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Bacillus Cereus Cereulide and Staphylococcus Aureus Enterotoxin Production in Lasagna

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Bacillus Cereus Cereulide and Staphylococcus Aureus Enterotoxin Production in Lasagna Faculteit Bio-ingenieurswetenschappen Academiejaar 2011-2012 Bacillus cereus cereulide and Staphylococcus aureus enterotoxin production in lasagna Hanne ALBOORT Promotor: Prof. dr. ir. Mieke Uyttendaele Co-Promotor en Tutor: Prof. dr. Andreja Rajkovic Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen:LEVENSMIDDELENWETENSCHAPPEN EN VOEDING Woord vooraf Nu deze thesis naar het einde loopt had ik graag enkele mensen bedankt die er mede voor gezorgd hebben dat deze thesis tot stand is kunnen komen. Hierbij had ik graag Prof. dr. ir. Mieke Uyttendaele bedankt voor het opnemen van het promoterschap. Een zeer grote dank gaat uit naar mijn co-promotor en begeleider, Prof. dr. Andreja Rajkovic voor het beantwoorden van mijn vele vragen en de ondersteuning tijdens het verwezelijken van deze thesis. Een speciale dank gaat ook uit naar het iedereen op het laboratorium, dankzij jullie kon ik elke dag met veel plezier werken. Ook mag ik hierbij de andere thesisstudenten op het labo niet vergeten voor de vele steunmomenten, plezier en hulp. Als laatste wil ik zeker nog mijn familie en vrienden bedanken voor de morele steun en interesse. Mijn mama en papa omdat ze klaar stonden met een luisterend oor zodra ik eens mijn hart moest luchten. Mijn broer en zus waarbij ik echt voor alles terecht kan. Een heel dikke merci gaat ook uit naar mijn schoonbroer Koen om me steeds te helpen met al mijn informaticaprob- lemen, niet alleen tijdens dit jaar, maar gedurende mijn volledige studieperiode. Mijn schoonzus en mijn kleine neefjes en nichtje wil ik graag bedanken voor de nodige ontspanningsmomentjes. Tot slot wil ik ook nog mijn vrienden danken voor de vele steunberichtjes net op de momenten dat ik het nodig had. Hanne Alboort, Gent, juni 2012 i Toelating tot bruikleen De auteur en de promotoren geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen ervan te kopi¨erenvoor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron te vermelden bij het aanhalen van resulaten uit deze scriptie. The author and the promotors give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws; more specifically the source must be specified when using results from this thesis. Gent, juni 2012 De auteur, De promotor, De co-promotor, Hanne Alboort Prof. dr. ir. Mieke Uyttendaele Prof. dr. Andreja Rajkovic ii Abstract In this thesis it was the goal to investigate the safety of ready-to-eat meals, exemplified by lasagna. These foods can be stored for a prolonged period of time at home in the refrigera- tor. As these types of foods are gaining in popularity, the safety concerning two relevant toxin producing bacteria was investigated. The bacteria investigated in this thesis were the emetic toxin (cereulide) producing B. cereus and the staphylococcal enterotoxin producing S. aureus. The toxins produced from these two bacteria cannot be removed anymore by a subsequent heat process once they are formed. Furthermore, B. cereus is a sporeforming bacteria and can thus survive the processes in the factory and remain as spore in these ready-to eat foods. On the other hand, S. aureus is often an issue of postheating contamination due to its presence on hair and skin of the workers in the processing environment. The possible growth and toxin production of these two bacteria was investigated during their storage. Two storage temperatures of 12°C and 22°C were chosen; 12°C indicating a severe temperature abuse at home and 22°C, the temperature if storage is at room temperature. These lasagnas were packaged in a modified atmosphere packaging, as ready-to-eat meals are conserved in a reduced oxygen environment. Additionally some lasagna was also stored at ambient atmo- sphere conditions to distinguish the influence of this reduced oxygen content on the growth and toxin production of both bacteria. Before the food based experiments started, the possible growth and toxin production of B. cereus and S. aureus was investigated in laboratory media at modified atmosphere. The laboratory media chosen were Tryptone Soya Agar and Tryptone Soya Broth, both general growth media. In these media, the growth and subsequent toxinproduction was very fast. Staphylococcal en- terotoxins were found after 20 hours and cereulide could be found after 43 hours. In the lasagna, staphylococcal enterotoxins could be found after 43 hours at 22°C. At 12°C, the staphylococcal enterotoxins were detected from day 10. At 12°C a significant decrease in growth could be seen if the lasagna was stored at modified atmosphere conditions, which resulted in a delay of onset of staphyloccal enterotoxins production. Cereulide could be found in lasagna at 22°C at day 3, if stored ambient. If the lasagna was stored in a modified atmosphere, the onset of toxin production was delayed with one day, showing the influence of the reduced oxygen on the growth and subsequent toxin production. Moreover, the reduced oxygen content had an influence on the critical density necessary to start the cereulide toxin production, resulting in a higher critical density necessary in the modified atmosphere stored lasagna. At 12°C, no cereulide could be detected in the lasagna. iii Contents Woord vooraf i Toelating tot bruikleen ii Abstract iii 1 Introduction 1 2 Literature review 2 2.1 Bacillus cereus ..................................... 2 2.1.1 General characteristics and background information . 2 2.1.2 B. cereus detection . 3 2.1.3 The emetic toxin cereulide . 3 2.1.4 Food sources . 7 2.1.5 Cereulide detection . 9 2.1.6 Prevalence . 10 2.2 Staphylococcus aureus ................................. 10 2.2.1 General characteristics and background information . 10 2.2.2 S. aureus detection . 12 2.2.3 S. aureus enterotoxins . 12 2.2.4 Food sources . 15 2.2.5 Staphylococcal enterotoxin detection . 16 3 Materials and methods 18 3.1 S. aureus and B. cereus strains used in the experimental setup . 18 3.2 Propagation of the strains . 18 3.3 B. cereus spore production . 18 3.4 Bacterial enumeration . 19 3.5 General experimental setup . 19 3.5.1 General setup with Tryptone Soya Broth . 19 3.5.2 General setup with Tryptone Soya Agar . 19 3.5.3 General setup with ready-to-eat lasagna . 20 3.5.4 Bacteria enumeration from samples . 21 3.6 Toxin tests . 22 3.6.1 Staphylococcal enterotoxin test . 22 3.6.2 B. cereus cereulide toxin test . 22 3.7 Result interpretation . 24 3.8 Overview of the different experimental setups . 24 iv CONTENTS v 4 Results and discussion 26 4.1 Maximum cell densities of tested strains in TSB-broth . 26 4.2 Spore forming by B. cereus .............................. 26 4.3 Staphylococcal enterotoxin production by S. aureus strain . 27 4.4 Cereulide production by B. cereus strains . 27 4.5 Cereulide extraction from lasagna . 27 4.6 Growth of S. aureus and B. cereus strains in modified atmosphere in TSB . 27 4.7 Experimental setup with TSA . 29 4.7.1 Growth of S. aureus and B. cereus on TSA . 29 4.7.2 Toxin production during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores on TSA . 30 4.8 Experimental setup with Lasagna . 32 4.8.1 pH and aw-value lasagna . 32 4.8.2 Measured gas concentration in fresh lasagna . 32 4.8.3 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 434 spores in lasagna . 32 4.8.4 Experiment with B. cereus LFMFP 434 vegetative cells in lasagna . 36 4.8.5 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 436 spores in lasagna . 38 4.9 Discussion . 42 4.9.1 Growth and toxin production in TSB-broth . 42 4.9.2 Growth and toxin production on TSA . 42 4.9.3 Growth and toxin detection in lasagna at 22◦C . 43 4.9.4 Growth and toxin detection in lasagna at 12◦C . 45 4.9.5 Effect of the combination of low residual oxygen content and temperature on the toxin production . 47 5 Conclusions 48 6 List of abbreviations 50 Bibliography 51 Chapter 1 Introduction Bacillus cereus is an omnipresent spore forming bacterium and therefore it is almost impossi- ble to completely avoid it in the food processing area or on raw materials. These spores are furthermore very heat resistant, so pasteurization heat treatment will not be able to eliminate the spores. The spores can thus survive processing conditions and if there is subsequent time / temperature abuse where the spores can germinate, they can multiply to alarming numbers. B. cereus has moreover the ability to form toxins during the growth phase. One of the important toxins of B. cereus is the emetic toxin named cereulide. This toxin is very heat and acid resistant and once it is formed, it cannot be eliminated by a heat treatment. Another important bac- terium is Staphylococcus aureus. This bacterium can come in a product as a cross contamination because it is present in 20-30% of the population. This bacterium has also the possibility to produce different toxins. Food relevant are the Staphylococcal enterotoxins which are also very heat and acid resistant, but to a smaller extent then cereulide. With the properties of these two bacteria species in mind it is important to investigate the safety of ready-to-eat meals. These are meals prepared with a mild heat treatment and can be stored for a long time at home at refrigerated conditions. These meals are gaining in popularity because the consumer nowadays doesn't want to spend a lot of time in preparing meals.
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