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Microbial Ecology and Food Safety of Fermented Carrot Juice

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Microbial Ecology and Food Safety of Fermented Carrot Juice MICROBIAL ECOLOGY AND FOOD SAFETY OF FERMENTED CARROT JUICE Cédric Verschueren Student number: 01611321 Promotor(s): Prof. dr. ir. Mieke Uyttendaele (Ghent University), Prof. dr. ir. Sarah Lebeer (University of Antwerp) Tutor: MSc. Wannes Van Beeck (University of Antwerp) Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioscience Engineering: Food Science and Nutrition Academic year: 2018 - 2019 MICROBIAL ECOLOGY AND FOOD SAFETY OF FERMENTED CARROT JUICE Cédric Verschueren Student number: 01611321 Promotor(s): Prof. dr. ir. Mieke Uyttendaele (Ghent University), Prof. dr. ir. Sarah Lebeer (University of Antwerp) Tutor: MSc. Wannes Van Beeck (University of Antwerp) Master’s Dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioscience Engineering: Food Science and Nutrition Academic year: 2018 - 2019 Permission of use De auteur en de promotor geven de toelating deze masterproef voor consultatie beschikbaar te stellen en delen van de masterproef te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron uitdrukkelijk te vermelden bij het aanhalen van resultaten uit de masterproef. The author and the promotor give 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 extensively specified when using results from this thesis. Ghent, September 2019 Promotor(s): Tutor: Author: Prof. dr. ir. Mieke Uyttendaele, MSc. Wannes Van Beeck Cédric Verschueren Prof. dr. ir. Sarah Lebeer ii Preface “non scholae sed vitae discimus” (Inversion of Annaeus Seneca, Lucius. Epistulae morales ad Lucilium, CVI) With the submission of this Master’s Dissertation my academic journey comes closely to an end. All the hard work is ultimately rewarded. Despite the hard work, this Master programme was very interesting and instructive in all its aspects. I am convinced that the acquired knowledge and experience only can bring added value to the next phases of my life. This is the perfect moment to thank the people who have supported me in recent years and also the people who have helped me bringing this Master’s Dissertation to a good end. First of all I would like to thank my promotors and tutor, Prof. dr. ir. Mieke Uyttendaele, Prof. dr. ir. Sarah Lebeer and MSc. Wannes Van Beeck. They were always there providing useful feedback and answers to my questions and managed this dissertation in the right directions. I would also like to thank the lab of environmental ecology and applied microbiology at the University of Antwerp and the lab of food microbiology and food preservation at Ghent University for the daily pleasant atmosphere. In particular dr. Inge Van der Linden and Ann Dirckx for their help in the lab and provision of necessary material for my research. Finally, I would also like to thank my parents, family and friends for their support during the past years, they made it possible to bring this chapter to a successful end. Ghent, September 2019 Cédric Verschueren iv List of contents Permission of use ___________________________________________________________ ii Preface __________________________________________________________________ iv List of Abbreviations _________________________________________________________ I Abstract (EN) _______________________________________________________________ 1 Abstract (NL) _______________________________________________________________ 2 Introduction _______________________________________________________________ 3 Literature__________________________________________________________________ 5 The art of fermentation __________________________________________________________ 5 1. Fermentation ______________________________________________________________ 7 Lactobacillus genus complex________________________________________________________ 7 Fermentation in general ___________________________________________________________ 8 Fermentation types ______________________________________________________________ 10 Lactic acid fermentation __________________________________________________________ 10 Vegetable fermentation __________________________________________________________ 11 1.5.1. Sauerkraut ________________________________________________________________ 13 1.5.1.1. Production process _______________________________________________________ 13 1.5.1.2. Microbiology ____________________________________________________________ 13 1.5.2. Kimchi ____________________________________________________________________ 13 1.5.2.1. Production process _______________________________________________________ 13 1.5.2.2. Whole-cabbage kimchi ____________________________________________________ 14 1.5.2.3. Microbiology ____________________________________________________________ 14 Fermented vegetable juices _______________________________________________________ 14 Spontaneous vs. Starter cultures ___________________________________________________ 15 2. Food safety _______________________________________________________________ 16 Food safety in general ___________________________________________________________ 16 2.1.1. Global ____________________________________________________________________ 16 2.1.2. Europe ____________________________________________________________________ 17 2.1.3. Belgium ___________________________________________________________________ 17 Food safety of vegetables _________________________________________________________ 18 Food safety of fermented foods ____________________________________________________ 19 Pathogens _____________________________________________________________________ 20 2.4.1. Listeria monocytogenes ______________________________________________________ 21 2.4.2. Salmonella spp. ____________________________________________________________ 22 2.4.3. Pathogenic STEC (Shiga-toxin E. coli): O157 and non-O157 __________________________ 22 2.4.4. Yersinia enterocolitica and pseudotuberculosis ___________________________________ 22 2.4.5. Opportunistic bacteria _______________________________________________________ 23 3. Why fermented carrot juice & what about the food safety? ________________________ 24 Material & methods ________________________________________________________ 25 1. Bacterial strains & culture conditions __________________________________________ 25 Strain selection _________________________________________________________________ 25 Stock culture ___________________________________________________________________ 25 Working culture _________________________________________________________________ 26 Selective media for pathogens _____________________________________________________ 26 1.4.1. Agar Listeria according to Ottaviani and Agosti (ALOA) _____________________________ 26 1.4.2. Xylose-Lysine-Desoxycholate (XLD) Agar _________________________________________ 26 1.4.3. Cefixime Tellurite - MacConkey Sorbitol agar (CT-SMAC) ___________________________ 27 Media for microbial community ____________________________________________________ 27 1.5.1. MRS (Man, Rogosa & Sharpe) _________________________________________________ 27 1.5.2. VRBG (Violet Red Bile Glucose) ________________________________________________ 28 1.5.3. YPD (Yeast Extract-Peptone-Dextrose) __________________________________________ 28 Good Laboratory Practices (GLP) ___________________________________________________ 29 Plating out _____________________________________________________________________ 29 2. Protocol optimisation _______________________________________________________ 29 Pathogen recognition & acid effect on culture media ___________________________________ 29 Pathogen survival in fermented carrot juice __________________________________________ 30 Cucumber juice mix ______________________________________________________________ 30 Whole carrot community _________________________________________________________ 30 3. Fermentation and cucumber/cooling __________________________________________ 32 Fermentation set-up _____________________________________________________________ 33 aw ____________________________________________________________________________ 34 Pathogen inoculation ____________________________________________________________ 34 Cucumber juice and cooling _______________________________________________________ 34 Fermentation progress analysis ____________________________________________________ 34 3.5.1. Plating ____________________________________________________________________ 34 3.5.2. Freeze-stocks ______________________________________________________________ 35 3.5.3. pH _______________________________________________________________________ 35 RNA-based 16S amplicon (V4) sequencing ____________________________________________ 35 3.6.1. RNA extraction and analysis___________________________________________________ 35 3.6.2. Routine DNase treatment ____________________________________________________ 35 3.6.3. First-strand cDNA Synthesis ___________________________________________________ 36 3.6.4. Barcoded PCR ______________________________________________________________ 36 3.6.5. PCR clean-up _______________________________________________________________ 37 3.6.6. Qubit _____________________________________________________________________ 37 3.6.7. Size selection using
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