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BACTERIAL CONTAMINATION of COMMERCIAL YEAST Susannah BACTERIAL CONTAMINATION OF COMMERCIAL YEAST Susannah Sara O’Brien A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Masters of Science. Gauteng, 2004 ________________________________________________________________________ DECLARATION I declare that this is my own, unaided work. It is being submitted for the degree of Master of Science in the University of the Witwatersrand, Gauteng. It has not been submitted before for any degree or examination in any other University. _____________________ Susannah Sara O’Brien _____ day of ___________, 2005. TABLE OF CONTENTS PREFACE .................................................................................. i ABSTRACT .................................................................................. iii LIST OF FIGURES .................................................................................. iv LIST OF TABLES .................................................................................. xi ACKNOWLEDGEMENTS .................................................................................. xiv CHAPTER 1 INTRODUCTION ................................................. 1 CHAPTER 2 ANALYSIS OF A COMMERCIAL YEAST PRODUCTION PROCESS .................................. 18 CHAPTER 2.1 Processing sources of bacterial contamination in cream, compressed and dry yeast production .... 19 CHAPTER 2. 2 The bacterial ecology of commercially manufactured yeast during processing ................ 43 CHAPTER 2.3 The presence of Listeria monocytogenes and other foodborne pathogens in commercially manufactured yeast ............................................... 59 CHAPTER 3 MICROBIOLOGICAL SHELF-LIFE STUDIES ON COMMERCIALLY MANUFACTURED YEAST................................. 76 CHAPTER 4 MICROBIOLOGICAL SURVEY AND BIOFILMS ASSOCIATED WITH THE POST- FILTRATION ENVIRONMENT IN BAKER’S COMPRESSED YEAST PRODUCTION............ 104 CHAPTER 5 SUMMARIZING DISCUSSION AND CONCLUSIONS .................................................... 137 REFERENCES .................................................................................. 148 PREFACE Some aspects of the work conducted for this dissertation have been or will be presented as papers or posters elsewhere: CHAPTER 2 O’Brien, S. S., Tessendorf, B. A., Brodie, M., Lindsay, D., von Holy, A. (2003). Bacterial contamination of commercial yeast. International Association for Food Protection (IAFP), 90th Annual Meeting, New Orleans, Louisiana. O’Brien, S. S., Tessendorf, B. A., Brodie, M., Lindsay, D., von Holy, A. (2003). Survey of a commercial yeast manufacturing process for bacterial contamination. South African Association for food Science and Technology (SAAFoST), 17th Annual Meeting, CSIR Conference Centre, Pretoria. O’Brien, S. S., Lindsay, D., von Holy, A. (2004). The presence of Enterococcus, coliforms and E. coli in a commercial yeast manufacturing process. International Journal of Food Microbiology 94, 23 – 31. O’Brien, S. S., Lindsay, D., von Holy, A. (2004). Survey of a commercial yeast manufacturing process for sources of spoilage and potentially pathogenic bacteria. International Association for Food Protection (IAFP), 91st Annual Meeting, Phoenix, Arizona, U.S.A. CHAPTER 3 O’Brien, S. S., Lindsay, D., von Holy, A. (2004). Bacterial populations associated with extended storage of commercially manufactured yeast. International Association for Food Protection (IAFP), 91st Annual Meeting, Phoenix, Arizona, U.S.A. CHAPTER 4 O’Brien, S. S., Lindsay, D., von Holy, A. (2004). Biofilm formation on yeast processing equipment surfaces as studied by scanning electron microscopy. Proceedings of the Electron Microscopy Society of Southern Africa 34, 39. O’Brien, S. S., Lindsay, D., von Holy, A. (submitted). In situ biofilms associated with Baker’s yeast processing equipment. International Association for Food Protection (IAFP), 92nd Annual Meeting, Baltimore, Maryland, U.S.A. ABSTRACT The bacterial contamination profile of a typical commercial yeast factory was assessed by three replicate microbiological surveys. In order to detect low-level contamination in samples, this study made use of a preliminary incubation technique (24h at 37°C), which boosted bacterial counts for the identification of sources of contamination. Numbers of bacteria were quantified by standard pour- and spread-plate techniques and various selective media. Raw materials were negligible in contributing to the bacterial contamination of commercial yeast, with the exception of soda ash, used to control the pH of fermentations, which contained 2 log CFU/ ml Enterococcus and aerobic bacteria. It was found that the scale up of seed yeast biomass was the primary site for contamination with Enterococcus, which progressively increased in number as the product passed down the production line. Coliforms were present at low levels, with significant increases (P < 0.05) observed during the storage of yeast cream; extrusion of compressed yeast; and packaging of dry yeast. The environment surrounding the compressed yeast production line was identified as a potential source of airborne contamination. Although Salmonella spp. and S. aureus were not detected, L. monocytogenes was isolated from compressed and dry yeast products. In addition, Bacillus spp. commonly associated with the rope-spoilage of bread, were isolated from 67% of all dry yeast product samples. Shelf-life investigations, showed that cream and compressed yeast samples were spoiled with lengthened storage periods, and especially at higher temperatures (>10°C), whilst vacuum-packaged dry yeast remained bacteriologically stable. During shelf-life studies, isolates from spoiled cream and compressed yeast samples were predominantly Lactobacillus (up to 78%), while populations of Enterococcaceae predominated in vacuum-packaged dry yeast samples (up to 68%). The use of stainless steel surfaces, attached to processing equipment used in the manufacturing of Baker’s compressed yeast, in conjunction with SEM illustrated the accumulation of yeast and bacterial cells with early stages of biofilm formation, with time. Where populations of Gram-positive members of the lactic acid bacteria family, Lactobacillus and Enterococcaceae, were isolated in the highest proportion from processing equipment surfaces used in the manufacturing of Baker’s compressed yeast (81-100%). LIST OF FIGURES FIGURE 1.1 Schematic diagram of the commercial yeast manufacturing process …………………………………………………………. 16 FIGURE 2.1.1 A commercial yeast manufacturing process with sampling areas for raw material analysis and seed, cream, compressed and dry yeast process analysis ………………………………………….. 34 FIGURE 2.1.2 Pre-treated raw materials [molasses (R1) and synthetic sugar (R2)]; raw materials [yeast inoculum (R3), sterile molasses (R4), defoamer (R5), soda ash (R6), vitamin mix (R7), mineral mix (R8), process water (R9), emulsifier – dry yeast production (R10) and emulsifier – compressed yeast production (R11)]; as well as process air (R12) were sampled during three replicate surveys of a commercial yeast factory … 36 FIGURE 2.1.3 WL Nutrient agar plus 1% cyclohexamide was used to determine total aerobic plate counts (A), Rapid’ E. coli 2 agar for coliform (green colonies) and E. coli (purple colonies) counts (B), and KF Streptococcus agar reinforced with 1% bacteriological agar plus 1% (v/v) 2-3-5-triphenyl-2H- tetrazolium chloride (TTC) for Enterococcus counts (C) associated with raw materials and yeast samples ……………… 37 FIGURE 2.1.4 Mean E. coli, coliform, Enterococcus and aerobic plate counts (Log CFU/ ml), before and after preliminary incubation at 37°C for 24 h, over three replicate surveys for seed yeast production (Lower detection limit = 0.7 log CFU/ ml). Mean pH over three replicate surveys is shown for seed yeast production. Mean bacterial counts followed by the same superscripts are not significantly different (P > 0.05) ………………………………. 39 FIGURE 2.1.5 Mean E. coli, coliform, Enterococcus and aerobic plate counts (Log CFU/ ml or g), before and after preliminary incubation at 37°C for 24 h, over three replicate surveys for cream and compressed yeast production (Lower detection limit = 0.7 log CFU/ ml or g). Mean pH over three replicate surveys is shown for cream and compressed yeast production. Mean bacterial counts followed by the same superscripts are not significantly different (P > 0.05) …………………………………………….. 40 FIGURE 2.1.6 Mean E. coli, coliform, Enterococcus and aerobic plate counts (Log CFU/ ml or g), before and after preliminary incubation at 37°C for 24 h, over three replicate surveys for dry yeast production (Lower detection limit = 0.7 log CFU/ ml or g). Mean pH over three replicate surveys is shown for dry yeast production. Mean bacterial counts followed by the same superscripts are not significantly different (P > 0.05) …………. 41 FIGURE 2.2.1 Modified characterization key after Fischer et al., (1986) for colonies isolated from aerobic plate count plates of WL Nutrient agar plus 1% cyclohexamide, for seed, cream, compressed and dry yeast samples …………………………….. 54 FIGURE 2.2.2 Light micrographs (x 1000 oil) of typical Gram-positive (purple) cocci (A), Gram-positive rods (B) and Gram-negative (red) rods (C) isolated from yeast samples …………………….. 55 FIGURE 2.3.1 Rapid L’ Mono agar, a selective chromogenic medium, was used for the detection of L. monocytogenes from cream, compressed and dry yeast products. This picture shows a Rapid L’ Mono agar plate with typical blue colonies
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