S rRNA and pheS gene sequences……………………………………...64 Fig. 3.1 Change in pH, as well as PCR-DGGE banding patterns representing the 16S rRNA gene fragments of the bacteria involved in the spontaneous fermentation of sorghum at 25°C……………………………………………………………………………………….....82 Fig. 3.2 Maximum likelihood (ML) phylogeny of species in the genera Lactobacillus and Weissella, based on 16S rRNA and pheS gene sequences.……………………………….…84 Fig. 3.3 Maximum likelihood (ML) phylogeny of species in the genera Lactococcus and Enterococcus based on 16S rRNA gene sequences………………………………………....86 Fig. 4.1 Changes in pH and titratable acidity in sorghum powder slurries during natural fermentation for 54 h and inoculated fermentations with different LAB starter cultures for 24 h.....................................................................................................................123 viii LIST OF TABLES Table 1.1 Examples of sorghum-based fermented foods, natural microflora and the methods used for characterization…………………………………………………………………...12 Table 1.2 Families and genera in the order Lactobacillales…………………………..……………...20 Table 2.1 Differential characteristics of nine LAB isolates from fermented sorghum, based on API 50 CHL analysis………………………………………………………………………62 Table 3.1 Microbial counts (cfu/g) at the start and end of spontaneous sorghum fermentations obtained on different selective media…………………………………………………..….80 Table 3.2 Likely species identities of bacteria isolated at the beginning and end of sorghum fermentations……………………………………………………………………………....81 Table 3.3 Comparison of the bacterial diversity in ting using culture-independent and culture- dependent approaches……………………………………………………………………...88 Table 4.1 Experimental protocol for the six sorghum fermentations……………………………….109 Table 4.2 Culture conditions used for microbiological analysis of sorghum samples……………...111 Table 4.3 Microbial counts (log cfu/g) of sorghum powder, fermented sorghum slurries and corresponding cooked fermented porridge (ting)…………………………………….118 Table 4.4 Occurrence of bacterial food-borne pathogens in sorghum powder, fermented sorghum slurries and cooked ting…...……………………………………………………120 Table 4.5 Antimicrobial interactions among lactic acid bacteria used as starter cultures for sorghum fermentation………..…………………………………………………………...121 Table 4.6 Acceptability of the sensory characteristics of ting produced using different starter cultures based on average hedonic ratings………………………………………………..125 Table 4.7 Pearson’s product-moment correlation coefficients (r) for pH, titratable acidity and acceptability of sensory attributes associated with ting produced using different starter cultures…………………………………………………………………………….126 ix LIST OF ABBREVIATIONS % percentage °C degrees Celsius bp base pair cfu colony forming units ClustalX cluster analysis version X cm centimetre DMSO dimethyl sulphoxide DNA deoxyribonucleic acid dNTP deoxyribonucleotide