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CHAPTER 1: Overview of the Literature: Lactic Acid Bacteria (LAB) and 1 Spoilage of Packaged Foodstuffs Stored Under Refrigeration Temperature

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CHAPTER 1: Overview of the Literature: Lactic Acid Bacteria (LAB) and 1 Spoilage of Packaged Foodstuffs Stored Under Refrigeration Temperature Promotors: Prof. Dr. ir. Frank Devlieghere Laboratory of Food Microbiology and Food Preservation Department of Food Safety and Food Quality Faculty of Bioscience Engineering Ghent University, Belgium Prof. Dr. Geert Huys Laboratory of Food Microbiology Department of Biochemistry and Microbiology Faculty of Sciences Ghent University, Belgium Dean: Prof. Dr. ir. Guido Van Huylenbroeck Rector: Prof. Dr. Anne De Paepe Examination Committee Chairman : Prof. Dr. Els Vandamme Prof. Dr. ir. Frank Devlieghere (Universiteit Gent) Prof. Dr. Geert Huys (Universiteit Gent) Prof. Dr. ir. Nico Boon (Universiteit Gent) Prof. Dr. Lieven De Zutter (Universiteit Gent) Prof. Dr. George-John Nychas (Agricultural University of Athens) Prof. Dr. Danilo Ercolini (Università degli Studi di Napoli Federico II) MSc. Vasileios Pothakos PSYCHROTROPHIC LACTIC ACID BACTERIA (LAB) AS A SOURCE OF FAST SPOILAGE OCCURING ON PACKAGED AND COLD-STORED FOOD PRODUCTS Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Thesis title in Dutch: Psychrotrofe melkzuurbacteriën als bron van snel bederf van gekoelde, verpakte levensmiddelen. Illustration on cover: Genome of type strain Leuconostoc gelidum subsp. gasicomitatum LMG 18811T (Johansson et al., 2011) Printer: University Press To refer to the thesis: Pothakos, V., (2014). Psychrotrophic lactic acid bacteria (LAB) as source of fast spoilage occurring on packaged and cold-stored food products. PhD dissertation, Faculty of Bioscience Engineering, Ghent University, Ghent. ISBN: 978-90-5989-689-5 The author and the promotors give the authorization to consult and copy parts of this work for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. FOREWORD As my journey in the Laboratory of Food Microbiology and Food Preservation reaches its end and my doctoral study its fulfillment, I feel a sincere need to reminisce all those who contributed to my work and therefore acknowledge and appraise their help. First and foremost, I would like to thank my promotor, Prof. Dr. ir. Frank Devlieghere. Not only did you give me the opportunity to come to Belgium and work in a laboratory of the highest standards but you also encouraged me with your trust, your support and your patience to perform this research study providing me as well with the financial means and stimulation. I am deeply grateful to you. My co-promotor, Prof. Dr. Geert Huys who initiated me in bacterial taxonomy and unfolded a new world before my eyes. Your knowledge and constructive comments as well as your personal involvement and commitment to our work is highly appreciated. My (ex-)colleagues in the Laboratory of Food Microbiology and Food Preservation and the Laboratory of Microbiology for all the moments that we shared together the last years. I am grateful for the warm environment you created around me, for accepting me right away and being my friends and family in Belgium. My thesis jury, Prof. Dr. George-John Nychas, Prof. Dr. Danilo Ercolini, Prof. Dr. Lieven De Zutter, Prof. Dr. ir. Nico Boon, Prof. Dr. Geert Huys and Prof. Dr. ir. Frank Devlieghere for all the critical comments in the manuscript. My thesis students Clarice and Yosi. Your hard work and your input made it all easier. I hope I did manage to stand up to your expectations. You were the best students anyone could wish for. My dear friends from the Faculty of Bioscience Engineering. Nothing would have been the same without you. Additionally, I would like to thank the State Scholarships Foundation of Greece for the honorary distinction and the support they provided throughout this entire period for the completion of my doctoral thesis. My friends back home who I kept missing. You are my backbone. Lastly, I would like to acknowledge two people who triggered my scientific curiosity. My first inspiration during my early years in the Agricultural University of Athens, Prof. Dr. Effie Tsakalidou for her encouragement and help. Prof. Dr. ir. Luc De Vuyst who during my Master thesis in the Vrije Universiteit Brussel, motivated me in continuing my studies. This whole dissertation is a product of hard, teamwork. Thank you all for your scientific help and contribution, you are all very dear to me. My family. My father, my mother Sophia and my siblings Kostas and Eirini for their moral support that propelled me to accomplish this. Thank you for believing in me, for your constant presence, encouragement and love, regardless of how far we may be. Την οικογένεια μος. Τον παηέπα μος, ηη μηηέπα μος Σοθία, ηα αδέπθια μος Κώζηα και Ειπήνη για ηην ςποζηήπιξη, ηην ενθάππςνζη και ηην αγάπη ηοςρ. TABLE OF CONTENTS ABBREVIATIONS i AIM OF THE STUDY – OVERVIEW OF THE THESIS iii CHAPTER 1: Overview of the literature: Lactic acid bacteria (LAB) and 1 spoilage of packaged foodstuffs stored under refrigeration temperature CHAPTER 2: Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled- 25 stored food products at the end of their shelf-life CHAPTER 3: Psychrotrophic members of Leuconostoc gelidum subsp. gasicomitatum, Leuconostoc gelidum subsp. gelidum and Lactococcus piscium 36 dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium CHAPTER 4: Source tracking of psychrotrophic lactic acid bacteria (LAB) in 51 a ready-to-eat (RTE) vegetable salad production environment CHAPTER 5: Spoilage potential of psychrotrophic lactic acid bacteria (LAB) species: Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, 76 on sweet bell pepper (SBP) simulation medium under different gas compositions CHAPTER 6: Production batch recalls and sporadic cases of product defects in Belgium, due to unexpected, early spoilage caused by psychrotrophic lactic 93 acid bacteria (LAB) in packaged and chilled-stored foodstuffs between 2010 and 2014 CHAPTER 7: Evaluation of possible cross-contamination routes of psychrotrophic Leuconostoc gelidum subsp. gasicomitatum through adhesion 104 to food contact surfaces CHAPTER 8: General discussion and perspectives 115 SUMMARY SAMENVATTING CURRICULUM VITAE ABBREVIATIONS (m/r/t)RNA (messanger/ribosomal/transfer) ribonucleic acid (SPME)GC-MS Solid Phase Microextraction Gas Chromatography Mass Spectrometry AFLP Amplified Fragment Length Polymorphism aw Water activity BA Biogenic amines BCCM Belgian Co-Ordinated Collection of Microorganisms BOPP Biaxially-Oriented Polypropylene C. Carnobacterium CFU Colony Forming Unit CSP(s) Cold shock protein(s) DNA Deoxyribonucleic acid E. Enterococcus EDTA Ethylenediaminetetraacetic acid EPS extracellular polymeric substances EVOH Ethylene vinyl alcohol GRAS generally regarded as safe HTS High-throughput sequencing ISO International Organization for Standardization K Reaction rate coefficients LAB Lactic acid bacteria Lb. Lactobacillus Lc. Lactococcus Le. Leuconostoc MA(P) Modified atmosphere (packaging) MLST Multi locus sequence typing MRS de Man-Rogosa-Sharpe Nmax maximum cell population OTR Oxygen transmission rate PA Polyamide PCA Plate Count agar PE Polyethylene PP Polypropylene PPS Peptone physiological solution i RCA Reinforced Clostridial agar rep-PCR Repetitive element polymerase chain reaction RH Relative humidity RTE Ready-to-eat SBP Sweet bell pepper SDS Sodium dodecyl sulfate SIFT-MS Selected Ion Flow Tube Mass Spectrometry SS Stainless steel SSO Specific Spoilage Organism TAE Tris base, acetic acid & EDTA TS(A/B) Trypton Soya (agar/broth) UPGMA Unweighted Pair Group Method with Arithmetic Mean UV Ultra violet VOC(s) Volatile organic compound(s) W. Weissella μmax maximum specific growth rate ii AIM OF THE STUDY During the past decades, great advances in the field of food preservation have paved the way towards ensuring food safety to a greater extent. Still many obstacles need to be surpassed and improvements to be made as microbial issues related to pathogen exposure incidents and spoilage still occur. In both cases the outcome is negative leading inevitably to foodborne illness outbreaks endangering susceptible consumers and food loss, respectively. Nowadays, the market is engaging to products, which are healthy and highly nutritious, convenient and easy to-use. At the same time, market demand is shifting towards products that contain less additives and preservatives, with functional properties and subjected to non invasive technological handlings. Balancing all these aspirations without compromising the safety aspects is challenging. The safety of foodstuffs constitutes priority for food industry, and hence legislation is strict and entails consequences for companies that do not conform. On the other hand, assessment of properties associated with the quality of the products is more flexible and in general subject to cultural background and personal preferences. However, shelf-life is determined by organoleptic parameters, which are evaluated through sensorial analysis and correlated to microbial activity. A food product is considered spoiled when the levels of microbial contamination result in alterations of the predetermined aroma, flavor, color or texture rendering it unfit for consumption. The quality of raw materials, cold chain maintenance, good hygiene practices and an effective hurdle technology prevent unexpected cases of spoilage within the anticipated shelf-life however, certain microbial groups still manage to proliferate and dominate in the food matrix. Food
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