University of Patras
School of Medicine
Msc Program
“Biomedical Sciences”
PhD Thesis
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
BIRMPA ANGELIKI
Agronomist (Food Scientist), MSc
Patras, 2014
Πανεπιστήμιο Πατρών
Τμήμα Ιατρικής
Μεταπτυχιακό Πρόγραμμα
«Βασικές Ιατρικές Επιστήμες»
Διδακτορική Διατριβή
Εναλλακτικές Τεχνολογίες Απολύμανσης Τροφίμων και εκτίμηση κινδύνου για την Δημόσια Υγεία.
ΜΠΙΡΜΠΑ ΑΓΓΕΛΙΚΗ
Γεωπόνος (Επιστήμης & Τεχνολογίας Τροφίμων), MSc
Πάτρα, 2014
True science teaches, above all, to doubt and be ignorant.
Miguel de Unamuno (1864-1936) Spanish writer and philosopher
3 Member Committee
Associate Professor Apostolos Vantarakis (Supervisor)
Professor Michalis Leotsinidis
Professor Chrissanthy Papadopoulou
7 Member Committee
Associate Professor Apostolos Vantarakis (Supervisor)
Professor Michalis Leotsinidis
Professor Chrissanthy Papadopoulou
Associate Professor Maria Kapsokefalou
Professor Iris Spiliopoulou
Professor John Zarkadis
Senior Lecturer James Lyng
Acknowledgements
I owe my deepest gratitude to my supervisor, Associate Professor Apostolos Vantarakis. I am heartily thankful to him for his warm encouragement, motivated guidance, and considerable support from the start to the completion in my research work. This thesis would not have been possible without his excellent insight and creative ideas as well as extreme patience and diligence during my dissertation.
I would also like to give my great appreciation to my committee members, Professor M. Leotsinidis, with his valuable comments and suggestions during the experimental part of the thesis as well as during writing of the PhD thesis, and Professor C. Papadopoulou, for her helpful suggestions and valuable comments in my thesis research.
I am also grateful to the members of my 7-member committee Associate Professor Maria Kapsokefalou, Professor Iris Spiliopoulou-Sdougkou and Professor John Zarkadis for their valuable suggestions in my study and research work. I would like to give my special thanks to the member of my 7-member committee Senior Lecturer Dr. James G.Lyng, who gave me the opportunity to have a great experience under his supervision in the laboratory in School of Agriculture Food Science and Veterinary Medicine of College of Life Sciences in UCD Dublin, during my Erasmus placement June-September 2012, in Dublin.
I would also like to extend my sincere appreciation to many people who have given me a huge help in my research progress. They are Postdoctoral Researchers Dr. Petros Kokkinos with his suggestions throughout the phD thesis as well as with his warm encouragement when it was needed. Thanks go also to Dr. Panos Ziros, MSc student Maria Bellou, PhD student Spiros Paparrodopoulos, Panagiotis Pitsos, Postodoctoral Researcher Dr. Eleni Sazakli and MSc student Efi Kougia. Furthermore, I am also grateful to my lab mates and many of my colleagues for their kindly helps and great support, MSc student Maria Tselepi and Gavriil Vasilopoulos. I would also like to give my special thanks to Dr. Paul Whyte (Senior Lecturer of School of Veterinary Medicine and Veterinary Science Centre, Dublin) who guided me and gave his special suggestions during my Erasmus placement June-September 2012, in Dublin.
Moreover, I would like to express my gratitude to Professor Petros Groumpos and his PhD student Antigoni Anninou for our collaboration to construct and implement the mathematical model of this PhD thesis.
i Thanks goes also to graduate students Wanda and Katell who gave me valuable help during my lab work in UCD, Dublin. I would also like to thank Assistant Professor Dr. Panagiotis Skandamis for his warm acceptance to the lab of Quality Assurance in Agricultural University of Athens as well as Dr .Vassiliki Sfika in Department of Quality Control, Regional Centre of Plant Protection and Quality Control Achaias, Ministry of Rural Development and Food.
Finally, I am forever indebted to my parents, my sister and Vassilis for their understanding, endless patience as well as their encouragement and support when it was most required.
Abstract
Fruits and vegetables are considered as part of a healthy diet and lifestyle. However, concerns have arisen regarding the microbiological safety of Ready To Eat (RTE) produces due to a number of foodborne outbreaks associated with pathogens. Although strict practices for controlling the safety of RTE produce have been implemented in the fresh produce industry, the current commercial operations rely on a wash treatment with water or with an antimicrobial agent as the only step for reducing microbial populations on fresh produce. However, washing with common sanitizers has been demonstrated to achieve no more than 1-2 log10 reduction in pathogen populations. Recently, much research effort has been put into development to provide multiple-hurdle techniques which enhance produce safety. Thus, non-thermal technologies for the inactivation of microorganisms are of increasing interest to the food industry for the control of spoilage and safety, thus for assuring public health.
In this study, the effects of non-thermal disinfection processes, Near UV-Visible light (NUV-Vis), Continuous Ultraviolet Light (UV 254 nm), High Intensity Light Pulses (HILP), Ultrasound (US), as well as conventional sodium hypochlorite (SH) disinfection solutions were used. The effect of the above technologies was tested against bacteria (Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis and Listeria innocua) and viruses (Human Adenovirus). More precisely, the bacteria that were used were: E. coli K12, E. coli NCTC 9001 (representative microorganisms for the Enterohaemorrhagic foodborne pathogen E. coli O157:H7), S. aureus NCTC 6571, L. innocua NCTC 11288 (as a surrogate microorganism for the common foodborne pathogen L. monocytogenes), S. Enteritidis NCTC 6676 and HAdV (indicator virus selected as a surrogate of HAV and norovirus).
The main scope of this work was to study the efficacy of three light technologies on liquid suspensions. Then, the effect of UV, US, SH and combined technologies were evaluated on their efficiency to disinfect inoculated romaine lettuce, strawberries and cherry tomatoes. Furthermore, the effect of the above technologies on quality (color) and physicochemical characteristics of the RTE produces was evaluated. The physicochemical characteristics tested were Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and Ascorbic Acid (AA) concentration.
This study demonstrates that the use of alternative non-thermal technologies is effective for inactivation of microorganisms in fresh RTE foods and could be used as an alternative to traditional chlorine immersions. However, the effect of UV and US on iii quality and nutritional quality retention of RTE foods should be considered before its use as a disinfection technique.
As far as non-thermal light technologies are concerned, HIPL treatment inactivated both E. coli and L. innocua more rapidly and effectively than either continuous UV-C or NUV-vis treatments. With HILP at a distance of 2.5 cm from the lamp, E. coli and L. innocua populations were reduced by 3.07 and 3.77 log10 CFU/mL respectively after a 5 sec treatment time, and were shown to be below the limit of detection (<0.22 log10 CFU/mL) following 30 sec exposure to HILP (106.2 J/cm2).
Treatment of lettuce with UV reduced significantly the population of E. coli, S.aureus, S.
Enteritidis and L. innocua by 1.75, 1.21, 1.39 and 1.27 log10 CFU/g, respectively.
Furthermore, more than a 2- log10 CFU/g reduction of E. coli, S. Enteritidis and L.innocua was achieved with US. In strawberries, UV treatment reduced bacteria only by 1–1.4 log10 CFU/g. The maximum reductions of microorganisms, observed in strawberries after treatment with US, were 3.04, 2.52, 5.24 and 6.12 log10 CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua, respectively. Finally, cherry tomatoes exhibited the best results when treated with non-thermal technologies. For instance,
3.16, 2.62, 3.29, 3.16 log10 CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua, respectively, were achieved when US treatment was used. UV treatment resulted in 2.39,
2.05, 2.62, 2.56 log10 CFU/g reduction of the above microorganisms. The combined technologies of alternative followed by conventional disinfection technologies resulted in 2-3.50 log10 CFU/g reduction for lettuce and strawberries. However, cherry tomatoes exhibited greater reductions (3.28-4.78 log10 CFU/g reduction). Finally, 1-2 log10 CFU/g log reduction was achieved for lettuce and strawberries when RTE foods were immersed in NaOCl 200ppm solutions, and greater reductions (3-4 log10 CFU/g) were achieved for cherry tomatoes.
It was observed that HAdV was inactivated faster when chlorine treatment was used. However, UV non thermal technology found to be more effective for disinfection of
HAdV compared to US, achieving a log10 reduction of 2.13, 1.25 and 0.92 for lettuce, strawberry and cherry tomatoes respectively when UV treatment for 30 minutes was implemented, whereas, US treatment for the same treatment period achieved a log10 reduction of 0.85, 0.53 and 0.36 log10 respectively. The sequential use of US and UV was found to be more effective and less time consuming, than when the treatments were used alone, indicating the existence of an additive effect.
Treatment with UV and US, for time periods (up to 30 min) did not significantly (p > 0.05) change the color of RTE foods tested. Moreover, it was indicated that no significant differences (p>0.05) were observed as far as TAC is concerned when conventional treatments at different treatment times were used. However, when alternative disinfection treatments were used, an increase in TAC concentration was obvious from the first minutes of treatment. TPC concentration remained constant or was slightly decreased when RTE foods were immersed in NaOCl solutions. However, TPC increased significantly (p<0.05) in all RTE foods when UV and US alternative disinfection technologies were used. The vit.C content of RTE foods did not exhibit any significant changes during different treatments. However, vit.C was slightly decreased (p<0.05) when treatments of more than 30 minutes for US, UV and combinations of UV+US occurred.
Furthermore, a computerized model was proposed based on critical points which are important during the production of lettuce. More precisely the development of a Decision Support System (DSS) using the theory of Fuzzy Cognitive Maps (FCMs), in order to diagnose the importance of critical control points (concepts) for the food safety and hygiene during the production of salad vegetables (lettuce), was implemented. The methodology described, extracts the knowledge from experts with different scientific background and exploits their experience on the process of lettuce production. The results of this study show that the present software tool can be explored and problems that can arise during the food production chain can be prevented.
Generally, it was noted that the effect of each disinfection method is dependent upon the treatment time tested and the type of food. Treatment with UV and US reduced the numbers of selected inoculated bacteria on lettuce, strawberries and cherry tomatoes, which could be good alternatives to other traditional and commonly used technologies such as chlorine and hydrogen peroxide solutions. These results suggest that UV and US might be promising, non-thermal and environmental friendly disinfection technologies for fresh RTE produce industry.
Taking everything into consideration, disinfection technologies play an important role in commercial practice in order to prevent the survival of pathogens and lower the risk of contamination thus assuring public health. However, nutritional and quality properties are essential as they can provide a protective role against the development and progression of many diseases and must be considered for the selection of disinfection process parameters.
v
ΠΕΡΙΛΗΨΗ
Εκτεταμένη Περίληψη στα Ελληνικά
Εισαγωγή
Η κατανάλωση φρούτων και λαχανικών αποτελεί μέρος μίας υγιεινούς δίαιτας και διατροφικού προφίλ γενικότερα. Η Μεσογειακή διατροφή αποτελεί ένα μοντέρνο τρόπο διατροφής η οποία έχει τις ρίζες της στις χώρες της Μεσογείου, όπως η Ελλάδα, η Ισπανία, η Πορτογαλία και η Νότια Ιταλία. Τα βασικά συστατικά που την απαρτίζουν είναι το λάδι, τα λαχανικά, τα δημητριακά, τα φρούτα, τα ψάρια, τα γαλακτοκομικά, και η μικρή κατανάλωση κρέατος (Noah and Truswell, 2001). Ως εκ τούτου, λαχανικά όπως το μαρούλι, οι τομάτες είναι κύρια συστατικά μιας ισορροπημένης διατροφής. Επίσης, φρούτα όπως οι φράουλες προτιμώνται από εκείνους που θέλουν να ακολουθούν την Μεσογειακή Διατροφή αλλά και από αυτούς που θέλουν να προσέχουν την υγεία τους.
Το μαρούλι (Lactuca sativa L.) καταναλώνεται κυρίως ως σαλάτα και αποτελεί μία πλούσια πηγή συστατικών ευεργετικών για την υγεία όπως τα φαινολικά, η βιταμίνη C, τα καροτενοειδή και οι χλωροφύλλες (Nicolle et al., 2004). Περιλαμβάνει πολλά μακροστοιχεία (π.χ K, Na, Ca και Mg) και ιχνοστοιχεία (π.χ Fe, Mn, Cu, Zn και Se), τα οποία αποτελούν σημαντικά συστατικά μια σωστής διατροφής (Kawashima & Soares, 2003). Το μαρούλι αποτελεί επίσης μία καλή πηγή φωτοσυνθετικών χρωστικών και άλλων φυτοχημικών τα οποία ωφελούν την διατροφή και διαδραματίζουν σπουδαίο ρόλο στην παρεμπόδιση πολλών οξειδωτικών- σχετιζόμενων με το στρες- ασθενειών (Llorach et al., 2008). Οι φράουλες, είναι πλούσιες σε μία σειρά φυτοχημικών, ιδιαίτερα των φαινολικών συστατικών, κατέχοντας υψηλή αντιοξειδωτική ικανότητα (Häkkinen et al., 1999, Koponen et al., 2007). Επίσης έχουν μεγάλη περιεκτικότητα σε βιταμίνη C (60-100 mg/100 g τροφίμου) και σε ανθοκυανίνες, ειδικά πελαργονιδίνη-3-γλυκοζίτη (pg-3-gluc) και κυανιδίνη-3-γλυκοζίτη (CyA-3-gluc). Ως εκ τούτου, η φράουλα θεωρείται ως μια σημαντική διαιτητική πηγή ενώσεων που προάγουν την υγεία (Koponen et al., 2007). Οι τομάτες αποτελούν μία καλή πηγή βιταμινών (βιταμίνη Α, βιταμίνη C, και άλλων βιταμινών) καθώς και μεταλλικών στοιχείων (νάτριο, ασβέστιο, φώσφορος, σίδηρος), και ινών, πρωτεϊνών και λιπών. Η τομάτα είναι μία καλή πηγή αντιοξειδωτικών όπως το λυκοπένιο. Είναι γνωστό ότι το λυκοπένιο και οι ίνες είναι ευεργετικές στην ανθρώπινη υγεία, όταν καταναλώνονται ως μέρος μια ισορροπημένης διατροφής (Canene-Adams et al., 2005). Σύμφωνα με μελέτες το λυκοπένιο έχει
vii χαρακτηριστεί για τις αντιφλεγμονώδεις, αντιμεταλλαξιγόνες και αντικαρκινικές ιδιότητές του (Boon et al., 2010). Επιπλέον, το λυκοπένιο είναι γνωστό για τη μείωση του κινδύνου αδενώματος, και την προώθηση λειτουργικότητας του ανοσοποιητικού συστήματος (Kun et al., 2006). Συνιστάται, 6-15 mg πρόσληψη λυκοπενίου για την βελτίωση της υγείας (Kun et al., 2006). Οι διαλυτές φυτικές ίνες ρυθμίζουν τη γλυκόζη στο αίμα και τα επίπεδα χοληστερόλης (Weickert and Pfeifer, 2008). Ενώ οι αδιάλυτες φυτικές ίνες προάγουν την κάθαρση και βοηθούν εναντίον πολλών καρκίνων όπως ο καρκίνος του παχέος εντέρου (Alvarado et al., 2001). Στα προϊόντα τομάτας, η βιταμίνη C και οι πολυφαινόλες έχουν αναφερθεί ότι είναι τα κύρια υδρόφιλα αντιοξειδωτικά συστατικά, ενώ η βιταμίνη Ε και τα καροτενοειδή αποτελούν κυρίως το υδρόφοβο κλάσμα (Hsu, 2008).
Πολλές επιδημιολογικές μελέτες έχουν συσχετίσει την κατανάλωση φρούτων και λαχανικών με τον προστατευτικό ρόλο τους ενάντια σε πολλές ασθένειες (Hannum, 2004). Ρίζες οξυγόνου, μπορεί να αντιδράσουν με λίπη, πρωτεΐνες και DNA. Ο ρόλος των αντιοξειδωτικών που υπάρχουν στα φρούτα και στα λαχανικά είναι να διατηρούν τα χαμηλά επίπεδα των ελευθέρων ριζών είτε παρεμποδίζοντας την εμφάνισή τους, είτε ευνοώντας την αποσύνθεσή τους (Hancock et al., 2007).
Παρόλα αυτά, το τελευταίο διάστημα, λόγω του αυξανόμενου αριθμού τροφιμογενών ασθενειών σε όλο τον κόσμο, επικρατεί ανησυχία σχετικά με την μικροβιολογική ασφάλεια των τροφίμων αυτών. Τρόφιμα «έτοιμα προς κατανάλωση (ready-to-eat)», θεωρούνται ότι ανήκουν στην κατηγορία «υψηλού κινδύνου». Τα συγκεκριμένα τρόφιμα δεν επιδέχονται κάποια θερμική ή άλλη επεξεργασία θανάτωσης παθογόνων μικροοργανισμών.
Η μικροβιολογική ασφάλεια των τροφίμων και των τροφιμογενών ασθενειών αποτελούν περίπλοκα ζητήματα, καθώς περισσότερες από 200 γνωστές ασθένειες είναι γνωστό ότι μεταδίδονται μέσω των τροφίμων. Οι κύριοι λόγοι μετάδοσης τροφιμογενών ασθενειών είναι η επιμόλυνση με βακτήρια, ιούς, παράσιτα, μύκητες.
Στις Η.Π.Α, το μέσο ετήσιο κόστος που σχετίζεται με βακτηριακές και παρασιτικές τροφιμογενείς λοιμώξεις εκτιμάται στα 6.5 δισεκατομμύρια δολάρια (Buzby & Roberts, 1996, Tauxe, 2002). Ο Tauxe (2002) αναφέρει ότι ανάμεσα στις καταγεγραμμένες τροφιμογενείς λοιμώξεις, οι βακτηριακές λοιμώξεις ευθύνονται για ένα περίπου 30% των περιπτώσεων, οι ιολογικές για το 67% και οι παρασιτικές για το 3%. Νοσηλεία στο νοσοκομείο πραγματοποιήθηκε λόγω λοιμώξεων από βακτήρια (60%), από ιούς (35%)
και από παράσιτα (5%). Τέλος, θάνατος είναι δυνατό να προέλθει από βακτήρια (72%), ιούς (7%) και παράσιτα (21%). Έχει αναφερθεί ότι πέντε τροφιμογενείς παθογόνοι μκροοργανισμοί (E. coli O157:H7, Salmonella, Campylobacter, Listeria, and Toxoplasma) είναι υπεύθυνοι για 3.5 εκατομμύρια κρούσματα, 33.000 νοσηλείες και 1.600 θανάτους ετησίως στις Η.Π.Α (Tauxe, 2002).
Μέχρι σήμερα στις βιομηχανίες τροφίμων εφαρμόζονται μία σειρά αυστηρών πρακτικών απολύμανσης για τον έλεγχο της ασφάλειας των έτοιμων προς κατανάλωση τροφίμων. Οι πρακτικές αυτές περιλαμβάνουν ξεπλύματα με τρεχούμενο νερό ή με αντιμικροβιακά διαλύματα. Επιστημονικές μελέτες όμως καταδεικνύουν την ανικανότητα επαρκούς απολύμανσης παθογόνων μικροοργανισμών που υπάρχουν στα τρόφιμα, με τις συνήθεις πρακτικές που εφαρμόζονται σήμερα. Για το λόγο αυτό, νέες τεχνικές πολλαπλών εμποδίων έχουν αρχίσει να εφαρμόζονται με σκοπό την διασφάλιση της δημόσιας υγείας.
Τα τρόφιμα επεξεργάζονται με διάφορες τεχνολογίες με σκοπό την μείωση και την απομάκρυνση πιθανών παθογόνων ή άλλων βιολογικών κινδύνων που μπορεί να υπάρχουν στα τρόφιμα. Οι κλασικές τεχνολογίες απολύμανσης όπως παστερίωση ή αποστείρωση, χρησιμοποιούνται με σκοπό την απενεργοποίηση ή τη θανάτωση των μικροβίων.
Η απολύμανση με χλώριο αποτελεί μία ευρέως διαδεδομένη και οικονομική μέθοδο απολύμανσης η οποία χρησιμοποιείται για την απολύμανση νερού και τροφίμων (EPA, 1999c). Διαφορετικές μορφές χλωρίου μπορούν να χρησιμοποιηθούν στην απολύμανση των τροφίμων όπως: διοξείδιο του χλωρίου, υποχλωριώδες νάτριο, υποχλωριώδες ασβέστιο, κ.α (Park et al., 2008). Παρόλα αυτά το χλώριο μπορεί να αντιδράσει με οργανικές ουσίες σχηματίζοντας έτσι τοξικές χημικές ουσίες όπως οργανοχλωρoπαράγωγα, δηλαδή ενώσεις που ανήκουν στην κατηγορία των τριαλογονοµεθανίων (THM’s) (McDonnel and Russell, 1999).
Οι εναλλακτικές, μη-θερμικές τεχνολογίες απολύμανσης έχουν αποδειχθεί ότι είναι ικανές να επιτύχουν απολύμανση μικροβίων χωρίς την έκθεση των τροφίμων σε θερμότητα. Έχει επίσης βρεθεί ότι οι τεχνολογίες αυτές διατηρούν τα διατροφικά και οργανοληπτικά χαρακτηριστικά των τροφίμων, επεκτείνοντας τον χρόνο ζωής τους και διατηρώντας την εξωτερική τους εμφάνιση (Butz and Tauscher, 2002).
ix Τέτοιες τεχνολογίες είναι τα παλλόμενα ηλεκτρικά πεδία (PEF), η υπεριώδης ακτινοβολία (UV), το παλλόμενο φως υψηλής έντασης (HILP), υπέρηχοι (US), εγγύς υπεριώδης φως (NUV light), ιονίζουσα ακτινοβολία, όζων, υψηλή υδροστατική πίεση (HPP)κ.α (Mohd. Adzahan and Benchamaporn, 2007).
Η ακτινοβολία στο εγγύς υπεριώδες 395± 5 nm, δρα διεγείροντας ενδογενή μόρια 1 πορφυρίνης παράγοντας μονήρες οξυγόνο ( O2), που καταστρέφει τα κύτταρα και έτσι θανατώνονται οι μικροοργανισμοί (Elman and J. Lebzelter, 2004, Feuerstein et al. 2005, Maclean et al. 2008b, Murdoch et al., 2012, Lipovsky et al. 2010).
Η υπεριώδης ακτινοβολία όταν διαπερνά την κυτταρική μεμβράνη των μικροοργανισμών και απορροφάται από τα κυτταρικά συστατικά τους (DNA, RNA), τους καθιστά ανίκανους να πολλαπλασιαστούν. Το κατάλληλο μήκος κύματος το οποίο μπορεί να προκαλέσει ζημιά στο μικροβιακό DNA ή RNA είναι περίπου 254 nm. Όταν το γενετικό υλικό των κυττάρων απορροφά την ενέργεια από την υπεριώδη ακτινοβολία σχηματίζονται διμερή πυριμιδίνης μεταξύ γενετικών βάσεων πυριμιδίνης στην ίδια αλυσίδα DNA. Χάρη σε αυτό το δεσμό διμερών στην αλυσίδα του DNA, οι μικροοργανισμοί προσβάλλονται με τέτοιο τρόπο ώστε ο διαχωρισμός των κυττάρων και επομένως ο πολλαπλασιασμός τους να είναι αδύνατος. Έτσι, ο μικροοργανισμός γίνεται αβλαβής και θανατώνεται (Guerrero- Beltrán and Barbosa-Cánovas, 2004). Αν και οι περισσότεροι μικροοργανισμοί προσβάλλονται από την υπεριώδη ακτινοβολία, η ευαισθησία τους ποικίλλει, καθώς εξαρτάται από την αντίσταση στη διείσδυση της υπεριώδους ακτινοβολίας. Η χημική σύνθεση του κυτταρικού τοιχώματος και το πάχος του καθορίζουν την αντίσταση των μικροοργανισμών στην υπεριώδη ακτινοβολία. Η αποτελεσματικότητα της απολύμανσης με υπεριώδη ακτινοβολία επηρεάζεται από την ποσότητα – δόση της υπεριώδους ενέργειας που απορροφάται από το μικροοργανισμό. Η δόση της ακτινοβολίας εξαρτάται από την ένταση της παρεχόμενης ακτινοβολίας (ενέργεια, mW), τον χρόνο κατά τον οποίο ο μικροοργανισμός εκτίθεται σε αυτήν (διάρκεια ακτινοβολίας, sec) και είναι αντιστρόφως ανάλογη με την επιφάνεια του υγρού στο οποίο εφαρμόζεται (cm2).
Το παλλόμενο φως υψηλής έντασης (HILP) αποτελεί μία αναδυόμενη μη-θερμική τεχνολογία απολύμανσης, η οποία χρησιμοποιεί μικρής διάρκειας (100–400 s) αλλά
υψηλής έντασης φως (200–1100 nm) (Marquenie et al., 2003, Woodling and𝜇𝜇 Moraru, 2007, Gomez-Lopez et al., 2007). Ο τρόπος δράσης βασίζεται στην φωτοχημική δράση της υπεριώδους ακτινοβολίας η οποία προκαλεί διμερισμό της θυμίνης οδηγώντας στον
θάνατο των κυττάρων (Muňoz et al., 2012, Gomez-Lopez et al., 2007, Rajkovic et al., 2010).
Εξ’ ορισμού οι υπέρηχοι συνιστούν κύματα υψηλής συχνότητας που μεταφέρουν πίεση κατά τη διέλευσή τους σε ένα μέσο. Αυτό έχει ως αποτέλεσμα τη δημιουργία περιοχών χαμηλής και υψηλής πίεσης. Η διακύμανση αυτή της πίεσης αναφέρεται ως πλάτος πίεσης (amplitude) και είναι ανάλογο της ποσότητας ενέργειας που εφαρμόζεται στο σύστημα. Στην περίπτωση που οι διακυμάνσεις της πίεσης είναι αρκετά υψηλές (3.000 ΜΡa), τότε ένα υγρό μέσο μπορεί να αποδομηθεί και να έχουμε το σχηματισμό μικροφυσαλίδων αερίου και ατμού. Το φαινόμενο αυτό είναι γνωστό ως σπηλαίωση (cavitation), ενώ οι φυσαλίδες είναι δυνατόν να διασπώνται και να επαναδημιουργούνται συνεχώς επιφέροντας αλλαγές στη δομή του μέσου που υφίσταται την επίδραση των υπερηχητικών κυμάτων, απενεργοποιώντας έτσι τους μικροοργανισμούς από την επιφάνεια των τροφίμων (Bilek and Turantas, 2013). Το κύριο πλεονέκτημα των υπερήχων για τη βιομηχανία τροφίμων είναι ότι θεωρούνται μια ευρέως αποδεκτή τεχνολογία από το ευρύ καταναλωτικό κοινό, λόγω της ασφάλειάς τους, της μη τοξικότητάς τους και της φιλικότητάς τους προς το περιβάλλον.
Σκοπός της παρούσας εργασίας
Πολλές επιδημίες που προέρχονται από τροφιμογενείς λοιμώξεις έχουν καταγραφεί τον τελευταίο καιρό, καθώς επίσης πολλές ανακλήσεις προϊόντων συμβαίνουν. Αποτελεί λοιπόν αναγκαιότητα η εξάλειψη των παθογόνων από τρόφιμα λόγω του υψηλού κινδύνου, του υψηλού ποσοστού θνησιμότητας καθώς και της οικονομικής επιβάρυνσης που προκαλούν οι ασθένειες (π.χ λιστερίωση, σαλμονέλλωση).
Στην παρούσα μελέτη μη-θερμικές, εναλλακτικές τεχνολογίες απολύμανσης ελέγχθηκαν όπως: Φως κοντά στην υπεριώδη ακτινοβολία σε μήκος κύματος 395±5 nm (NUV-Vis light), συνεχής υπεριώδης ακτινοβολία σε μήκος κύματος 254nm (Continuous UV light), υψηλής έντασης παλμοί φωτός (HILP), υπέρηχοι (ultrasound). Επίσης, η συμβατική και κλασική μέθοδος της εμβάπτισης σε υποχλωριώδες νάτριο (sodium hypochlorite solution) εφαρμόστηκε. Τέλος, συνδυασμοί εναλλακτικών, καθώς και εναλλακτικών με κλασικές μεθόδους πραγματοποιήθηκαν. Ο σκοπός ήταν ο έλεγχος της εφαρμογής των τεχνολογιών αυτών στα τρόφιμα με σκοπό τη διασφάλιση της δημόσιας υγείας των καταναλωτών.
xi Όλες οι παραπάνω μέθοδοι εφαρμόστηκαν σε τρόφιμα έτοιμα προς κατανάλωση όπως μαρούλι, φράουλες και τοματίνια τύπου cherry, τα οποία αγοράστηκαν από τοπικό σουπερμάρκετ και εμβολιάστηκαν με παθογόνους μικροοργανισμούς οι οποίοι αποτέλεσαν το αρχικό μικροβιακό φορτίο. Οι μικροοργανισμοί οι οποίοι εμβολιάστηκαν ήταν βακτήρια που έχουν συναντηθεί στα εν λόγω τρόφιμα όπως E. coli, S. aureus, S. enteritidis και L. innocua καθώς και ο αδενοιός (HAdV35). Πιο συγκεκριμένα τα στελέχη που χρησιμοποιήθηκαν ήταν: E. coli K12, E. coli NCTC 9001 (ως μικροοργανισμοί δείκτες για το εντεροαιμοραγικό παθογόνο E. coli O157:H7), S. aureus NCTC 6571, L. innocua NCTC 11288 (ως μικροοργανισμοί δείκτες για το παθογόνο Listeria monocytogenes), S. Enteritidis NCTC 6676 και HAdV (ιός δείκτης για τους ιούς HAV και norovirus).
Πιο συγκεκριμένα, στο πρώτο μέρος της διατριβής, τρεις τεχνολογίες απολύμανσης (NUV-Vis, Continuous UV, HILP) χρησιμοποιήθηκαν όσον αφορά την απολύμανση των μικροοργανισμών δεικτών (E. coli και L. innocua), τα οποία εμβολιάστηκαν σε υγρά διαλύματα (MRD Buffer). Ο σκοπός ήταν να ελεγχθεί η απολυμαντική δράση των τεχνολογιών αυτών, χρησιμοποιώντας διαφορετικές εντάσεις φωτός.
Στο δεύτερο μέρος της διατριβής, τα τρόφιμα εμβολιάστηκαν με διάφορα βακτήρια (E. coli, S. aureus, S. Εnteritidis, L. innocua) και έναν αδενοϊό με σκοπό να ελεγχθεί η απολύμανσή τους, έπειτα από τη χρήση του χλωρίου, του υπεριώδους φωτός και των υπερήχων, καθώς επίσης και συνδυασμών τους. Επίσης, πραγματοποιήθηκαν πειράματα με διαφορετικές αρχικές συγκεντρώσεις βακτηρίων, με σκοπό τον περεταίρω έλεγχο της απολυμαντικής δράσης των παραπάνω τεχνολογιών. Τέλος, τα αποτελέσματα της συγκέντρωσης των ιών που προήλθαν από τη χρήση αλυσιδωτής αντίδρασης πολυμεράσης σε πραγματικό χρόνο (Real-Time PCR), επιβεβαιώθηκαν με τη χρήση καλλιεργειών κυττάρων. Τέλος, έπειτα από την επεξεργασία των τροφίμων με τις μεθόδους απολύμανσης, αποθηκεύτηκαν τα τρόφιμα στο ψυγείο για διάστημα 15 ημερών, και ελέγχθηκε το μικροβιακό τους φορτίο έπειτα από 3, 7 και 15 ημέρες.
Στο τρίτο μέρος της διατριβής, η επίδραση των παραπάνω μεθόδων σε επιλεγμένες διατροφικές παραμέτρους και παραμέτρους ποιότητας μελετήθηκε. Για το σκοπό αυτό, ελέγχθηκαν πριν και έπειτα από την χρήση των τεχνολογιών: η ολική αντιοξειδωτική τους ικανότητα, η περιεκτικότητά τους σε ολικά φαινολικά, η συγκέντρωση ασκορβικού οξέος και η ένταση του χρώματός τους.
Στο τέταρτο μέρος της διατριβής, χρησιμοποιήθηκε ένα μοντέλο πρόβλεψης για την ασφάλεια των τροφίμων. Το μοντέλο αποτελεί ένα μοντέλο λήψης απόφασης το οποίο χρησιμοποιήθηκε στην παρούσα διατριβή με σκοπό την λήψη απόφασης σε ένα πολύπλοκο σύστημα μιας καθετοποιημένης εταιρείας μαρουλιού. Στην συγκεκριμένη περίπτωση, 9 κρίσιμα σημεία κατά τη διάρκεια παραγωγής-επεξεργασίας και διάθεσης λαχανικών επιλέχθηκαν για το σύστημα. Τα σημεία αυτά ήταν: εργατικό δυναμικό- προσωπικό, συστήματα ποιότητας και ασφάλειας τροφίμων, τοποθεσία-περιβάλλων χώρος της μονάδας παραγωγής τροφίμων, φυτώριο μαρουλιού, έδαφος παραγωγής μαρουλιού, διαδικασία συγκομιδής, διαδικασία μετά τη συγκομιδή, μεταφορά, πώληση. Στη συνέχεια, τρεις ειδικοί βαθμολόγησαν τα 9 κριτήρια αυτά μεταξύ τους, ως προς την ύπαρξη ή απουσία σχέσης τους με σκοπό την πρόβλεψη ασφάλειας του τελικού προϊόντος. Το συγκεκριμένο μοντέλο βασίζεται στη θεωρία των ασαφών γνωστικών δικτύων.
Στο τελευταίο κομμάτι της διατριβής, τα αποτελέσματα από τις μεθόδους απολύμανσης συγκεντρώθηκαν, και με βάση τη μολυσματική δόση κάθε μικροοργανισμού στο τελικό προϊόν που έχει καταγραφεί στην βιβλιογραφία, εξάχθηκαν συμπεράσματα σχετικά με την αποτελεσματικότητα των μεθόδων σε σχέση με την διασφάλιση της δημόσιας υγείας.
Αποτελέσματα
Τα αποτελέσματα της διατριβής απέδειξαν ότι οι εναλλακτικές, μη-θερμικές τεχνολογίες απολύμανσης είναι αποδοτικές για την απενεργοποίηση των μικροοργανισμών σε φρέσκα έτοιμα προς κατανάλωση τρόφιμα και μπορούν να εφαρμοστούν ως εναλλακτικές στην διαδεδομένη απολύμανση με τη χρήση χλωρίου. Ιδιαίτερη έμφαση αξίζει να δοθεί στα διατροφικά χαρακτηριστικά, πριν την επιλογή της τεχνολογίας απολύμανσης, έτσι ώστε να μην υποβαθμίζονται τα ευεργετικά συστατικά των τροφίμων αυτών για την ανθρώπινη υγεία.
Από τις μη-θερμικές τεχνολογίες φωτός, οι παλμοί υψηλής έντασης (HILP) απενεργοποίησαν τους μικροοργανισμούς E. coli και L. innocua σε συντομότερο χρονικό διάστημα σε σύγκριση με τις άλλες δύο τεχνολογίες απολύμανσης (Continuous UV και NUV-Vis). Όταν το δείγμα τοποθετήθηκε σε μικρή απόσταση από την πηγή φωτός (2.5 cm), οι μικροοργανισμοί E. coli και L. innocua μειώθηκαν κατά 3.07 and
3.77 log10 CFU/mL αντίστοιχα μετά από χρόνο επεξεργασίας 5 δευτερόλεπτα. Έπειτα
xiii από χρόνο επεξεργασίας 30 δευτερολέπτων με την ίδια τεχνολογία και έντασης 106.2 2 J/cm , οι μικροοργανισμοί ήταν κάτω από το όριο ανίχνευσής τους (<0.22 log10 CFU/mL).
Η επεξεργασία με τη μη-θερμική τεχνολογία του υπεριώδους φωτός στο μαρούλι, μείωσε σημαντικά τους πληθυσμούς των παρακάτω μικροβίων E. coli, S.aureus, S.
Enteritidis and L. innocua κατά 1.75, 1.21, 1.39 και 1.27 log10 CFU/g, αντίστοιχα. Όταν η μη-θερμική τεχνολογία των υπερήχων εφαρμόστηκε, μία λογαριθμική μείωση της
τάξης των 2 log10 CFU/g για τους πληθυσμούς E. coli, S. Enteritidis και L. innocua καταγράφηκε. Η τεχνολογία υπεριώδους ακτινοβολίας μείωσε το μικροβιακό φορτίο
κατά 1–1.4 log10 CFU/g. Οι μέγιστες λογαριθμικές μειώσεις που παρατηρήθηκαν στη
φράουλα μετά την επεξεργασία με υπερήχους, ήταν 3.04, 2.52, 5.24 και 6.12 log10 CFU/g για τους μικροοργανισμούς E. coli, S. aureus, S. Enteritidis και L. innocua, αντίστοιχα. Τέλος, στα τοματίνια cherry, η απολύμανση με τη χρήση μη-θερμικών τεχνολογιών, είχε τα καλύτερα αποτελέσματα. Πιο συγκεκριμένα η απολύμανση με τη
χρήση υπερήχων μείωσε το μικροβιακό φορτίο κατά 3.16, 2.62, 3.29, 3.16 log10 CFU/g για τους μικροοργανισμούς E. coli, S. aureus, S. Enteritidis και L. innocua, αντίστοιχα. Αξίζει να αναφερθεί ότι η τεχνολογία υπεριώδους ακτινοβολίας, είχε ως αποτέλεσμα
την μείωση κατά 2.39, 2.05, 2.62, 2.56 log10 CFU/g των παραπάνω μικροοργανισμών αντίστοιχα. Στη συνέχεια εφαρμόστηκαν και στα τρία έτοιμα προς κατανάλωση τρόφιμα συνδυασμοί των παραπάνω μη-θερμικών εναλλακτικών τεχνολογιών. Η μείωση του
μικροβιακού φορτίου με τη χρήση υποχλωριώδους νατρίου 200ppm ήταν 1-2 log10
CFU/g log για το μαρούλι και τις φράουλες, ενώ μεγαλύτερες μειώσεις (3-4 log10 CFU/g) καταγράφηκαν όταν τα τοματίνια απολυμάνθηκαν με χλώριο. Τέλος, οι συνδυασμοί εναλλακτικών-συμβατικών τεχνολογιών είχαν ως αποτέλεσμα μία μείωση
της τάξεως των 2-3.50 log10 CFU/g, για το μαρούλι και τις φράουλες, ενώ μείωση 3.28-
4.78 log10 CFU/g πραγματοποιήθηκε για τα τοματίνια τύπου cherry.
Κατά την απολύμανση του αδενοϊού, η πιο αποτελεσματική μέθοδος ανάμεσα σε όλες οι οποίες εφαρμόστηκαν, αποδείχθηκε η χρήση χλωρίου. Από τις εναλλακτικές μη- θερμικές τεχνολογίες, η χρήση υπεριώδους φωτός ήταν πιο αποδοτική σε σχέση με τους
υπερήχους, μειώνοντας το ιϊκό φορτίο κατά 2.13, 1.25 και 0.92 log10 για τα μαρούλια, τις φράουλες και τα τοματίνια αντίστοιχα όταν τα τρόφιμα αυτά εκτέθηκαν σε υπεριώδη ακτινοβολία για χρονικό διάστημα 30 λεπτών. Αντιθέτως η μείωση του ιϊκού φορτίου
όταν εφαρμόστηκαν υπέρηχοι για 30 λεπτά ήταν 0.85, 0.53 και 0.36 log10 για τα τρία τρόφιμα αντίστοιχα. Αξίζει να αναφερθεί ότι η συνδυαστική χρήση υπεριώδους
ακτινοβολίας και υπερήχων ήταν πιο αποδοτική και λιγότερο χρονικά δαπανηρή, σε σχέση με τη μεμονωμένη χρήση των δύο παραπάνω τεχνολογιών, όσον αφορά την απολύμανση των τροφίμων από βακτήρια και ιούς, αποδεικνύοντας την αθροιστική τους δράση.
Όσον αφορά τις παραμέτρους ποιότητας των τροφίμων, η χρήση εναλλακτικών τεχνολογιών απολύμανσης (UV, US) για χρονικό διάστημα μικρότερο των 30 λεπτών, δεν άλλαξε σημαντικά (p>0.05) το χρώμα των τροφίμων. Επίσης, δεν παρατηρήθηκαν στατιστικά σημαντικές διαφορές (p>0.05) στην ολική αντιοξειδωτική ικανότητα των τροφίμων όταν χρησιμοποιήθηκε η συμβατική τεχνολογία απολύμανσης με χλώριο. Όταν οι εναλλακτικές τεχνολογίες χρησιμοποιήθηκαν, μία αύξηση στην συγκέντρωση των ολικών αντιοξειδωτικών ήταν εμφανής από τα πρώτα λεπτά της απολύμανσης. Η περιεκτικότητα σε ολικά φαινολικά παρέμεινε σταθερή ή μειώθηκε ελαφρώς όταν τα τρόφιμα απολυμάνθηκαν με την χρήση χλωρίου. Αντιθέτως, όταν τα τρόφιμα απολυμάνθηκαν με τις εναλλακτικές τεχνολογίες απολύμανσης η περιεκτικότητά τους σε φαινολικά συστατικά αυξήθηκε σημαντικά (p<0.05). Τέλος, η περιεκτικότητα σε βιταμίνη C δεν μεταβλήθηκε κατά τη διάρκεια των διαφόρων τεχνολογιών. Όταν ο χρόνος απολύμανσης με τις διάφορες τεχνολογίες ξεπέρασε τα 30 λεπτά ή όταν συνδυάστηκαν οι εναλλακτικές τεχνολογίες μεταξύ τους για συνολικό διάστημα 30 λεπτών, σημαντική μείωση της περιεκτικότητας της βιταμίνης C παρατηρήθηκε (p<0.05).
Το υπολογιστικό μοντέλο που χρησιμοποιήθηκε βασίστηκε σε κρίσιμα σημεία τα οποία θεωρούνται σημαντικά σε μια καθετοποιημένη μονάδα παραγωγής μαρουλιών. Πιο συγκεκριμένα, αναπτύχθηκε ένα σύστημα λήψης απόφασης με τη χρήση των ασαφών γνωστικών δικτύων. Ο σκοπός ήταν η διάγνωση και ο έλεγχος των κρίσιμων σημείων ελέγχου σε μία παραγωγική μονάδα, έτσι ώστε να διασφαλιστεί η υγιεινή και η ασφάλεια των τροφίμων. Η μεθοδολογία που εφαρμόστηκε, χρησιμοποιεί την αληθινή γνώση και εμπειρία ειδικευμένων στην παραγωγική διαδικασία της παραγωγής των μαρουλιών. Αποδείχθηκε ότι η χρήση ενός τέτοιου μοντέλου μπορεί να προβλέψει και να αποτρέψει προβλήματα που μπορεί να συμβούν κατά τη διάρκεια της παραγωγικής διαδικασίας, έτσι ώστε να διασφαλιστεί η υγεία και η ασφάλεια των καταναλωτών. Το μοντέλο λήψης απόφασης εφαρμόστηκε σε τρεις διαφορετικές περιπτώσεις της ίδιας μονάδας παραγωγής μαρουλιών, όπου εξάχθηκαν αποτελέσματα σχετικά με την ασφάλεια του τελικού προϊόντος.
xv Στο τελευταίο μέρος των αποτελεσμάτων της διατριβής, βιβλιογραφικά δεδομένα σχετικά με τη μολυσματικότητα των παραπάνω βακτηρίων και ιών συλλέχθηκαν από διάφορους φορείς (FDA, PHAC, European pathogen fact sheet). Στη συνέχεια, με βάση τα αποτελέσματα των μεθόδων απολύμανσης που εφαρμόστηκαν στα τρόφιμα, συμπεράσματα εξήχθηκαν για την ικανότητα των μεθόδων αυτών να εφαρμοστούν στα τρόφιμα και να διασφαλίσουν την δημόσια υγεία. Έτσι στο μαρούλι, παρατηρήθηκε ότι η συνδυαστική τεχνολογία των υπερήχων ακολουθούμενη από χλώριο αποτέλεσε την καλύτερη τεχνολογία απολύμανσης των βακτηρίων, ενώ η υπεριώδης ακτινοβολία και το χλώριο είναι αποτελεσματικές τεχνολογίες για την απολύμανση των ιών. Για τις φράουλες, οι υπέρηχοι, και η συνδυαστική τεχνολογία US+NaOCl, ήταν ικανές να απολυμάνουν επαρκώς τα βακτήρια, ενώ η υπεριώδης ακτινοβολία αποδείχθηκε ως η πιο αποτελεσματική τεχνολογία για την απολύμανση του αδενοιού στη φράουλα. Στα τοματίνια τύπου cherry σχεδόν όλες οι τεχνολογίες ήταν ικανές να τα απολυμάνουν ικανοποιητικά. Η συνδυαστική τεχνολογία US+NaOCl αποδείχθηκε αποδοτική για την απολύμανση των βακτηρίων ενώ το χλώριο για τους ιούς.
Σε γενικές γραμμές παρατηρήθηκε ότι η επίδραση των τεχνολογιών απολύμανσης εξαρτάται από την τεχνολογία, το χρόνο επεξεργασίας και το είδος του τροφίμου. Τα αποτελέσματα της διατριβής αυτής απέδειξαν τις εναλλακτικές τεχνολογίες ως ικανές και φιλικές προς το περιβάλλον τεχνολογίες που μπορούν να εφαρμοστούν στα τρόφιμα με σκοπό την διασφάλιση της υγείας των καταναλωτών.
Συμπεράσματα
Τα αποτελέσματα της παρούσας μελέτης απέδειξαν ότι οι εναλλακτικές, μη-θερμικές τεχνολογίες απολύμανσης, επέδρασαν σε μία αποτελεσματική μείωση του μικροβιακού πληθυσμού τόσο των υγρών διαλυμάτων όσο και των τροφίμων.
Από τις εναλλακτικές τεχνολογίες φωτός, το παλλόμενο φως υψηλής έντασης αποτελεί την πιο αποδοτική μέθοδο για την απολύμανση των βακτηρίων E.coli και L.innocua. Επίσης αυτή η τεχνολογία είχε ως αποτέλεσμα την πιο γρήγορη και εντατική απολύμανση σε σχέση με τις άλλες δύο τεχνολογίες φωτός που εφαρμόστηκαν. Η εξαιρετική απόδοση της τεχνολογίας αυτής πιθανόν οφείλεται στην υψηλότερη διεισδυτική ικανότητα της συγκεκριμένης πηγής φωτός καθώς και της μεγαλύτερης ισχύος εκπομπής σε σχέση με τη συνεχή υπεριώδης ακτινοβολία και την εγγύς υπεριώδη
ακτινοβολία (NUV-vis). Η υψηλή ένταση του φωτός από την τεχνολογία παλλόμενου φωτός, απέδιδε μία ένταση φωτός κατά 100 φορές μεγαλύτερη από τις άλλες τεχνολογίες, στον ίδιο χρόνο λειτουργίας. Περισσότερη μελέτη χρειάζεται να πραγματοποιηθεί σε τρόφιμα ώστε να διεξαχθούν συμπεράσματα εάν η τεχνολογία αυτή δημιουργεί παραπροϊόντα στα τρόφιμα αυτά. Μπορεί να εξαχθεί το συμπέρασμα ότι η εγγύς υπεριώδη ακτινοβολία είναι μία υποσχόμενη μέθοδος για τη χρήση της σε βιομηχανίες τροφίμων, αυξάνοντας έτσι την παραγωγικότητα τους.
Οι εναλλακτικές τεχνολογίες (UV, US) που εφαρμόστηκαν στην απολύμανση των τροφίμων αποτελούν εναλλακτικές στις ήδη υπάρχουσες διαδεδομένες τεχνολογίες απολύμανσης. Μπορούν δηλαδή να εφαρμοστούν από τις βιομηχανίες τροφίμων, ως τεχνολογίες χαμηλού κόστους και χαμηλής κατανάλωσης ενέργειας, καθώς δεν απαιτούν ιδιαίτερο εξοπλισμό. Η απόδοση των τεχνολογιών αυτών εξαρτάται από την δόση, τον χρόνο έκθεσης, την επιφάνεια του τροφίμου. Οι υπέρηχοι ήταν αποδοτικότερη μέθοδος για την απολύμανση των βακτηρίων, ενώ η υπεριώδης ακτινοβολία πιο αξιόπιστη για την απολύμανση των ιών. Κάποιες πιθανές μεταβολές του χρώματος των τροφίμων μπορούν να ελεγχθούν, εφόσον χρησιμοποιηθούν οι κατάλληλες συνθήκες των τεχνολογιών, διατηρώντας έτσι αναλλοίωτα τα χαρακτηριστικά των τροφίμων. Οι συνδυαστικές τεχνολογίες είναι υποσχόμενες, εφόσον επιβεβαιωθεί η απουσία παραπροϊόντων χλωρίου στα τελικά προϊόντα.
Επίσης, πρέπει να σημειωθεί ότι δεν μπορεί να γίνει μία απευθείας σύγκριση τεχνολογιών απολύμανσης εάν δεν ληφθούν υπόψη και άλλες παράμετροι όπως το χρώμα και διατροφικές παράμετροι. Στην παρούσα μελέτη αποδείχθηκε ότι υπάρχει θετική ή μηδαμινή επίδραση στην ποιότητα των τροφίμων έπειτα από τις περισσότερες τεχνολογίες που εφαρμόστηκαν.
Οι μικροβιολογικές και οι μοριακές αναλύσεις μπορούν να εφαρμοστούν στον ποιοτικό έλεγχο μιας διατροφικής αλυσίδας. Ωστόσο, τα αποτελέσματα των αναλύσεων αυτών είναι χρονοβόρα (μικροβιολογικές αναλύσεις) και πολλές φορές οικονομικά ασύμφορα (μοριακές αναλύσεις). Επίσης, πολλές φορές τα αποτελέσματα των αναλύσεων εξαρτώνται από την ακρίβεια καθώς και το καλιμπράρισμα του εξοπλισμού που χρησιμοποιείται. Για τον σκοπό αυτό προτάθηκε και το θεωρητικό μοντέλο το οποίο μπορεί να παρέχει ένα πρώτο έλεγχο-εκτίμηση της ποιότητας του τροφίμου που παράγεται σε μία μονάδα παρασκευής-επεξεργασίας. Το μοντέλο αυτό εφαρμόστηκε στα πλαίσια της παρούσας διατριβής πρώτη φορά σε μονάδα επεξεργασίας μαρουλιών.
xvii Βασίζεται στη θεωρία των ασαφών γνωστικών δικτύων και αποτελεί μία απλή, φθηνή, φιλική, πραγματικού χρόνου και εύκολη προσέγγιση για την πιθανή εκτίμηση της ποιότητας-ασφάλειας του μαρουλιού. Επιπροσθέτως, το συγκεκριμένο μοντέλο θα μπορούσε να αξιοποιηθεί από τις Αρχές Ελέγχου Τροφίμων, με σκοπό να αποκτούν μία πρώτη εκτίμηση των προϊόντων που πρόκειται να επιθεωρήσουν.
Οι τροφιμογενείς λοιμώξεις παρουσιάζουν μία διαρκώς αυξανόμενη τάση, και βασίζονται στην αυξανόμενη παραγωγή και ζήτηση τροφίμων και ιδιαίτερα έτοιμων προς κατανάλωση τροφίμων. Οι καταναλωτές αντιλαμβάνονται την ασφάλεια των τροφίμων ως «δεδομένο», και για το λόγο αυτό η βιομηχανία τροφίμων πρέπει να εξασφαλίσει την «ποιότητα» των παραγόμενων προϊόντων. Η αυξημένη ζήτηση των καταναλωτών για τα συγκεκριμένα τρόφιμα βασίζεται στην προμήθεια τροφίμων με «μηδενικό» ή «ανύπαρκτο» κίνδυνο και το κόστος για την επίτευξη του γεγονότος αυτού δεν θα πρέπει να λαμβάνεται υπόψη εφόσον ο στόχος παραμένει πάντα η διασφάλιση της δημόσιας υγείας.
Λαμβάνοντας υπόψη όλα τα αποτελέσματα της παρούσης διατριβής, οι εναλλακτικές τεχνολογίες απολύμανσης διαδραματίζουν ένα σπουδαίο ρόλο και αποτελούν βιώσιμες τεχνολογίες οι οποίες μπορούν να ενταχθούν στην καθημερινή πρακτική με σκοπό να παρεμποδίσουν την ανάπτυξη των μικροοργανισμών και να μειώσουν το κίνδυνο μόλυνσης, διασφαλίζοντας έτσι την δημόσια υγεία. Ιδιαίτερη προσοχή πρέπει να δοθεί στην επιλογή των κατάλληλων συνθηκών των τεχνολογιών ώστε να διατηρηθούν τα απαραίτητα διατροφικά και ποιοτικά χαρακτηριστικά των τροφίμων, τα οποία έχουν ευεργετικές επιδράσεις στην υγεία των καταναλωτών.
Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Table of Contents
Abbreviations ...... 7
List of Figures ...... 9
List of Graphs ...... 14
List of Tables ...... 15
INTRODUCTION ...... 17
Chapter 1: Literature Review ...... 19
1.1 Fresh produce and Mediterranean diet ...... 19
1.1.1 Lettuce ...... 20
1.1.2 Strawberries ...... 22
1.1.3 Tomatoes ...... 23
1.2. Nutritional, Quality and Health Aspects of fresh ready-to- eat (RTE) fruits and vegetables ...... 25
1.2.1 Antioxidant Compounds...... 27
1.2.1.1 Determination of Antioxidants ...... 28
1.2.2 Phenolic Compounds ...... 31
1.2.2.1 Determination of TPC (total phenolic content) ...... 31
1.2.3 Ascorbic Acid ...... 33
1.2.3.1 Determination of ascorbic acid...... 34
1.2.4 Quality Aspects of Foods ...... 36
1.2.4.1 External Quality-Color ...... 36
1.3 Foodborne Pathogens and Foodborne diseases ...... 37
1.3.1 Global challenge and increased frequency of foodborne diseases...... 37
1.3.2 Hazards during the food production chain ...... 39
Page 1 Table of Contents
1.3.3 Microbial colonization on fresh produce surfaces ...... 40
1.3.4 Foodborne illness and foodborne disease outbreaks ...... 42
1.3.5 Foodborne Bacteria ...... 44
1.3.5.1 Escherichia coli ...... 44
1.3.5.2 Staphylococcus aureus ...... 47
1.3.5.3 Salmonella spp...... 49
1.3.5.4 Listeria spp...... 51
1.3.6 Foodborne Viruses ...... 53
1.3.6.1 Noroviruses ...... 55
1.3.6.2 Hepatitis ...... 56
1.3.6.3 Adenoviruses ...... 57
1.3.7 Pathogens and Infectious Dose ...... 59
1.3.8 Methods for detection of foodborne pathogens ...... 61
1.4 Infection and Disinfection ...... 64
1.4.1 The Bacterial Cell and Antimicrobial Interaction ...... 65
1.4.2 The virus genome and infectivity ...... 66
1.4.3 Conventional Food Processing/Preservation Technologies ...... 67
1.4.3.1 Chemical Methods ...... 67
1.4.3.1.2 Organic Acids ...... 69
1.4.3.1.3 Peroxyacetic Acid ...... 70
1.4.3.1.4 Hydrogen Peroxide ...... 71
1.4.3.2 Physical Methods ...... 71
1.4.3.2.1 Heat Processing ...... 71
1.4.3.2.2 Radio Frequency (RF) and Microwave Heating (MH) ...... 72
Page 2 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.4.3.2.3 Ohmic Heating ...... 72
1.4.4 Non-thermal Technologies (Alternative Technologies) ...... 73
1.4.4.1 Ultraviolet Light (UV)...... 73
1.4.4.2 High Intensity Light Pulses (HILP)...... 77
1.4.4.3 Near UV-Vis Light (NUV-Vis) ...... 78
1.4.4.4 Ultrasound ...... 79
1.4.5 Other Methods ...... 82
1.4.5.1 Ozone...... 82
1.4.5.2 Pulsed Electric Fields ...... 83
1.4.5.3 High Pressure Processing ...... 84
1.4.5.4 Electrochemical (Cold Plasma) Method...... 85
1.4.6 Biological control ...... 86
1.5 Control of foodborne diseases ...... 86
1.5.1 Public Health Surveillance ...... 87
1.5.2 Food Legislation ...... 89
1.5.3 Guidelines for the microbiological quality of RTE foods in Greece ...... 90
1.5.4 Predictive Models-Risk Assessment Support Systems and Public Health ...... 91
AIM OF THE STUDY ...... 95
Chapter 2. MATERIALS AND METHODS ...... 97
2.1 In Vitro Experiments with 3 Light Technologies ...... 98
2.1.1 Equipment ...... 98
2.1.2 Disposables- Plasticwares ...... 98
2.1.3 Culture Media ...... 99
2.1.4 Solutions for microbiological analysis ...... 99
Page 3 Table of Contents
2.1.5 Disinfection Light Treatments ...... 100
2.1.6 Microbiological analysis ...... 103
2.2 Food Disinfection ...... 104
2.2.1 Equipment ...... 104
2.2.2 Disposables- Plasticwares ...... 104
2.2.3 Culture Media ...... 105
2.2.4 Solutions for microbiological analysis ...... 106
2.2.5 Solutions for virus concentration ...... 107
2.2.6 Bacterial Strains ...... 107
2.2.7 Cell lines and virus Adeno-35 stock ...... 108
2.2.8 Bacterial Preparation ...... 108
2.2.9 Sample Selection ...... 108
2.2.10 Sample preparation ...... 109
2.2.11 Bacterial Cocktail ...... 109
2.2.12 Sample Inoculation ...... 109
2.2.13 Virus inoculation ...... 110
2.2.14 Disinfection Treatments ...... 110
2.2.15 Storage conditions ...... 112
2.2.16 Microbiological Analysis ...... 113
2.2.17 Bacteria Enumeration ...... 113
2.2.18 Analysis for Detection of Viruses ...... 114
2.2.19 Evaluation of disinfection with different initial bacteria cocktail ...... 118
Page 4 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.2.20 Culture Assay for HAdV35 ...... 119
2.3 Food Quality parameters ...... 120
2.3.1 Color Measurement ...... 120
2.3.2 Physicochemical Parameters ...... 121
2.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain ...... 125
2.4.1 Selection of critical points ...... 125
2.4.2 Decision Making Support System in lettuce’s safety using fuzzy cognitive maps...... 129
STATISTICS ...... 130
Chapter 3. RESULTS ...... 133
3.1 In Vitro Experiments with 3 Light Technologies ...... 135
3.2 Food Disinfection ...... 141
3.2.1 Bacteria Disinfection ...... 141
3.2.2 Adenovirus Disinfection...... 156
3.2.3 High and Low Initial Load Disinfection Treatments ...... 161
3.2.4 Storage Conditions ...... 164
3.3 Food Quality parameters ...... 169
3.3.1 Color ...... 169
3.3.2 Physicochemical Parameters ...... 185
3.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain ...... 195
3.5 Assessment of disinfection technologies based on infectivity doses ...... 202
Chapter 4. DISCUSSION ...... 204
4.1 In Vitro Experiments with 3 Light Technologies ...... 206
4.2 Food Disinfection ...... 209 Page 5 Table of Contents
4.2.1 Bacteria Disinfection ...... 210
4.2.2 Adenovirus Disinfection ...... 221
4.2.3 High and Low Initial Load Disinfection Treatments ...... 225
4.2.4 Storage Conditions ...... 226
4.3 Food Quality parameters ...... 229
4.3.1 Color ...... 229
4.3.2 Physicochemical Parameters ...... 231
4.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain ...... 238
4.5 Assessment of disinfection technologies based on infectivity doses ...... 242
Chapter 5. CONCLUSIONS AND FUTURE RECOMMENDATIONS ...... 246
5.1 Conclusions...... 246
5.2 Future Recommendations ...... 248
References ...... 252
APPENDIX ...... 302
Page 6 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Abbreviations
AA: Antioxidant Activity
ABTS: 2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid
ANOVA: Analysis of Variance
AW: Water Activity
CDC: Center for Disease Control and Prevention
CFU: Colony Forming Units
DPPH: 2,2-diphenyl-1-picryl-hydrazyl assay
EFSA: European Food Safety Authority
ELISA: Enzyme-linked Immunosorbent assay
EPR: Electronic Paramagnetic Resonance
FAO: Food and Agriculture Organization
FDA: Food and Drug Administration
Fe3+-TPTZ: ferric tripyridyltriazine
FRAP: Ferric Reducing Antioxidant Power
GA: Gallic Acid
GAP: Good Agriculture Practice
GHP: Good Hygienic Practice
GMP: Good Manufacturing Practice
HACCP: Hazard Analysis Critical Control Point
HAV: Hepatitis A Virus
HEV: Hepatitis E Virus
ID: Infective/Infectious Dose
ORAC: Oxygen-Radical Absorbance Capacity
RTE: Ready-To-Eat
SEs: Staphylococcal Enterotoxins
SSC: Soluble Solid Contents
Page 7 Table of Contents
TEAC: Trolox Equivalent Antioxidant Capacity
TPC: Total Phenolic Content
TP: Total Phenolics
TRAP: Total Radical-Trapping Antioxidant Parameter
TSST: toxic-shock syndrome toxin
USDA: United States Department of Agriculture
US: Ultrasound
UV: Ultraviolet
WHO: World Health Organization
Page 8 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki List of Figures
Chapter 1
Figure 1.1: Reported Foodborne Outbreaks in vegetables (on the left), and in fruits (on the right)………………………………...………………………..……………………..20
Figure 1.2.3.1 Oxidation of L-ascorbic to dehydro-L-ascorbic acid followed by evolution into products lacking biological activity…………………………………………………………...……………………...33
Figure 1.3.2.1:Ways of contamination of raw fruits and vegetables with pathogenic microorganisms………………………………..……………..………...……………….39
Figure 1.3.4.1 Number of multistate foodborne disease outbreaks, by year and pathogen — Foodborne Disease Outbreak Surveillance System, United States, 1998– 2008………………………………..…………..…………..……………………………43
Figure 1.3.5.1: E. coli on fresh ready to eat lettuces...... 46
Figure 1.3.5.3.1: Taxonomic scheme of Salmonella serovars………………………….49
Figure 1.3.6.3.1: Adenovirus…………………………………………………………....58
Figure 1.4.1.1: Schematic illustration of cell wall structures of microbial pathogens. (a) Gram-negative bacteria, (b) Gram-positive bacteria………………………..…….…….66
Figure 1.4.2.1: Effects of capsid of virus when different situations occur…….………..67
Figure 1.4.4.1.1: Electromagnetic Spectrum……….…………..……………………….74
Figure 1.4.4.1.2:Structure of DNA before and after absorbing a photon of UV light………………………...……………………………………………………………76
Figure 1.4.4.1.3: UV chamber……………………………………………..…………....76
Figure 1.4.4.2.1: Equipment of HILP...... 78
Figure 1.4.4.2.2: Internal Part of pulsed Light with a Data Logger……………...……..78
Figure 1.4.4.3.1: High intensity near ultraviolet/visible (NUV–vis) 395±5 nm light unit …………………………………………………………………………………………..79
Figure 1.4.4.4.1: Ultrasonic Cleaning Bath………………………………………..…....80
Figure 1.4.4.4.2: Ultrasonic Probe or horn…………………………………….………..80
Figure 1.4.4.4.3: Ultrasound cup-horn………………………………………………….80
Figure 1.4.4.6.1: Schematic Representation of PEF equipment……….………………..84
Figure 1.4.4.7.1: High pressure processing Unit……………………….……………….85
Page 9 Table of Contents
Figure 1.5.1.1: Cycle of the public health prevention (Tauxe, 2002)………...…………88
Figure 1.5.1.2: Surveillance pyramid………………………………………….….….....89
Chapter 2
Figure 2.1.5.1.1: NUV Vis Equipment……………………………………….………100
Figure 2.1.5.2.1: Layout of UV treatment unit I, housing for W lights; 2, safety interlock; 3, treatment chamber with dimensions (length, width, and height) of 790 by 390 by 345 mm; 4, UV lights (95 W) 500 mm in length………………………………..…..……101
Figure 2.1.5.2.2: Custom made UV equipment……………………………...……….101
Figure 2.1.5.3.1: HILP Unit……………………………………………..…………...102
Figure 2.2.14.3.1: Ultrasound equipment……………………………...………………112
Figure 2.2.18.4.1: Schematic Presentation of the procedure of Nucleic Acid Extraction……………………………………………………………….……………...117
Figure 2.4.1.1 Flow Chart of Lettuce/ Leafy Greens Production...... 128
Chapter 3
Figure 3.1.1: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 3cm (∆), 12cm (☐), 23 cm (○) and L. innocua placed at: 3cm (▲), 12cm (■) and 23cm (●) from the high intensity near ultraviolet/ visible (NUV–vis) 395±5 nm light source (Results expressed as mean log10 CFU/mL)……………………………………………………………………………....135
Figure 3.1.2: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 6.5 cm (∆), 17cm (☐), 28.5 cm (○) and L. innocua placed at: 6.5cm (▲), 17cm (■) and 28.5cm (●) from continuous UV light source (Results expressed as mean log10 CFU/mL)……………………………………………………………..…………136
Figure 3.1.3: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 2.5 cm (∆), 8cm (☐), 11.5 cm (○), 14 cm (◊) and L. innocua placed at: 2.5cm (▲), 8cm (■), 11.5cm (●) and 14cm (♦) from high Intensity pulsed light source (Results expressed as mean log10 CFU/mL)…………………………………...………….……136
Figure 3.1.4: Mean Log cfu/mL E. coli on MRD after treatment at the same dosages at shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High Intensity Light Pulses (■). ………………………………………...……….137
Figure 3.1.5: Mean log cfu/mL L. innocua on MRD after treatment at the same dosages at shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High Intensity Light Pulses (■)……………………………………...……………138
Figure 3.1.6: Mean Temperature increase (ΔΤ ᵒC) for NUV-Vis light technology at distances : 3cm (▲), 12cm (■) and 23cm (●)………………………………...……….139
Figure 3.1.7: Mean Temperature increase (ΔΤ ᵒC) for UV light technology at distances: 6.5cm (▲), 17cm (■) and 28.5cm (●)……………………………………….……...... 139 Page 10 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Figure 3.1.8: Mean Temperature increase (ΔΤ ᵒC) for HILP light technology at distances: 2.5cm (▲), 8cm (■), 11.5cm (●) and 14cm (♦)…………………………….139
Figure 3.2.1.1.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. enteritidis, L. innocua inoculated on fresh romaine lettuce……...…141
Figure 3.2.1.1.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. enteritidis, L. innocua inoculated on fresh romaine lettuce………..…………………..142
Figure 3.2.1.1.3: Disinfection Efficiency of combined alternative and conventional disinfection technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm) on E.coli, S.aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce…………………………………………………………….……..……143
Figure 3.2.1.1.4: Disinfection Efficiency of combined alternative disinfection technologies on E.coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce……………………………………………………………………….………….144
Figure 3.2.1.2.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries………..….146
Figure 3.2.1.2.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries……………………...…….147
Figure 3.2.1.2.3: Disinfection Efficiency of combined alternative and conventional technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm) on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries…………………………………………………………………...….148
Figure 3.2.1.2.4: Disinfection Efficiency of combined alternative technologies on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries…………..…..149
Figure 3.2.1.3.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes…….…150
Figure 3.2.1.3.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes………………….……..151
Figure 3.2.1.3.3: Disinfection Efficiency of combined alternative and conventional technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm) on E. coli, S. aureus, S. Enteritidis, L.innocua inoculated on fresh cherry tomatoes……………………………………………………...…………..……..153
Figure 3.2.1.3.4: Disinfection Efficiency of combined alternative technologies on E. coli, S. aureus, S. Enteritidis, L.innocua inoculated on fresh cherry tomatoes…………...154
Figure 3.2.2.1: Standard Curve based on the entire hexon region of Ad35 cloned into pBR322…………………………………………………………………………...……156
Figure 3.2.2.2: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and single step conventional Disinfection Treatments…………………………………………………………………..…………158
Page 11 Table of Contents
Figure 3.2.2.3: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and single step Alternative Disinfection Treatments…………………………………………………………….……………….159
Figure 3.2.2.4: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and combined Disinfection Treatments...... 160
Figure 3.2.2.5: The results of Real Time PCR were also evaluated with cell cultures observed under an Epifluorescence microscope…………………………….…………163
Figure 3.2.4.1: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on romaine lettuce before and after selected disinfection treatments during storage for 15 days at 6°C……………………...... 164
Figure 3.2.4.2: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on strawberries before and after selected disinfection treatments during storage for 15 days at 6°C…………………………………………………………………………………..166
Figure 3.2.4.3: Populations of E. coli, S. aureus, S. Enteritidis and L.innocua inoculated on cherry tomatoes before and after selected disinfection treatments during storage for 15 days at 6°C…………………………………………………………..……………168
Figure 3.3.2.1: TAC of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies…………………………………………….……..185
Figure 3.3.2.2: TPC of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies……………………………………………….…..186
Figure 3.3.2.3: AA of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies……………………………………….……..……187
Figure 3.3.2.4: TAC of Strawberries before and after conventional, alternative and combined disinfection technologies……………………………………………...……188
Figure 3.3.2.5: TPC of Strawberries before and after conventional, alternative and combined disinfection technologies………………………………………………...…189
Figure 3.3.2.6: AA of Strawberries before and after conventional, alternative and combined disinfection technologies……………………………………………...……190
Figure 3.3.2.7: TAC of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies…………………………………………….……..191
Figure 3.3.2.8: TPC of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies…………………………………...………………192
Figure 3.3.2.9: AA of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies……………………………….…………………..193
Figure 3.4.1. The FCM Model…………………………………………...... …………197
Figure 3.4.2: Fuzzy Cognitive Map………………………………………..…………..197
Figure 3.4.3: Subsequent values of concepts till convergence of 1st case……………199
Page 12 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Figure 3.4.4: Subsequent values of concepts till convergence of 2nd expert…….…....200
Figure 3.4.5: Subsequent values of concepts till convergence of 3rd expert…...... …..201
Page 13 Table of Contents
List of Graphs
Graph 1.3.5.3.1. Time trend of salmonellosis notification rate, Mandatory Notification System, Greece, 2004-2012.Hellenic Center for Disease Control and Prevention ……50
Graph 1.3.5.3.2. Annual notification rate (cases/100,000 population) of salmonellosis by age group, Mandatory Notification System, Greece, 2004- 2012…………………………………………………………………………………….50
Graph 1.3.5.4.1. Notification rate of listeriosis by age group and gender in Greece, Mandatory Notification System, 2004-2012…………………………………………...53
Page 14 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki List of Tables
Chapter 1
Table 1.1 Most Commonly Recognized Foodborne Pathogens……………….…..……17
Table 1.3.4.1: Number of reported foodborne diseases-outbreaks, cases and deaths in United States, 1998-2002………………………………………………………..……...43
Table 1.3.4.2: Number of multistate foodborne disease outbreaks, by year and pathogen- Foodborne Disease Outbreak Surveillance System, Greece, 2004- 2012……………………………………………………………………………….…….44
Table 1.3.5.1 : Five categories of the diarrheagenic E. coli…………………….………45
Table 1.3.5.3.1 Number of notified cases of salmonellosis per year, Mandatory Notification System, Greece, 2004-2012………………………………………...……..50
Table 1.3.5.4.1. Annual number of notified cases and notification rate of listeriosis in Greece, Mandatory………………………..……………………...……………….…….53
Table 1.3.6.1: Enteric viruses and clinical syndromes………………...………………..54
Table 1.4.4.1.1: Ranges, Wavelengths and Characteristics of different types of UV)………………………………………………………………………………….…..74
Table 1.4.4.4.1: Advantages-Disadvantages of electrochemical ultrasonic apparatuses………………………………………………………………………....…....80
Chapter 2
Table 2.1.5.3.1: Calculated exposure time (sec) of non-thermal light technologies at selected distances from the light source.(NUV-Vis: NUV–vis light; UV: Ultraviolet Light; HILP: High Intensity Light Pulses, *Samples that are not analyzed due to high temperature, NT: Not tested samples)……………………..……………….…………102
Table 2.2.14.1.1: Sodium Hypochlorite Treatments…………………………………..110
Table 2.2.14.2.1: UV treatments……………………………………….………….…..111
Table 2.2.14.3.1: Various ultrasound treatments…………………………….……..….111
Table 2.2.14.4.1: Combined Treatments……………………………………....…...….112
Table 2.2.16.1: ISO Methods…………………………………………………….…….113
Table 2.2.18.5.1: Working solutions of primers and probe……………..……………..117
Table 2.2.18.5.2: Volumes of reagents for PCR mix…………...……………………..118
Page 15 Table of Contents
Chapter 3
Table 3.2.3.1: High and Low Inocula on lettuce and disinfection with selected treatments………………………………………………………..……………………..161
Table 3.2.3.2: High and Low Inocula on strawberries and disinfection with selected treatments……………………………………………………………………………....161
Table 3.2.3.3: High and Low Inocula on cherry tomatoes and disinfection with selected treatments ………………………………………………………………………..…....162
Table 3.3.1: Values are average ± standard deviation of at least three experiments and represent the color parameters of romaine lettuce after each processing time with each disinfection method: NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm, UV+US………………………………………………………….……………………..173
Table 3.3.2: Values are average ± standard deviation of at least three experiments and represent the color parameters of strawberries after each processing time with each disinfection method : NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm, UV+US…………………………………………………………………………….…..178
Table 3.3.3: Values are average ± standard deviation of at least three experiments and represent the color parameters of cherry tomatoes after each processing time with each disinfection method: NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm, UV+US…………………………………………………………..…………………….183
Table 3.4.1: Evaluation of three experts, where W: Weak, M: Medium, S: Strong, VS: Very Strong………………………………………………………………….………....196
Table 3.5.1: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in lettuce. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***)…………………………..……….202
Table 3.5.2: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in strawberries. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***)………………………..203
Table 3.5.3: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in cherry tomatoes. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***)…………………...... …203
Page 16 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki INTRODUCTION
The market of fresh produce is increasing constantly and this can be attributed to the consumers’ tendency for healthy and convenient foods, due to the fact that positive effects to human health have been attributed to consumption of fresh produce (Gilbert, 2000, Ragaert et al., 2004). Organizations such as the World Health Organization (WHO), Food and Agriculture Organization (FAO), United States Department of Agriculture (USDA), and European Food Safety Authority (EFSA) recommended an increase of consumption of fresh ready-to-eat (RTE) fruit and vegetables as they are correlated with a decrease in the risk of cardiovascular diseases and cancer (Allende et al., 2006). Moreover, the Mediterranean diet is followed by many people nowadays, due to its positive effects on health. Many epidemiological studies suggest that food involved in Mediterranean diet may be linked to a reduction in coronary heart disease risk. There is also evidence that the antioxidants present in selected foods, improve cholesterol regulation and LDL cholesterol reduction, and moreover anti-inflammatory and anti- hypertensive effects are correlated with Mediterranean diet (Covas, 2007).
RTE produce is defined as washed, bite-size, and packaged fresh fruit and vegetables, which allow consumers to eat healthy on the run and to save time on food preparation. In fact, the availability of fresh-cut fruits in automated vending machines existing in public places constitutes an excellent strategy to improve the nutritional quality of snacks and convenience of foods in a time when obesity and nutrition-related illnesses affect large percentages of the population (Olivas and Barbosa-Cánovas, 2005).
Microbiological safety of foods and foodborne illness are complex issues since there are more than 200 known diseases that are transmitted through foods. Primary causative agents of foodborne illness are bacteria, viruses, parasites and molds (table 1.1).
Bacteria Viruses Parasites Molds Listeria Monocytogenes Norovirus Giardia lamblia Aspergillus spp. Salmonella spp. Rotavirus Cryptosporidium parvum Penicillium spp. Campylobacter spp Astrovirus Toxoplasma gondii Fusarium spp. Escherichia coli O157:H7 Hepatitis A virus Cyclospora cayetanensis Staphylococcus aureus Trichinella spiralis Clostridium perfrigens
Table 1.1 Most Commonly Recognized Foodborne Pathogens (Parikh, 2007)
Page 17 Introduction
In United States, the annual patient-related costs of the principal bacterial and parasitic foodborne infections have been estimated at $6.5 billion or more (Buzby and Roberts, 1996, Tauxe, 2002). Tauxe (2002) reported that among the established foodborne infections, bacterial infections accounted for an estimated 30% of cases, viral infections count for 67% and parasitic infections for 3%. Hospitalizations are attributed to bacteria (60%), viruses (35%) and parasites (5%). Finally, bacteria accounted for 72%, viruses for 7% and parasite for 21% of deaths respectively. It is reported that five foodborne pathogens – E. coli O157:H7, Salmonella, Campylobacter, Listeria, and Toxoplasma – together cause an estimated 3.5 million cases, 33,000 hospitalizations and 1600 deaths each year in United States (Tauxe, 2002).
The symptoms of foodborne illnesses range from mild gastroenteritis to life threatening neurologic, hepatic, and renal syndromes. Center for Disease Control and Prevention (CDC) has estimated approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the U.S. each year, out of which approximately 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths were due to known foodborne pathogens. Moreover, CDC recognized Salmonella, Listeria monocytogenes, and Toxoplasma gondii as leading causes of death since they were responsible for 1,500 deaths each year. For instance, more than 75% of deaths were caused by known pathogens however they accounted for only approximately 11% of total cases of foodborne illness (Mead et al., 1999).
In the environment, bacteria and viruses can be found in contaminated animal or water sources. Animal droppings may be used to fertilize crops while a contaminated water source may be used to irrigate or wash plants (Solomon et al., 2003). Contamination can also be initiated by infected servers (Berrang et al., 2008).
Microorganisms have been shown to enter fruits and vegetables through various pathways such as through the stomata, stem, stem scar, or calyx (Zhuang et al., 1995, Seo and Frank, 1999). They can enter physically damaged fruits and vegetables through punctures, wounds, cuts, and splits during maturation, harvesting, or processing. Bacterial soft rot of fruits and vegetables can also increase the likelihood of contamination with pathogens (Zhuang et al., 1995).
Page 18 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Chapter 1: Literature Review
1.1 Fresh produce and Mediterranean diet
The Mediterranean diet is a modern nutritional pattern inspired by the traditional dietary patterns of Greece, Spain, Portugal and Southern Italy. The Mediterranean diet pyramid is based on food patterns typical of Crete, much of the rest of Greece, and southern Italy in the early 1960s, where adult life expectancy was among the highest in the world and rates of coronary heart disease, certain cancers, and other diet-related chronic diseases were among the lowest (Willett et al., 1995). The principal ingredients of this diet are based on high consumption of olive oil, legumes, cereals, fruits, and vegetables, moderate to high consumption of fish, dairy products (mostly cheese and yogurt), moderate wine consumption, and low consumption of meat and meat products (Noah and Truswell, 2001). Thus, lettuce and tomatoes are important ingredients of a balanced every day diet, as well as strawberries, which are selected among other fruits and vegetables, by those who adhere to Mediterranean diet and are interested in following a healthy lifestyle.
Flavonoids from vegetable and fruits intake appear to be inversely related to coronary heart disease (CHD) mortality (Hertog et al., 1995, Rimm et al., 1996). Furthermore, catechin, a naturally occurring flavonoid, has been linked to the prevention of human plasma oxidation and to inhibition of oxidation of low-density lipoprotein (Lotito and Fraga, 1998). Thus, flavonoids as well as other antioxidants are responsible for the protective effects of the Mediterranean diet, rich in vegetable, fruit and wine against CHD (Evans et al., 1995, Mangiapane et al., 1992). Moreover, Mediterranean diet has also been linked with reduced obesity. The potential role of fiber-rich diets, and of components other than fiber but included in the same foods (fruits and vegetables) such as antioxidants are known to exert the important effect on weight regulation (Park et al., 2005). Thus, Mediterranean diet is recommended for people who face problems with obesity (Bes-Rastrollo et al., 2006).
However, contamination of fresh fruits and vegetables is of special concern, because they are consumed raw, without any type of microbiologically lethal processing, thus are prone to a number of pathogens. Ramos et al. (2013) has extensively studied the outbreaks that have been reported for fresh produce (figure 1.1).
Page 19 Literature Review
Figure 1.1: Reported Foodborne Outbreaks in vegetables (on the left), and in fruits (on the right) (Ramos et al., 2013)
The majority of pathogens implicated in produce-related outbreaks are transmitted via the fecal-oral route (Johnston et al., 2006). Microbial contamination of fruits and vegetables can occur during plant growth, harvesting, transport, processing, distribution and marketing, or during processing at home (Beuchat, 1998). Produce contaminated with pathogens cannot be completely disinfected by washing or rinsing the product in an aqueous solution (Rodgers and Ryser, 2004). The prevention of produce contamination with human pathogens is the only practical and effective means of ensuring safe produce for human consumption. Therefore a growing need for effective sanitizer treatments to reduce the number of microbial pathogens on produce to safer levels is raised.
1.1.1 Lettuce
Lettuce is one of the most consumed vegetable worldwide with a global production of about 24 million tons in 2011 (FAOSTAT, 2011). Mean daily consumption of lettuce in Europe is 22.5 g, which represents about 6.5% of the total dietary intake of vegetables (WHO, 2003). In Greece 84000 tons of lettuce was produced in 2008, in a cultivation area of 5500 hectares (EL.STAAT, 2014). In Peloponnesus half of the above production occurs (around 37000 tons) (EL.STAAT, 2014).
Lettuce (Lactuca sativa L.) is one of the most popular vegetable for human consumption, especially in salad, and is considered as a good source of health-promoting compounds such as phenolics, vitamin C, folates, carotenoids and chlorophylls (Nicolle et al., 2004). It contains several macrominerals (e.g. K, Na, Ca and Mg) and trace elements (e.g. Fe, Mn, Cu, Zn and Se) which are essential for human nutrition (Kawashima and Soares,
Page 20 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2003). It is also known as a good source of photosynthetic pigments (chlorophylls and carotenoids) and other phytochemicals that benefit nutrition and have a significant role in the prevention of several oxidative stress-related diseases (Llorach et al., 2008).
Romaine lettuce is consumers’ favorite leafy vegetable for its crispness, good aroma, tender appearance and high concentration in many health-promoting compounds (Llorach, et al., 2008). Lettuce leaves consists of vascular and photosynthetic tissues (Toole et al., 2000), where a thick white mid-rib (vascular tissue) constitutes the majority of the leaf (photosynthetic tissue). The leaves are tightly wrapped and interlocked providing a crispy texture to lettuce (Toole et al., 2000). Toole et al. (2000), exhibited that photosynthetic and vascular tissues possess different phenolic metabolism as well as textural characteristics. Vascular tissue has lower polyphenol oxidase, peroxidase and phenylamonium lyase activity than photosynthetic tissue (Fukumoto et al., 2002). However the potential for the development of “browning” is higher in vascular tissues, especially in the outer than the inner leaves probably due to the fact that vascular tissues have a higher total volume and cut area (Fukumoto et al., 2002).
Lettuce can be found as whole-heads and as fresh-cut lettuce. Indeed, in the fresh-cut industry, fresh-cut lettuce is one of the most important products. However, the physical damage or wounding caused during preparation, increases the rate of biochemical reactions responsible for changes in visual quality (color, texture and browning) and phytochemicals (vitamin C content and phenolic content) (Saltveit, 2003).
In the specie Lactuca sativa there are seven main groups of cultivars: butterhead lettuce, iceberg or crisphead lettuce, romaine/cos lettuce, cutting lettuce, stalk lettuce, latin lettuce and oilseed lettuce. Among them, romaine lettuce is the most popular minimally processed leafy vegetable. Its consumption has increased dramatically in recent years due to the fact that consumers need more convenient and less time consuming products, which also seem fresh and possess healthy characteristics (Ragaert et al., 2004, Rico et al., 2007).
Lettuce head has been classified as a commodity with moderate respiration rate. However, cutting lettuce for minimally processing enhances its respiration rate (Kader and Saltveit, 2003). Respiration rate has been associated with the perishability of the product. Therefore it is assumed that higher respiration rate reduces the shelf-life of lettuce. It has been demonstrated that the increase in respiration rate is higher in
Page 21 Literature Review transverse than longitudinal cut sections. In addition, other methods of preparation such as shredding lettuce increases the respiration rate in comparison to cutting lettuce with a sharp knife or tearing by hand due to less damage that is caused to the tissue (Kader and Saltveit, 2003, Lopez et al., 2014).
1.1.2 Strawberries
Greece has increased its exports in strawberries, as an increasing demand all over the worlds exists. The largest percentage of strawberries is exported to Germany, Italy, Hungary, Romania, Bulgaria, Russia, Serbia (EL.STAAT, 2014). In 2013, 34.118.653 kg of strawberries produced in Greece, were exported (EL.STAAT, 2014). Moreover, the strawberry is an attractive fruit, with benefits to human health, due to its high content of vitamin C, anthocyanins and flavonols, and high antioxidant activity (Odriozola- Serrano et al., 2010, Tiwari et al., 2009, Alexandre et al., 2012).
The garden strawberry (Fragaria × ananassa) comes from the genus Fragaria (collectively known as the strawberries) belonging to the family of Rosaceae. It is cultivated worldwide for its fruit. The fruit has a characteristic aroma, bright red color, juicy texture, and sweetness. It is consumed in large quantities, either fresh or prepared in foods as fruit juice, pies, ice creams, milkshakes, and chocolates. Strawberry is rich in soluble sugars such as glucose, fructose and sucrose. Organic acids and soluble pectins also contribute to soluble solid content of strawberry (Pelayo-Zaldívar et al., 2005). The Soluble Solid Contents (SSC) continually increase in the fruit during its development but the main changes occur between 21 and 28 days after fruit set, during fruit ripening period (Montero et al., 1996). SSC is used as an indicator for fruit taste. However, the relative proportions of sugar components such as glucose, fructose and sucrose may influence the perception of sweetness.
Compared to other fruits, berries possess high antioxidant capacity and are rich in a variety of phytochemicals, such as phenolic compounds (Häkkinen et al., 1999, Koponen et al., 2007). Furthermore, strawberries are extremely rich in vitamin C (60- 100 mg/100 g FW) and in anthocyanins, especially pelargonidin-3-glucoside (pg-3-gluc) and cyanindin-3-glucoside (cya-3-gluc). However, variability in concentration of the fruit components is often observed. Therefore, strawberry is considered as an important dietary source of health promoting compounds (Koponen et al., 2007).
Page 22 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Numerous epidemiological studies have shown that consumption of fruits and vegetables has a protective effect against degenerative diseases (Hannum, 2004). Oxygen radicals which are produced with ageing of the fruit can react with lipids, protein and DNA. The antioxidants which exist in fruits and vegetables are able to maintain low cellular levels of oxygen radicals by preventing their formation, scavenging them or promoting their decomposition (Hancock et al., 2007).
However, strawberries are highly susceptible to mechanical injury, physiological disorders, fungal activity and water loss (Romanazzi et al., 2013). The storage period and the shelf life of strawberries are very short due to perishability and susceptibility to rot-causing pathogens (Aday, 2014). The most severe postharvest diseases of strawberries are gray mould (Botrytis cinerea Pers.ex.Fr.) and Rhizopus rot (Rhizopus stolonifer Ehrenb.Fr.Vuill.) which cause severe losses during storage and transport from cold store to the market (Vardar and Ilhan, 2012).
1.1.3 Tomatoes
Tomato (Solanum Lycopersicum) is an herbaceous fruiting plant and is consumed worldwide with an annual production mass of more than 140 million tons (Faostat, 2009). Greece is selected among the 14 processing countries which represent 92% of global production. Greece total exports in tomatoes were around 20.000.000 kg in year 2013 (EL.STAAT, 2014). In Greece tomatoes are grown from Autumn until Spring and Summer in colder climates. Tomato has become one of the most widely grown vegetables with the ability to survive in diverse environmental conditions. Moreover, tomato grows better in fertile, well-drained soils, with pH 6 and ambient temperatures of 25°C (Rice et al., 1987).
Tomato fruit is a good source of vitamins (vitamin A, vitamin C, folate and other trace vitamins) and minerals (potassium, calcium, phosphorus, iron), also containing a fibre and a very small amount of protein and fat. Tomato is also a good source of antioxidants, such as lycopene. Lycopene and fibres are beneficial to health when consumed in a diet (Canene-Adams et al., 2005). According to researches, lycopene has anti-inflammatory, antimutagenic and anticarcinogenic properties (Boon et al., 2010). Moreover, lycopene is also known for reducing the risk of adenoma, and promoting immune system functionality (Kun et al., 2006). The aforementioned health benefits are related to the singlet oxygen and free radical scavenging properties of lycopene (Canene-Adams et al.,
Page 23 Literature Review
2005). It is recommended, 6-15 mg lycopene intake for improved health (Kun et al., 2006). Soluble fibre modulates blood glucose and cholesterol levels (Weickert and Pfeifer, 2008). Insoluble fibre promotes laxation and helps against many cancers such as the cancer of colon (Alvarado et al., 2001). In tomato products, vitamin C and polyphenols are reported to be the major antioxidant hydrophilic components, whereas vitamin E and carotenoids mainly constitute the hydrophobic fraction (Hsu, 2008).
Cherry tomato is a very small variety of tomato. Cherry tomatoes range in size from a thumbtip up to the size of a golf ball, and can range from being spherical to slightly oblong in shape. The oblong cherry tomatoes often share characteristics with plum tomatoes, and are known as grape tomatoes. The berry tomato is regarded as a botanical variety of the cultivated berry Solanum lycopersicum var. cerasiforme (Raffo et al., 2006).
In Mediterranean countries, cherry tomatoes are largely used for fresh consumption, thus their commercial importance is continuously increasing (Leonardi et al., 2000a). Cherry tomatoes are grown in unheated greenhouses, which have no climate control systems and are covered with plastic film. As a consequence, the development and the ripening of cherry tomatoes happen under varying climatic conditions. It has been reported that cherry tomato plants grown in greenhouse under high light, exhibited an approximately two-fold greater soluble phenols content (rutin and chlorogenic acid) than low-light plants (Raffo et al., 2006). The temperature and the light intensity are factors that influence the quality attributes of tomato, such as appearance, firmness, texture, dry matter and sensory properties. Furthermore, environmental factors can also affect the antioxidant content of tomatoes (Dumas et al., 2003, Lee and Kader, 2000).
Page 24 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.2. Nutritional, Quality and Health Aspects of fresh ready-to- eat (RTE) fruits and vegetables
The nutritional quality and health-beneficial properties of plant products have been studied by many research groups because of the growing consumer’s interest in recent years in including healthy foods in their everyday diet. Fruits and vegetables are rich in ingredients such as phytosterols, vitamins, minerals, fibers, water, isoflavones, lycopene, etc. (Hasler et al., 2004). Eating fruits and vegetables also reduces blood pressure, boosts the immune system, detoxifies contaminants and pollutants, and reduces inflammation in the human body (Wang and Lin, 2000). Thus, researchers support the recommendation of consuming five or more servings of fruits and vegetables daily (Crecente-Campo et al., 2012, Hung et al., 2004). In fact, the nutritional profile of many fruits and vegetables is influenced by many factors, such as cultural practices, preharvest conditions (climate, temperature), maturity, post-harvest handling and food processing (Rekika et al., 2005, Wang et al., 2002).
Many epidemiological studies have shown the inverse relationship between the consumption of fresh RTE fruits and vegetables and the incidence of chronic diseases such as cardiovascular diseases and certain types of cancer (Maynard et al., 2003, Temple and Gladwin, 2003, Trichopoulou et al., 2003). The above effect is attributed to bioactive or phytochemical compounds that are present in the foods and are involved in certain biological actions, resulting in health beneficial effects (Prior and Cao, 2000). Therefore, the consumption of fruits and vegetables includes not only nutrients essential to life (carbohydrates, proteins, fats, vitamins, etc.), but also phytochemical or bioactive compounds (carotenoids, phenolics, vitamins A, C, and E, fiber, glucosinolates, organosulfur compounds, sesquiterpenic lactones, etc.) (Duthie et al., 2000, Eastwood and Morris, 1992, Knekt et al., 2000, Lampe and Peterson, 2002, Ling and Jones, 1995, Piironen et al., 2000, Plaza et al., 2006a, 2006b, Sanchez-Moreno et al., 2006a, 2006b, Simon et al., 2001, Skimola and Smith, 2000).
Other researchers also emphasize that the health benefits of lettuce have been attributed to the presence of phenolic compounds, fiber and vitamin C content (Nicolle et al., 2004). A regular intake of antioxidant compounds from lettuce is useful to improve the lipid status and to prevent lipid peroxidation in tissues. Generally, differences in chlorophyll and anthocyanin concentrations, as well as in phenolics and antioxidant
Page 25 Literature Review properties in the lettuce leaves have been observed in different lettuce cultivars that range in color (green and yellow to deep red) (Ozgen and Sekerci, 2011). Generally red lettuce has been found to have a higher content in total phenolics (TP) and in total antioxidant capacity (TAC) than green lettuce (Llorach et al., 2008). Studies have shown that, the cultivar, the planting date and the growing conditions may alter the phenolic content and antioxidant capacity of lettuce (Liu et al., 2007). Moreover, it is known that phytochemicals and antioxidant capacity in lettuce may differ from outer to inner leaves. Thus, consumers tend to eat from middle part of lettuce head towards to inner since these parts look fresher, crispier and tender without knowing the real phytonutrient contents of these parts (Ozgen and Sekerci, 2011).
Strawberries are a rich source of antioxidants and a common and important fruit consumed in the Mediterranean diet because of their high content in essential nutrients and beneficial phytochemicals, and also due to healthy substances that are observed in human health (Giampieri et al., 2012). These phytochemicals are essentially flavonoids (mainly anthocyanins, with flavonols providing a minor contribution), hydrolysable tannins (ellagitannins and gallotannins) phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), and also condensed tannins (proanthocyanidins) (Cerezo et al., 2010). The color expression of strawberry fruits is associated with concentration and composition of anthocyanins. Anthocyanins in strawberries are the major known polyphenolic compounds. Pelargonidin-3-glucoside is the main anthocyanin in strawberries, whereas cyanidin-3-glucoside exists in smaller proportions. However, the composition of strawberries varies according to genotype (Tulipani et al., 2008). Research that has been conducted with strawberry extracts, exhibited a high level of antioxidant capacity against free radical species (Li et al., 2014). Thus, strawberries may have an effective role in decreasing the risk of cancer and in preventing various human diseases caused by oxidative stress. On the other hand, strawberries are highly susceptible to infection by pathogens and spoil rapidly after harvesting. Thus, low temperature storage is effective in order to reduce decay and maintain the overall acceptable quality of strawberries. However, there is strong evidence that the levels of anthocyanin and aroma compounds in strawberries remain low at lower temperatures than at higher temperature storage conditions (Jin et al., 2011).
Tomatoes (Solanum lycopersicum L.) commonly used in the Greek diet, are a major source of antioxidants and contribute to the daily intake of a significant amount of these molecules. In fact, tomato fruit is rich in diverse antioxidant molecules, such as Page 26 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki carotenoids (especially lycopene), phenolics, flavonoids, vitamin C, vitamin E and tocopherols (Mitchell et al., 2007). Lycopene (the red pigment of tomato), phenolics and flavonoids have received great interest during the last few years because of their antioxidant properties in relation to free radicals, suggesting protective roles in reducing risk of chronic diseases, such as cancer and cardiovascular disease (Rice-Evans et al., 1996). It is known that the skin of some tomato fruits contains significantly higher levels of phenolics, flavonoids, lycopene, ascorbic acid and antioxidant activity than pulp and seed fractions (Toor and Savage, 2005). These results led researchers to propose the peel enrichment of tomato-based products as a means of increasing the nutritional value of tomato pastes and enhancing carotenoids intake (Reboul et al., 2005). The tomato skin is not only a valuable source of nutrients, but also acts as a protective organ by helping to preserve the nutritional value of the remaining parts of the fruit. It maintains the physical integrity of tomato and prevents flesh deterioration, by avoiding a direct contact with air and, thus, preventing both dehydration and oxidation of sensitive chemical compounds. Capanoglu et al. (2010) have showed that the direct contact of the tomato pulp with oxygen can have detrimental effects to ascorbic acid, lycopene and phenolic content and this is why nitrogen-conditioned packaging for tomato derivatives is used. Moreover, the skin also prevents the direct incidence of light on the pulp, which has been linked to the deterioration of bioactive compounds (Lee and Chen, 2002). Finally, tomato seeds are edible and rich in bioactive compounds and minerals (Toor and Savage, 2005), nevertheless they are usually discarded specially in the preparation of tomato derivatives. Recently, studies have shown that consumption of the natural gel found in tomato seeds can help to maintain a healthy blood circulation by preventing blood from clotting (Vinja et al., 2014).
1.2.1 Antioxidant Compounds
An antioxidant compound delays or inhibits oxidation of an oxidizable substrate (lipids, proteins, and DNA). Among the most important antioxidants present in foods are vitamins C and E, carotenoids, and phenolic compounds (Martin-Belloso and Soliva- Fortuny, 2011).
Augmented antioxidant activity (measured using the ABTS•+ or DPPH radical scavenging methods) has been reported in tissues of various vegetables in response to stress caused by mechanical damage (peeling and cutting). This has been associated with an increase or decrease in concentrations of phenolic compounds more than vitamin C Page 27 Literature Review concentration (Reyes et al., 2007). Phenolic compounds confer antioxidant capacity (Proteggente et al., 2002), and as a consequence this capacity is one of the major reasons why increased consumption of fruits and vegetables has been recommended as beneficial to health (Prior and Cao, 2000).
Many methods (FRAP, DPPH, TEAC, TRAP, ORAC, EPR) are available for analyzing antioxidant activity, with different concepts, mechanisms of action, ways of expressing results, and applications (Huang et al., 2005, Frankel and Meyer, 2000).
1.2.1.1 Determination of Antioxidants
1.2.1.1.1 FRAP Method (Ferric Reducing Antioxidant Power)
This method is based on the reducing ability of an iron complex Fe+3-TPTZ (2,4,6-Tri(2- Pirydil)-s-triazine) which is colorless, to a blue colored ferrous iron when antioxidants- and phenolic compounds are present. This conversion is owned to the transfer of an electron from the antioxidant complex. Frap values are obtained by comparing the absorbance change at wavelength of 593 nm in test reaction mixtures with those containing ferrous ions of known concentration. The bigger the difference of absorbance is, the greater the capacity of the antioxidant to reduce TPTZ-Fe+3 to TPTZ-Fe+2 is. The results are expressed in μmol Fe+2/l and the absorbance is measured with a UV-Vis spectrophotometer (Benzie and Strain, 1996).
The scheme that follows represents the above reaction that takes place:
Antioxidant compound
TPTZ-Fe+3 (colorless) Fe+2-TPTZ
T=37°C, pH=3.6
The measurement of the difference of absorbance indicates the antioxidant capacity of the sample.
1.2.1.1.2 DPPH Method (2,2-diphenyl-1-picryl-hydrazyl assay)
This method is based on the measurement of capacity of antioxidant compounds, such as phenolic compounds, to bind to the stable radical DPPH. This radical is converted to hydrazine which reacts with compounds such as phenolic compounds that can give one
Page 28 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki hydrogen atom from its molecule. The solutions’ reaction takes place in the presence of DPPH solution for a period of time. The absorbance is then measured at 515-528 nm. The reduction of absorbance of the DPPH solution is correlated with color alteration, which indicates the enhanced capacity of DPPH, thus the enhanced capacity of being bound from the antioxidant compound. This “effectivity” can be calculated:
%DPPH radical scavening = ((Αbs initial – Αbs sample)/Αbs initial) * 100, where ABS initial is the DPPH absorbance, ABS sample is the absorbance of both DPPH and of the sample after a certain period of time.
The higher the reduction of the absorbance gets, the bigger the antioxidant capacity of a sample is, binding a larger amount of DPPH radicals. The DPPH method is useful, offers repeatability, and is mainly used for the TAC determination in food and drink extracts (Sánhez-Moreno, 2002).
1.2.1.1.3 ΤΕΑC Method (Trolox Equivalent Antioxidant Capacity)
TEAC or ABTS method is based on the antioxidant’s inhibition in the absorbance of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) which shows characteristic absorption at 734 nm. ABTS+ is a green-blue chromophor, which is reduced to ABTS, when antioxidants are present. The stable solution ABTS+ is based on the reaction of aqua solution ABTS with potassium persulfate solution. Then the mix must remain in dark conditions for 12-16 h. The initial method is based on the activation of metmyoglobin with Η2Ο2 through the creation of ferrymyoglobin, which after oxidize ABTS to form ABTS+.
In this method the forming ABTS+ are mixed with the sample (which contains polyphenols) and the inhibition’s percent of absorbance is measured at 734 nm, which determines the quantity of polyphenols. Trolox is used as a standard, and this name has been attributed to the method TEAC. The results are stated as “equivalents Trolox”, which are defined as the solution concentration Trolox (mmol/l), with a dynamic antioxidant equivalent in 1mmol/l of sample’s solution. This method has been used not only in vitro but also in vivo (Sánhez-Moreno, 2002).
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1.2.1.1.4 TRAP Method (Total Radical-Trapping Antioxidant Parameter)
This method was first introduced by Wayner et al. (1985) in order to determine the antioxidant level of human’s plasma. R-phycoerythrin (R-PE) is a phycobiliprotein of red color. A correlation exists between the antioxidant activity and the damage of fluorescence of R-PE. This method is based on the protection that antioxidants offer to the damage of fluorescence of R-PE (lag-phase) during controlled peroxidase reaction. The kinetic reaction takes place in 38οC and is recorded during 1h with a spectrophotometer. TRAP values (values in μΜ), are calculated from the length of lag phase and are expressed as molecules Trolox, which have the same antioxidant capacity in 1l of plasma (Sánhez-Moreno, 2002).
1.2.1.1.5 ORAC Method (Oxygen-Radical Absorbance Capacity)
This method is commonly used for the determination of antioxidant capacity in plasma and tissues. It is based on the initial experiment that was carried out by Delange and Glazer (1989) and it is based on unique properties of phycoerythrin, which is used as a target of free radicals. According to this method, TAC is determined through the calculation of area under curve, until the moment of flection of fluorescence of phycoerythrin curve, in the presence of antioxidants. For the creation of hydroxyl radicals, radical AAPH is used. ORAC combines the inhibition time and the inhibition rate of free radicals from antioxidants by using area under curve technique. In other words, the calculation of the area, is used in order to have a quantitative analysis. The results are expressed as ORAC units or Trolox equivalents. (Sánhez-Moreno, 2002). This method shows a sensitivity against hydrogen transfer, whereas FRAP against electron transfer and TEAC against both (Aguilar-Garcia et al., 2007).
1.2.1.1.6 ΕPR Method (Electronic paramagnetic resonance)
This is a spectroscopic method which is used for the tracing of free radicals and the determination of antioxidant capacity in vitro, in fruits, vegetables, olive oil, juices and other plant foods. This method is based on microwave irradiation and tracing of free radicals with niter-oxides directly, or indirectly when resonance happens. Free radicals are traced after determination of height and breadth of spectrum vertices, and from this area, quantification of these radicals occurs. Antioxidant capacity of these foods is calculated through the use of standard radical TEMPOL and the method is standardized using Trolox (Papadimitriou et al., 2005).
Page 30 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.2.2 Phenolic Compounds
Phenolic compounds are secondary metabolites produced by vegetables, which possess a benzene ring in their chemical structure with hydroxyl groups that are responsible for their activity. The polyphenols present a wide variety of structures including simple molecules (monomers and dimers), and polymers (tannins). Abundant are hydrocinnamic acids (C6-C3), benzoic acids (C6-C1), flavonoids (C6-C3-C6), proanthocyanidins (C6-C3-C6)n, stilbenes (C6-C2-C6), lignanes (C6-C3-C3-C6), and lignines (C6-C3)n (Scalbert et al., 2005). Phenolics have been widely studied for their relationship with the quality characteristics of plant foods such as color, because many of them are pigments (such as anthocyanins) responsible for the color of grapes, cherries, plums, strawberries, raspberries, etc. Moreover, phenolic compounds when oxidized cause enzymatic browning, which is responsible for a large percentage of loss of quality in plant foods during processing and storage. They are also widely known for their contribution to the flavor and aroma of plant foods. Flavones, flavonols, flavanols, flavanones, anthocyanins, and isoflavones are common in fruits and vegetables. The basic characteristic of most existing flavonoids in fruits and vegetables is the antioxidant and radical scavenger activity (Rice-Evans et al., 1996).
It is well known that the intake of foods rich in phenolics, and most particularly in flavonoids, are correlated with a low incidence of cardiovascular diseases and some types of cancer (Duthie et al., 2000, Skibola and Smith, 2000). The more widely studied foods include tea, spices, and grape derivatives, and also some fruits such as apples and berries (strawberries, raspberries, blackberries, etc.). Furthermore, flavonoids are known for their anti-inflammatory, antiallergic, antiviral, hypocholesterolemic, and anticarcinogenic activities (Knekt et al., 2000, Skibola and Smith, 2000). Maas and Galleta, (1991) found that red grapes, blackberries, raspberries, and strawberries are rich in hydroxybenzoic acids, particularly in ellagic acid that has been shown to have a protective effect against cancer. 1.2.2.1 Determination of TPC (total phenolic content)
Several analytical methods have been proposed for the determination of flavonoids. Due to the importance of phenolics in prevention of a multitude of diseases, a series of in vitro methods have been developed in order to be able to detect phenolics accurately.
Page 31 Literature Review
1.2.2.1.1 Spectrophotometric Method (Folin-Ciocalteau)
With this method the quantitative determination of total phenolics takes place. An Ultraviolet-visible (UV-vis) spectrophotometer is used and is based on a colorimetric oxidation/reduction reaction. The oxidizing agent is Folin-Ciocalteau reagent. The Folin–Ciocalteu phenol reagent is used to obtain a crude estimate of the amount of phenolic compounds present in an extract. Phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids present in the reagent to phosphotungstic/phosphomolybdic phenolic complex, which has blue color and exists in alkaline conditions.
Phenolics + alkaline + FC reagent blue colored product, Abs 765 nm
With this quantitative determination, total phenolics can be calculated through a gallic acid standard curve, as it is not always feasible to find every single phenolic compound separately. The results are expressed as mg gallic acid/g of food weight (Skerget et al., 2004, Sokmen et al., 2005).
1.2.2.1.2 Standard curve of catechin
This method is based on the creation of standard solutions of catechin of different concentrations. Then, a standard curve of catechin is prepared, which shows the concentration of catechin in correlation with absorbance. After that, the absorbance of the sample is determined and from the standard curve of catechin, the concentration of total phenolics in mg catechin/g is finally calculated. For the determination of total phenolics in different plant extracts, vegetables and fruits, the standard solutions of catechin are prepared in distilled water or in the same extract of plant (Kivits et al., 1997).
1.2.2.1.3 Terbium sensitized fluorescence Method
This method is based on the fluorescence sensitization of terbium (Tb3+) by complexation with flavonols (quercetin as a reference standard) (at pH 7.0), which fluoresces intensely with an emission maximum at 545 nm. Quercetin and terbium cations (at pH 7.0) form a stable complex and the resulted emission at 545 nm can be used for the determination of the total phenols concentration expressed in terms of “quercetin equivalent”. This fluorimetric method is very simple, precise, rapid and more sensitive than the other methods that have been used for the determination of total Page 32 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki phenols (Benzie and Strain, 1996). The developed method was easily applied to the determination of total phenols in tea infusions, tomato and apple juice with excellent reproducibility (Shaghaghi et al., 2008).
1.2.3 Ascorbic Acid
Vitamin C belongs to the group of water-soluble vitamins and is one of the most important micronutrients, 90% of which derive from the intake of fruit and vegetables. Structurally, vitamin C is composed of chenodiol conjugated with the carbonyl group in the lactone ring. In the presence of oxygen, ascorbic acid is oxidized to dehydroascorbic acid. The latter has the same vitamin activity but is more unstable, so that the activity can readily be lost through lactone hydrolysis and formation of 2,3-diketogulonic acid (figure 1.2.3.1). Numerous epidemiological studies have shown a strong correlation between the health effects of consuming fruit and vegetables and their content in vitamin C. Vitamin C (ascorbic acid, ascorbate) is an effective antioxidant against free radicals. Vitamin C (ascorbic acid-AA + dehydroascorbic acid-DAA) content of fruit and vegetables depends on the species, cultivar, climatic conditions, agricultural practices, ripeness, and of course postharvest handling (Lee and Kader, 2000).
The vitamin C concentration increases in vegetable tissues through the action of light during growth of the plant. On the other hand, the principal cause of vitamin C degradation in vegetables is storage at high or inappropriate postharvest temperatures.
Vitamin C content starts to decline as soon as the product is harvested. Levels depend significantly on the type of vegetable and the processing and storage conditions. The amount of vitamin C that is degraded increases with storage temperature and time (Davey et al., 2000).
Figure 1.2.3.1: Oxidation of L-ascorbic to dehydro-L-ascorbic acid followed by evolution into products lacking biological activity (Martin-Belloso and Soliva-Fortuny, 2011).
Page 33 Literature Review
1.2.3.1 Determination of ascorbic acid
Various methods have been employed for the analysis of ascorbic acid in food, including electrochemical (Calokerinos and Hadjiioannou, 1983), spectrophotometric (Liu et al., 1982), spectrofluorimetric (Sánchez-Mata et al., 2000) and chromatographic methods.
1.2.3.1.1 HPLC method
High-performance liquid chromatography (HPLC) is a very efficient method in ascorbic acid analysis of fruits, vegetables or beverages (Quirós et al., 2009) and it also has some advantages, such as the increase of selectivity, sensitivity and the elimination of interferences, specificity, easy operation, high accuracy, good repeatability and reproducibility, a relative short analysis time, unambiguous identification of AA and isoAA (Valente et al., 2014). Reversed-phase, bonded-phase NH2, ion-exchange or ion- pair reversed columns have been the most commonly employed columns for ascorbic acid analysis. Regarding the way of detection, AA can be easily detected by UV at wavelengths between 245 nm and 254 nm. Although, UV detectors are usually included in HPLC systems and are simpler and faster than others, UV-HPLC methods have been validated to be used for vitamin C determination in foods. Most of these methods have been validated in beer, wine and fruit beverages. Reliability has been satisfactory for all the evaluated UV-HPLC methods, reducing agents and fruits. In every case, suitable linearity, sensitivity, precision and accuracy through recovery for AA and vitamin C analysis in strawberries, tomatoes and apples were obtained (Odriozola-Serrano et al., 2007).
1.2.3.1.2 2,6 Dichlorophenol-indophenol titration method
Of the many methods that have been proposed for the direct chemical determination of ascorbic acid in plants and animal tissues, those based upon the reduction of 2,6 dichlorophenol indophenol have been adopted. The indophenol method depends upon the fact, that ascorbic acid is the major or only natural tissue component which reduces the dye rapidly in an acid solution (e.g. pH 2 to 4). It is essential that four major precautions should be observed if accurate results are to be obtained in the titration against 2,6-Dichlorophenolindophenol method. These are: (1) representative sampling, (2) complete extraction, (3) prevention of oxidation, (4) rapid performance of the titration itself (Kallner, 1986).
Page 34 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.2.3.1.3 Analytical Voltammetry
Analytical voltammetry is an electrochemical method in which the changes of electrolysis current are measured when a gradually increasing voltage is applied to the cell. Conditions are adjusted so that the analyte is oxidized or reduced selectively at one of the electrodes in the cell. Voltammetric analysis using different electrodes (conventional electrodes, microdisc electrodes, microband and multiple microband electrodes and carbon paste electrodes). A wax-impregnated graphite electrode (WIGE) is used as the indicator electrode. During the electrolysis, ascorbic acid donates electrons to the indicator electrode and the voltammetric experiment exhibits an oxidation (anodic) current step. The quantity of electricity involved in the oxidation peak is directly proportional to the concentration of ascorbic acid. Therefore, an unknown concentration of ascorbic acid can be determined (Arya et al., 2000).
1.2.3.1.4 Bromatometric method
The determination of ascorbic acid is also performed by a direct bromatometric method.
During titration, KBrO3 standard solution is added drop wise to the acidic solution of vitamin C and KBr and the liberated bromine reacts quantitatively. Methyl orange is used to indicate the end point. Because it has a higher standard redox potential than that of L-ascorbic acid the liberated bromine first oxidizes the L-ascorbic acid and when the end point of the titration is reached, the first drop of excess amount of bromate forms elementary bromine which oxidizes the indicator (decolorization), too (Arya et al., 2000).
1.2.3.1.5 Oxidation-reduction reaction
Furthermore, ascorbic acid can be determined in food by using an oxidation-reduction reaction. The redox reaction is preferable to an acid-base titration because a number of other species in juice can act as acids, but relatively few interfere with the oxidation of ascorbic acid by iodine. The solubility of iodine is increased by complexation with iodide to form triiodide. Triiodide then oxidizes ascorbic acid to dehydroascorbic acid. The endpoint is indicated by the reaction of iodine with starch suspension, which produces a blue-black product. As long as ascorbic acid is present, the triiodide is quickly converted to iodide ion, and no blue-black iodine-starch product is observed. However, when all the ascorbic acid has been oxidized, the excess triiodide (in
Page 35 Literature Review equilibrium with iodine) reacts with starch to form the expected blue-black color (Arya et al., 2000).
1.2.4 Quality Aspects of Foods
1.2.4.1 External Quality-Color
External quality is the most important and direct sensory quality attribute of food products. In general terms, the external quality of fruits and vegetables is evaluated by considering their color, texture, size, shape, and the visual defects (Costa et al., 2011). The outer appearance of fruits and vegetables affects their point-of-sale value and consumers’ buying behavior, and sometimes the defective, infected and contaminated fruits and vegetables can spread the infection or contamination to the sound products even the whole batch, thus causing great economic losses, and safety problems (Gomez- Sanchis et al., 2008). Visual observation can give information about the color of different foodstuffs. The limitations of visual observation may be overcome by the instrumental evaluation of color and color differences according to the system of color measurement established by the Commission International d' Eclairage (CIE) (Clydesdale, 1978). This system is based on the reflectance spectrophotometric instrumentation (Clydesdale, 1978, Hunter and Harold, 1987). In this CIE system surface color of fruits and vegetables can be easily measured and may be regarded in a three-dimensional space in which each color has a unique location. As a consequence, colors are measured in terms of their fundamental tristimulus values (X, Y and Z). These values, can be further used to calculate L*, a* and b* (CIE-Lab) color space values (Hunter and Harold, 1987). L* indicates lightness, a* indicates hue on a green (-) to red (+) axis, and b* indicates hue on a blue (-) to yellow (+) axis (Clydesdale, 1978). Moreover, CIE-Lab space measures two important functions: (a) the hue angle, H°= (tan b/a)-l, which is recommended by the CIE as the psychometric correlation of the visually perceived attribute of hue. (b) the total color difference, which can be calculated as ΔE =(ΔL2+ Δa2+ Δb2) 1/2, which expresses a color difference between two colors by a single function number (Hunter and Harold, 1987).
The above measurements of total color difference have been found to be quantitative, repeatable and reproducible. The above instrumental system has been employed to assess colors in fruits and in the food industry (Yang et al., 1990). A hand-held
Page 36 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki tristimulus reflectance colorimeter (Minolta CR-200) can be used, in order to measure the color on different foodstuffs, after its calibration with a white standard tile (L =97.92, a =-0.45, b=2.12).
1.3 Foodborne Pathogens and Foodborne diseases
1.3.1 Global challenge and increased frequency of foodborne diseases.
The occurrence and epidemiology of foodborne diseases in a population is the result of complex interactions among environmental, cultural and socioeconomic factors (Johnston et al., 2006).
Growing international trade in food industries, migration and travel are factors that accelerate the spread of dangerous pathogens and contaminants in food products thus increasing our universal vulnerability (Tauxe et al., 2010).
It must be mentioned that through globalization of food distribution, the health of people in different countries can be affected, by consuming contaminated food. Once the food is contaminated, a recall of literally tons of food products is inevitable. This can lead to considerable economic losses in production, as well as damage to tourist industry development at a country level. It must be emphasized that many global foodborne diseases have resulted from foods that are produced in developed countries. For example, the global spread of Salmonella serotype Enteritidis (now the most important Salmonella serotype in many countries) and potentially also the spread of multiply antibiotic-resistant strains of Salmonella serotype Typhimurium, has initially started from developed countries (Hendriksen et al., 2011).
Furthermore, changing food consumption trends (e.g. eating more meals away from home, including greater use of salad bars), increased global trade in raw fruits and vegetables, as well as increased international travel in general, could also increase the risk of produce-associated foodborne diseases (Stine et al., 2005, Beuchat, 1998, Beuchat et al., 2001). Finally, the susceptibility of the public to foodborne diseases, is changing due to increased numbers of people who are elderly, immunocompromised or suffer from chronic diseases. This change in social demographics is likely to lead to
Page 37 Literature Review increased risk of illness associated with the consumption of raw produce that otherwise may contain levels of pathogens innocuous to healthy individuals (Beuchat, 1998).
The current need for a healthier way of life has led to an increasing demand for fresh RTE foods, without additives but with high nutritional value, included antioxidants and free radicals. The last decades an increasing number of industries produce packaged foods, which can be consumed easily not only at house but also at work. Taking also into account their accessible-low price, minimally processed fresh RTE fruits and vegetables offer big advantages to consumers (Artés et al., 2009).
While the prevention of contamination of fruits and vegetables during production, transport, processing and handling, still remains an important issue, much improvement is needed in some parts of the world, in order to ensure hygienic production of fruits and vegetables. Furthermore, many microbial contaminants are present in the nature and are part of the environment, thus fruits and vegetables may be inadvertently contaminated (Beuchat, 2001).
The high quality of minimally processed foods is mainly related to 5 main characteristics: color, texture, flavour, nutritional value and finally food safety. However, it must be emphasized that food safety constitutes the most critical point (Artés et al., 2009).
The steps required to prepare fresh cut produce can lead to a rapid increase of microorganisms, some of which may be potentially harmful to human health. Although RTE foods are processed minimally, the destroyed plant structure and thus the increase of the aging rate of plant tissues can lead to an increase of plant resistance and to microbial spoilage (Artés-Hernández et al., 2007).
Moreover, the shelf-life of freshly cut products is influenced by several factors. These are: pre-processing factors (crop varieties, cultivation, harvesting and ripening stage), processing factors (chilling, cutting, cleaning, conditioning, cutting, peeling, decoring, washing, disinfection, drainage, irrigation, drying, packaging) and distribution conditions (temperature, relative humidity) (Artes, 2004).
In addition, during the production of safe fresh produces, the materials that enter the processing chain (processing and packaging treatments) result in higher microbial load. The suppression of microbial growth during processing, as well as the prevention of
Page 38 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki infection after processing, must be taken seriously into account (Artés and Allende, 2005).
In developing countries, the use of untreated wastewater and manure as fertilizers for the production of fruits and vegetables are important factors, which contribute to food contamination that causes numerous foodborne disease outbreaks (Hedberg et al., 1994). The increased application of improperly composted manures to soils in which fruits and vegetables are grown, changes in packaging technology such as the use of modified or controlled atmosphere and vacuum packaging, extended time between harvesting and consumption, also play an important role (Beuchat et al., 2002). The use of properly composted manure and properly treated irrigation and spray waters, as well as safe and pathogen-free water for washing, can minimize the risk of contamination of fruits and vegetables with microbial pathogens. Moreover, good hygienic practice during production and transport, everyday sanitizing of harvesting equipment and transport vehicles, as well as the application of good hygienic practice during processing and preparation are important factors that must be taken into account (Beuchat, 2001).
1.3.2 Hazards during the food production chain Prevention of contamination of fruits and vegetables with pathogenic microorganisms should be the goal of everyone involved in both the pre-harvest and post-harvest phases of delivering produce to the consumer. However, this seems a very difficult task, since some pathogens normally exist in the soil and may therefore be present on the surface of fruits and vegetables when they are harvested (figure 1.3.2.1) (Beuchat, 1996). Extremely important is the elimination of animals and insects from processing, storage, marketing and food-service facilities. The highest level of hygiene must be maintained and practiced by all handlers (including consumers) of fresh RTE foods, from the field to the table.
Figure 1.3.2.1: Ways of contamination of raw fruits and vegetables with pathogenic microorganisms (Beuchat, 1996).
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Agricultural systems are rich in microorganisms (soil inhabitants) that are observed on whole fruit or vegetable surfaces and are responsible for maintaining a dynamic ecological balance. The dissemination of these microbes is facilitated by vectors, like soil particles, airborne spores, and irrigation water (figure 1.3.2.1). In most cases, bacteria and fungi that arrive on the developing crop plant are responsible for crop damage (Andrews and Harris, 2000). The microorganisms normally present on the surface of raw fruits and vegetables may consist of chance contaminants from the soil or dust of the environment, or bacteria or fungi that have grown and colonized by utilizing nutrients that exist in the surface of plant tissues. Among the groups of bacteria that are commonly found on fruits and vegetables are coliforms or faecal coliforms e.g. Klebsiella and Enterobacter, Salmonella, Listeria monocytogenes, S. aureus, C. botulinum, Yersinia enterocolitica, Campylobacter, Bacillus cereus (Beuchat, 1996).
1.3.3 Microbial colonization on fresh produce surfaces
Generally, microorganisms contaminate the surfaces of fresh produce mostly via water or soil. Each type of microbe has its own method of attachment and can vary even within different strains of the same bacteria (Katsikogianni and Missirlis, 2004, Rivas et al., 2005, Takeuchi and Frank, 2000). The factors that are responsible for an effective attachment of bacterial cells to a surface are: bacterial hydrophobicity, surface charge, cell surface structures (flagella, pili, curli), outer membrane proteins, and bacterial growth conditions (Van Houdt and Michiels, 2005). The produce surface exhibits hydrophobicity, due to epicuticular wax of produces (Koch et al., 2008). Thus the water has a reduced access to the produce, due to the hydrophobicity (Wang et al., 2009). Surface composition, surface charge, and surface free energy of plant also play a role in bacteria adhesion. Enteric pathogens prefer to grow or survive, in special distinct and localized spots on plant surfaces which are rich in nutrients like sucrose, amino acids and nitrates (Jaeger et al., 1999). The viability of microorganisms also is dependent on changes in temperature and osmotic conditions within the same day, and on poor nutrition (Heaton and Jones, 2008). There seems to be an optimal adhesion temperature for each microorganism (Gorski et al., 2003). Finally, different bacteria have exhibited different attachment to different fresh produce surfaces (cut edge and stomata versus intact tissue) or plants (Barak et al., 2008, Seo and Frank, 1999).
A large number of fruits and vegetables present nearly ideal conditions for the survival and growth of many types of microorganisms. Their internal tissues are rich in nutrients Page 40 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki and vegetables and have a neutral pH. Their structure is comprised mainly of polysaccharides, such as cellulose, hemicellulose, and pectin. Microorganisms have the ability to exploit the host using extracellular lytic enzymes that degrade these polymers to release water and the plant’s other intracellular constituents for use as nutrients for their growth. Moreover, microbes are capable of colonizing and creating lesions on healthy, undamaged plant tissues, they also can enter plant tissues during fruit development, either through the calyx (flower end) or along the stem, or through various specialized water and gas exchange structures of leafy matter (Tournas, 2005).
Moreover, in order to achieve a successful establishment to fruits and vegetables, microbes need to overcome multiple natural protective barriers. Fruits and vegetables possess an outer protective epidermis, which is covered by a natural waxy cuticle layer containing the polymercutin. The microorganisms firstly identify and recognize the plant surface, secondly employ strategies to achieve irreversible attachment to the plant surface, and finally internalize and colonize to the tissue. It must be mentioned that external damage such as bruising, cracks, and punctures facilitates the establishment and growth conditions of the microbes to fresh produce. Thus, microorganisms will arrive within open wound sites at the packing facility, and through shedding from the asymptomatic wound, cross-contamination during different treatments (handling, culling, washing, sorting, and packing before storage) occurs (Barth et al., 2009).
Food surface topology plays a major role in how and where bacteria attach to fresh produce. Studies show bacteria to prefer to attach to cuts in the leaves, lenticels, trichomes, locations around veins on leaves, and stomata in plants (Burnett et al., 2000, Kroupitski et al., 2009, Seo and Frank, 1999).
In general terms, rough, highly-textured surfaces with deep crevices can easier harbour soil, thus an increased number of microorganisms can be hosted. On the other hand, smooth surfaces of fruits and vegetables can host a smaller amount of microorganisms. The presence of a pathogen is minimized when the rind, skin or peel is removed before consumption. Furthermore, microorganisms may have become trapped on the inner complicated surfaces of leaves of certain vegetables, thus they can not be removed by routine cleaning practice (Barth et al., 2009).
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1.3.4 Foodborne illness and foodborne disease outbreaks
Foodborne disease can be defined as any infectious disease or toxic nature disease caused by consumption of food. The number of foodborne disease outbreaks, by year and pathogen is recorded by Foodborne Disease Outbreak Surveillance System (table 1.3.4.1, table 1.3.4.2, figure 1.3.4.1) Foodborne disease outbreak can be defined as: a) the disease that is observed, when the number of cases of a particular disease exceeds the expected number and as b) the occurrence of two or more cases of a similar foodborne disease, which results from the ingestion of a common food. Sporadic case can be defined as the case that cannot be linked epidemiologically to other cases of the same illness (WHO, 2008). Foodborne diseases outbreaks can be further divided to common- source outbreaks, propagated outbreaks or mixed outbreaks (HCDCP, 2012).
Foods are implicated for transmitting infections through foodborne infections and foodborne poisoning. Foodborne infections such as cholera, typhoid fever, dysentery are transferred through food. Food plays the key role of the first step in the chain of transmission of infection. In foodborne poisoning, the pathogen is usually multiplied inside the food, and this step is necessary in order to reach the adequate infective dose and/or to produce toxin. Surveys of minimally-processed fruit and vegetables have demonstrated that fresh-cut produce could harbor high counts of bacteria and also foodborne pathogens such as Salmonella, L. monocytogenes, Aeromonas hydrophila and E. coli O157:H7 (table 1.3.4.1) (Abadias et al., 2008, Beuchat, 1996).
S. aureus is related to staphylococcal food poisoning by ingestion of preformed toxins generated in food (Fueyo et al., 2001). The incubation time of staphylococcal infection may be only 30-min while salmonellosis needs an incubation time of 15-48 hours depending on the infecting dose. Microorganisms that cause food poisoning are different from those that cause food spoilage. Foods that contain pathogens have pleasant organoleptic properties, compared to foods that contain spoilage microorganisms which have unpleasant taste, smell and appearance without necessarily being hazardous to health.
To minimize the risk of infection or intoxication associated with the consumption of raw fruits and vegetables, potential sources of contamination from the environment to the table should be identified and specific measures and interventions to prevent and/or
Page 42 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki minimize the risk of contamination should be considered and correctly implemented (Beuchat, 1998).
Table 1.3.4.1: Number of reported foodborne diseases-outbreaks, cases and deaths in United States, 1998-2002 (CDC, 2013).
Figure 1.3.4.1: Number of multistate foodborne disease outbreaks, by year and pathogen - Foodborne Disease Outbreak Surveillance System, United States, 1998–2008 (CDC, 2013).
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DISEASE 2004 2005 2006 2007 2008 2009 2010 2011 2012
Salmonellosis (non- 1327 1062 886 709 810 406 299 471 405 typhic-paratyphic)
Hepatitis A 52 160 120 282 119 89 58 41 74
Sighellosis 62 26 28 48 19 38 33 47 91
Typhic-Paratyphic 20 20 16 18 11 4 10 7 6 Fever
Listeriosis 3 8 7 10 1 4 10 9 11
Infection by EHEC 2 0 1 1 0 0 1 1 0 (STEC/VTEC)*
Allantiasis 0 0 0 0 0 1 0 0 0
Table 1.3.4.2: Number of multistate foodborne disease outbreaks, by year and pathogen - Foodborne Disease Outbreak Surveillance System, Greece, 2004–2012 (HDCP, 2012).
1.3.5 Foodborne Bacteria
1.3.5.1 Escherichia coli
Escherichia coli are Gram-negative, non-spore-forming, facultatively anaerobic rods bacteria of family Enterobacteriaceae. They are typically mesophilic and grow from 7- 10°C up to 50°C (optimum 37°C). The minimum aw for growth is 0.95 and pH 4.4-8.5 (WHO, 2008). Several different pathotypes of pathogenic E. coli have been categorized based on their virulence properties and disease-causing mechanisms (table 1.3.5.1) (Kaper et al., 2004).
Page 44 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Table 1.3.5.1: Five categories of the diarrheagenic E. coli. (Zhou, 2010).
Escherichia coli are classified as a genetically diverse species with the majority of its members being nonpathogenic and part of the natural gut microflora of humans and animals. Fruits and vegetables can be contaminated with E. coli in the field or during post-harvest handling. E. coli O157:H7 is a strain of enterohemorrhagic E. coli. The growth of this strain in the human intestine produces a large quantity of toxins that can cause severe damage to the lining of the intestine and other organs of the body. Many outbreaks have been linked to lettuce (Ackers et al., 1996), apple cider (CDC 1996a), radish sprouts, (Nathan, 1997) and alfalfa sprouts (CDC, 1997). Enterohemorrhagic E. coli can grow on fruits and vegetables like cantaloupe and watermelon cubes, shredded lettuce, sliced cucumbers and apple cider (Abdul-Raouf et al., 1993), causing health problems. An outbreak was linked to contaminated spinach in which 200 people were affected, more than half of which were hospitalized, and three died (Anonymous, 2006). The world’s largest reported E. coli O157:H7 outbreak occurred in Japan in 1996 and was linked to the consumption of white radish sprouts, where approximately 6,000 school children were infected and 17 died (Michino et al., 1999). In 1997, white radish sprouts were once again implicated in another E. coli O157:H7 outbreak in Japan, affecting 126 people and resulting in one death (Taormina et al., 1999).
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Figure 1.3.5.1: E. coli on fresh ready to eat lettuces (Bermúdez-Aguirre and Barbosa-Cánovas, 2013).
Enterohemorrhagic E. coli (EHEC) was first recognized as a human pathogen in 1982 when it was identified as the cause of two outbreaks of hemorrhagic colitis. Those outbreaks were associated with undercooked hamburgers served at fast food restaurants (Smith, 2005). E. coli O157:H7 produces toxins that cause a mild non-bloody diarrhea or an acute grossly bloody diarrhea which is known as hemorrhagic colitis (Doyle, 1991). In patients such as children and elderly people, E. coli O157:H7 infection can progress to hemolytic uremic syndrome (HUS), which is a severe postdiarrheal systemic complication. The toxins of E. coli have been referred to as shiga-like toxins (verotoxin, verocytotoxin). E. coli O157:H7 has been isolated from undercooked ground beef and from other products including fresh RTE fruits and vegetables (Griffin and Tauxe, 1991, Smith et al., 2003). However, it must be stated that outbreaks related with fresh produce are obvious in restaurants which are accounted for 40% of the E. coli O157:H7 outbreaks associated with fresh produce, and approximately 47% was responsible for cross-contamination during food preparation (Rangel et al., 2005). For instance, Stine et al., (2005) found that the survival of E. coli O157:H7 was enhanced under high humidity conditions. E. coli O157:H7 was found to survive for 12 days at 4°C on lettuce (figure 1.3.5.1), bean sprouts and dry coleslaw (Francis and O'Beirne, 2002). E. coli O157:H7 inoculated on lettuce increased significantly when it was kept at 12°C for 14 days. Whereas, modified atmosphere packaging had little or no effect on the survival or growth of E. coli O157:H7 (Abdul-Raouf et al., 1993).
Page 46 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Enterohaemorrhagic E. coli infection is the rarest stated foodborne illness notifiable in Greece. The declared average annual incidence of infection with EHEC for the period 2004-2011 was 0.06 / 1,000,000 people per year (total declared 6 cases), whereas the average declared suspicious cases in countries of both the European Union and EEA / EFTA (European Economic Area / European Free Trade Association), was consistent with the latest published data, 6.60 cases per 1,000,000 inhabitants. Serotype O157: H7 has been implicated in the largest percentage epidemics worldwide, however different serotypes have emerged, such as the recent large outbreak in Germany in May 2011 with the responsible serotype O104:H4 (HCDCP, 2012).
1.3.5.2 Staphylococcus aureus
S. aureus is a principal cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of staphylococcal enterotoxins prior existed in the food (Trudeau, 2012). Lettuce, parsley, radish, salad vegetables and seed sprouts are among the RTE foods that have been reported for Staphylococcus outbreaks (Abadias et al., 2012, FAO/WHO, 2008, Olaimat and Holley, 2012, Ramos et al., 2013).
S. aureus is a facultative anaerobic Gram-positive coccal bacterium, also known as "golden staph" and Oro staphira. Optimal temperature for Staphylococcus growth is within the range of 30 - 37°C. Moreover, growth can be observed at 7 - 48°C and at pH values between 4.0 and 9.8 (Vilhelmsson, 2000). S. aureus produces a wide variety of extracellular proteins that may play an important role in its virulence as a human pathogen. These include hemolysins, nucleases, proteases, lipases, hyaluronidase, collagenase, leukocidins, exfoliative toxins, and pyrogenic toxin superantigens which include toxic shock syndrome toxin-1 (TSST-1), epidermolysins and the staphylococcal enterotoxins (Vilhelmsson, 2000).
S. aureus is a major human pathogen and is potentially able to infect any tissue of the human body, causing from skin infections to life-threatening diseases. Methicillin resistant S. aureus (MRSA) has been a topic of concern for several years, being a large burden for most healthcare institutions around the world, with higher mortality, morbidity and financial costs compared to methicillin-susceptible S. aureus (MSSA) (Gould, 2006). The MRSA rates have been increased rapidly worldwide during the last decades (Stefani et al., 2012).
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Staphylococcal food poisoning is reported as the third most prevalent cause of foodborne illness worldwide (Normanno et al., 2005, Zhang et al., 1998). There are two major aggravations to its presence: the toxins production and the antimicrobial resistance. S. aureus produces more than 30 different extracellular byproducts and staphylococcal toxins like: pyrogenic toxin superantigens (PTSAgs), exfoliative toxins, leukocidins and other toxins. The family of PTSAgs includes staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, exfoliative toxins and toxic shock syndrome toxin-1 (TSST-1) (Lina et al.,
2004). Generally, five classical antigenic SE types (SEA, SEB, SEC, SED, and SEE) are known. Recently, new types of SEs toxins (SEG, SEH, SEI, SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElR, SElU, SElU2, and SElV) have been also reported (Thomas et al., 2006, Peles, 2007). SEs are the main cause of food poisoning that occur after ingestion of foods contaminated with S. aureus by improper handling and subsequent storage at elevated temperatures. Human handling of food products as well as infection/colonization of livestock or farm workers have been described as mechanisms for the contamination of food with S. aureus (Greig et al., 2007). S. aureus can contaminate food by direct contact through body, through skin fragments, or through respiratory droplets produced when people cough and sneeze. Most S. aureus foodborne illness results from food contamination by food handlers, meat grinders, knives, storage containers, and cutting blocks. While low levels of the S. aureus bacterium exist in many foods, proper food handling techniques can prevent further contamination or growth in the food, thus preventing toxin production. Illness is caused by enterotoxin-producing S. aureus. About 100 - 200 ng of the enterotoxin are adequate to produce illness, and because the toxins are heat-resistant, cooking is not capable of maintaining the food safe (Anderson et al., 1996).
Symptoms are of rapid onset and include nausea and violent vomiting, with or without diarrhea, and abdominal pain within 1-6 h post-consumption of contaminated foods. The illness is usually self-limiting and only occasionally is severe enough to lead to hospitalization (Argudín et al., 2010).
Foodborne outbreaks attributed to S. aureus include a variety of foods such as meat, milk, and cheese (Asao et al., 2003, Guven et al., 2010, Rall et al., 2008), dairy products (Normanno et al., 2007) and RTE foods (Oh et al., 2007) .
Page 48 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.3.5.3 Salmonella spp.
The genus Salmonella belongs to the family of Enterobacteriaceae. Salmonella are Gram negative, facultative anaerobe bacteria that diverged from E. coli approx. 100 – 160 million years ago and acquired the ability to invade host cells (Müller, 2012). Based on DNA relatedness, Salmonella is today divided into the two species S. enterica and S. bongori (figure 1.3.5.3.1). Major clinical symptoms associated with humans are enteric (typhoid) fever caused by S. enterica serovar Typhi and Paratyphi, and gastroenteritis caused by non typhoidal Salmonellae (NTS). Salmonella may be divided in three groups based on their association with human and animal hosts (Armon et al., 1997, Chavant et al., 2007). The first group is characterized by specificity for the human host. The second group consists of organisms that are usually adapted to specific animal hosts. The third group consists of un-adapted Salmonellae that cause disease in humans and a variety of animals. Most Salmonellae are included into the third group with Salmonella enterica serovar Typhimurium (S. Typhimurium) being the most common. There are over 2,463 recognized serovars of Salmonella (Brenner et al., 2000) with Typhimurium and Enteritidis being the most prevalent nontyphoidal Salmonella serovars isolated from human salmonellosis cases in the U.S.A (Andrews-Polymenis et al., 2009).
Figure 1.3.5.3.1: Taxonomic scheme of Salmonella serovars (Müller, 2012).
Outbreaks of salmonellosis have been linked to a wide variety of fresh fruits and vegetables including apple, cantaloupe, alfalfa sprout, mango, lettuce, unpasteurized orange juice, tomato, melons, celery and parsley, bean sprouts (Krause et al., 2001, Mahon et al., 1997). Two outbreaks caused by S. Typhimurium in 2006 and 2008 in USA were linked to contaminated tomatoes and peanut butter, respectively, led to
Page 49 Literature Review enormous economic loss and 714 confirmed cases of salmonellosis according to CDC (CDC, 2013).
The average annual incidence of disease of Salmonelosis declared in Greece, based on the mandatory declaration system diseases (HCDCP), for the years 2004-2012, was 6.6 cases per 100,000 population (table 1.3.5.3.1, graph 1.3.5.3.1). A higher incidence is observed in children, especially in the age group 0-4 years (graph 1.3.5.3.2) (HCDCP, 2012).
Table 1.3.5.3.1: Number of notified cases of Graph 1.3.5.3.1: Time trend of salmonellosis salmonellosis per year, Mandatory notification rate, Mandatory Notification Notification System, Greece, 2004-2012. System, Greece, 2004-2012.Hellenic Center (http://www.keelpno.gr - 7/5/2013 ) for Disease Control and Prevention (HCDCP, 2012)
Graph 1.3.5.3.2: Annual notification rate (cases/100,000 population) of salmonellosis by age group, Mandatory Notification System, Greece, 2004-2012 (HCDCP, 2012).
The routes of transmission of Salmonella to humans are the environment and the contact with animals or the person-to-person contact but in industrialized countries, Salmonella are mostly transmitted through contaminated food (Müller, 2012). Common food products that enhance the Salmonella transmission are fresh meat and eggs (Wegener et Page 50 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki al., 2003). Salmonella in the food can directly originate from the farm reservoirs. However, Salmonella can enter at any stage of the food chain through contamination from the environment, other foods, animals or humans. The endpoint of the food chain is the kitchen of the consumers, restaurants or canteens. The main risk factors for Salmonella outbreaks have been associated to undercooking, improper storage and cross-contamination. Cross-contamination is one of the most important factors and needs special attention, in order to eliminate the spread of the bacteria thus protecting the contamination of RTE foods (Müller, 2012).
1.3.5.4 Listeria spp.
Listeria monocytogenes is a short, Gram-positive, non-sporeforming rod, with tumbling end-over-end motility at room temperature. It is a catalase positive, oxidase negative, facultative anaerobe with slight β–hemolysis on blood agar. L. monocytogenes has been known to survive refrigeration, freezing, heating, and drying, which creates obstacles for the food industry (CFSAN, 2001). It has optimum growth at 32-35°C, but can survive and multiply at refrigeration temperatures. It has been found in raw foods, such as fruits, vegetables, and uncooked meats, and has been associated with outbreaks in raw milk, ice cream, raw meats, and RTE meat and cheese products.
The genus Listeria, together with the genus Brochotrix, belongs to the Listeriaceae family, the order Bacillales, the class Bacilli and the phylum Firmicute (Wheeler et al., 2000). Today the genus comprises the following seven species: L. monocytogenes, L. innocua, L. ivanovii subsp. ivanovii and, L. ivanovii subsp. londoniensis, L. seeligeri, L. welshimeri,and L. grayi. Recently described specie is L. marthii (Graves et al., 2009).
L. monocytogenes has been linked to infection due to consumption of contaminated prepackaged salads, cabbage, lettuce, celery, and tomatoes (Berrang et al., 1989). L. monocytogenes is a major public health concern due to its high mortality rate ( 20%) and its ability to grow at refrigeration temperatures (Gandhi and Chikindas, 2007,∼ Mead et al., 1999). Food safety is one of the top eleven priorities of the World Health Organization, which has called for systematic and more aggressive steps to reduce the risk of foodborne diseases due to microbial contamination (WHO, 2000).
L. monocytogenes is responsible for approximately 2,500 illnesses and 500 deaths in the United States each year (CDC, 2000). Most healthy adults have few or no symptoms, as the disease generally affects those with compromised immune systems. In at-risk Page 51 Literature Review populations, flu-like symptoms including fever, headache, nausea, vomiting, and diarrhea appear about 12 hours or more after ingestion. After several days, the more serious symptoms appear, including meningitis, encephalitis, septicemia, and intrauterine/cervical infections that may result in spontaneous abortion in pregnant women (CFSAN, 2001). Generally, mortality rates for listeriosis may be as high as 80% for neonatal infections, and 50-70% for meningitis and septicemia patients (CFSAN, 2001). The infective dose of L. monocytogenes is currently unknown, although it appears to be above 100 viable cells, depending on pathogen strain and susceptibility of the host (Roberts, 1994). While pasteurization and cooking methods used by processors can kill L. monocytogenes, post-processing contamination may occur because the organism is so resilient in the environment.
Listeria innocua is a Gram-positive bacterial strain closely related to L. monocytogenes. It is non-sporeforming short rod, catalase positive, oxidase negative, and facultatively anaerobic. However, L. innocua does not produce β-hemolysis on blood agar (Poysky et al., 1993). This characteristic has been one of the few tests available for the differentiation of Listeria species. The apparent difference between L.innocua and L. monocytogenes is the lack of pathogenicity of L. innocua. There have been over 1000 worldwide cases of human foodborne illness associated with L. monocytogenes in the last 35 years (CFSAN, 2001). On the other hand, no cases of human illness associated with L. innocua have been reported until recently. However, the presence of L. innocua indicates the potential for L. monocytogenes contamination. Thus, L. innocua strain is used to model the behavior of L. monocytogenes. The ubiquitous nature of L. monocytogenes makes it nearly impossible to completely eradicate from an environment, thus many researchers and processors are understandably hesitant to introduce a pathogen into their working environment for fear of future contamination problems. (Benech et al., 2002, Dykes et al., 2003, Olasupo et al., 2004, Scannel et al., 2001, Sommers et al., 2002).
Listeriosis is the foodborne disease caused by L. monocytogenes. The case fatality rate of listeriosis is high compared to other foodborne diseases. It mainly affects pregnant women, newborns, the elderly and immunocompromised adults (HCDCP, 2012). In total, 64 cases of listeriosis were reported in Greece from 2004 to 2012 (table 1.3.5.4.1). The highest mean annual notification rate of the disease regarded the age group of ≥ 65 years old (2.41/1,000,000 population) and the age group of 0-4 years old (0.62/1,000,000 population) (graph 1.3.5.4.1). Generally, notification rate of listeriosis is low in Greece. Page 52 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki The mean notification rate in the EU and EEA/EFTA countries was 3.5 cases per 1,000,000 population for the year 2009 (HCDCP, 2012). Outbreaks from L. monocytogenes are not so common compared with those caused by pathogens like Salmonella. However, they receive considerable attention when they do occur because they usually have some seriously affected cases and even deaths (Todd and Notermans, 2011). Moreover, foods related to listeria outbreaks are bean sprouts, cabbage, chicory, cantaloupe, eggplant, lettuce, potatoes, radish and lettuce (Ramos et al., 2013).
Graph 1.3.5.4.1: Notification rate of Table 1.3.5.4.1: Annual number of listeriosis by age group and gender in notified cases and notification rate of Greece, Mandatory Notification listeriosis in Greece, Mandatory System, 2004-2012 (HCDCP, 2012). Notification System, 2004-2012 (http://www.keelpno.gr - 7/5/2013)
1.3.6 Foodborne Viruses
Fruits and vegetables are vehicles for viral infection. In Europe, viruses were responsible for 10.2% of the foodborne outbreaks during 2006 and were pointed out as the second most common causative agent, after Salmonella (EFSA, 2007). Lettuce (Rosenblum et al., 1990), diced tomatoes and raspberries (Reid and Robinson, 1987) as well as strawberries (CDC, 1997) have been contaminated with Hepatitis A. Moreover, Hernandez et al. (1997) have declared that lettuce contaminated with sewage could be a vehicle for hepatitis A virus and rotavirus. An outbreak caused by hepatitis A, associated with green salad onions was reported in the United States in 2003 (Chancellor et al., 2006). It must be stated that cases of foodborne disease caused by Norwalk-like viruses (i.e. Small Round Structured Viruses, or SRSV) have been also reported (Bean and Griffin, 1990). NoV outbreaks have been reported linked to leafy greens (Ethelberg et al., 2010, Gallimore et al., 2005). Soft red fruits have also been implicated in NoV outbreaks (Maunula et al., 2009). Rapid alerts related to the detection of NoV in lettuce and soft red fruits have been noted (RASSF, 2010). Hepatitis A and Norwalk-like
Page 53 Literature Review viruses are the most commonly documented viral food contaminants (FDA, 2001). Numerous viruses can be found in the human intestinal tract (table 1.3.6.1).
Table 1.3.6.1: Enteric viruses and clinical syndromes (Koopmans et al., 2002)
Studies have shown that viruses may present for weeks or even months on vegetable crops and in soils that have been irrigated or fertilized with sewage wastes. For instance, viruses introduced onto green onions remained stable for over 14 days (Kurdziel et al., 2001). In general, Rotaviruses, astroviruses, enteroviruses (polioviruses, echoviruses and coxsackie viruses), parvoviruses, adenoviruses and coronaviruses have been reported to be transmitted by foods on occasion (Cliver, 1994).
The food- and waterborne viruses can be divided into three disease categories:
1. viruses that cause gastroenteritis (e.g. astrovirus, rotavirus, the enteric adenoviruses, and the two genera of enteric caliciviruses, i.e. the small round structured viruses or ‘Norwalk-like viruses’ (NLV), and typical caliciviruses or ‘Sapporo-like viruses’ (SLV),
2. fecal orally transmitted hepatitis viruses: hepatitis A virus (HAV), hepatitis E virus (HEV) .
3. viruses which cause other illnesses, e.g. enteroviruses (Koopmans et al., 2002).
More precisely, human noroviruses and hepatitis A virus (HAV) are the most important human foodborne viral pathogens with regard to the number of outbreaks and people affected (Bozkurt et al., 2014). Scallan et al. (2011) reported that an estimated 80-90%
Page 54 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki of all non-bacterial outbreaks of gastroenteritis reported each year are due to human noroviruses and HAV. These viruses are generally environmentally stable, survive adverse conditions and are resistant to extreme pH conditions and enzymes of the gastrointestinal tract (D’Souza et al., 2006). They are known to have low infectious doses, of as few as 10 infectious particles, which can cause illness (CDC, 2012).
1.3.6.1 Noroviruses
Norovirus is a genus of the Caliciviridae family. The NoV genome is approximately 7.5 kb in length and contains three open reading frames. NoV infection causes acute vomiting, diarrhea, fever and abdominal cramps (Koopmans, 2008). Cases typically become symptomatic 24–48 h after infection, and the illness typically resolves after 48- 72 h (Teunis et al., 2008).
Foods can become contaminated with pathogens at any point during production, processing, and preparation (Greig et al., 2007). NoV outbreaks, are associated with food handlers and poor personal hygiene practices (Baert et al., 2009b, Dominguez et al., 2010).
The stability and persistence of NoV is also a contributing factor to food and waterborne outbreaks. Food products provide varying degrees of protection or antiviral activity, depending on their properties. FCV has been shown to survive for 7 days on ham, 3–5 days on lettuce, 1–5 days on cantaloupe, 3–4 days on bell peppers, and 1 day on strawberries (Mattison et al., 2007, Stine et al., 2005), although it is rapidly inactivated in the acidic environment of mussels (Hewitt and Greening, 2004). Temperature control is a key parameter for control of bacterial pathogens in food but not for eliminating NoV (Baert et al., 2009).
Fresh fruits and vegetables may also be contaminated with NoV during production, processing or distribution. Contaminated irrigation water or wash water is the vehicle for transferring NoV to fresh products (Cheong et al., 2009, Mara and Sleigh, 2010), and surrogate viruses have been shown to attach and persist on fruit and vegetable surfaces (Mattison et al., 2007, Urbanucci et al., 2009, Wei et al., 2010). NoV has been implicated as the cause of outbreaks of gastroenteritis from salads, cantaloupe and frozen raspberries (Allwood et al., 2004, Bowen et al., 2006, Ethelberg et al., 2010, Gallimore et al., 2005b, Maunula et al., 2009). NoV genomes have also been detected in
Page 55 Literature Review up to 6% of prepackaged salads (Mattison et al., 2010). Cooking could be an effective control measure for NoV contamination but is not applicable to the fresh RTE fruit and produce category. Washing in clean water can reduce levels of NoV contamination from
1 to 3 log10 (Baert et al., 2008b). The most effective intervention is the prevention measures (Mattison et al., 2010). Moreover, appropriate treatment of irrigation and wash water can inactivate NoV (Baert et al., 2009). Surveillance networks may detect point source foodborne outbreaks, and this information can be used to prevent or limit the further spread of disease (Koopmans et al., 2003).
1.3.6.2 Hepatitis
The viruses which cause hepatitis can be divided into enterically transmitted viruses (HAV, HEV), and parenterally transmitted hepatitis viruses (hepatitis B, C, D, G) (Koopmans et al., 2002).
HAV are small, non-enveloped spherical viruses, measuring between 27 and 32 nm in diameter. They contain a single (positive) stranded RNA genome of approximately 7.5 kb in length (Koopmans et al., 2002). The majority of infections occur in early childhood and virtually all adults are immune. However, young children generally remain asymptomatic. In developed countries, however, HAV infections become less common as a result of increased standards of living. Infection with HAV can produce asymptomatic or symptomatic infection after an incubation period of 30 days. The illness caused by HAV infection is characterized by symptoms that can include fever, headache, fatigue, nausea and abdominal discomfort (Koopmans et al., 2002, Koopmans et al., 2004).
Outbreaks associated with food, particularly raw produce, contaminated before reaching the food service establishments have been recognized increasingly in recent years (CDC, 1997). Fresh RTE produce appears to be contaminated during harvest, which could occur from handling by virus-infected humans (Koopmans et al., 2002). Approximately 10% of the virus particles can easily be transferred from faecally contaminated fingers to foods and surfaces (Bidawid et al., 2000b). Furthermore, a low relative humidity favours the survival of HAV and human rotavirus (HRV) (Mbithi et al., 1991). In another study it was stated that HAV remained infectious in dried faeces for 30 days when stored at 25 °C and 42% relative humidity. HAV vaccination is recommended for foodhandlers,
Page 56 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki although the use of stringent personal hygiene is better preferred, in order to prevent infections (Koopmans et al., 2004).
Many produce items have been implicated in HAV linked outbreaks including blueberries, strawberries, lettuce, green onions, raspberries, and semi-dried tomatoes (Calder et al., 2003, Donnan et al., 2012, Rosenblum et al., 1990). In 2003, the largest documented HAV outbreak in the United States was associated with contaminated green onions consumed at a restaurant in Pennsylvania causing 601 cases of illness including 3 deaths and 124 hospitalizations (Wheeler et al., 2005). More recently, a multi- jurisdictional outbreak in Australia was linked to semi-dried tomatoes with over 562 reported cases (Donnan et al., 2012).
1.3.6.3 Adenoviruses
Adenoviruses are large, non enveloped particles that constitute a double-stranded deoxyribonucleic acid (DNA) genome totally packaged in an icosahedral capsid, or protein coat (figure 1.3.6.3.1). They have been isolated from mammalian species. There are 51 HadV serotypes classified originally on the basis of their ability to be neutralized by specific animal antisera (Crawford-Miksza and Schnurr, 1996). These can be further subdivided into six species -or subgroups- (A to F) based on their G-C content of their DNA and their capacity to agglutinate erythrocytes of human, rat and monkey as well as on their oncogenicity in rodents (Crawford-Miksza and Schnurr, 1996, Wadell, 1984). Species B is further subdivided into B1 and B2 (Segerman et al., 2003a). Species B1, C and E mainly cause respiratory disease, whereas species B, D and E can induce ocular disease. Species F is responsible for gastroenteritis and B2 viruses infect the kidneys and urinary tract (Russell, 2005). Subgenus F includes Ad40 and Ad41, which are considered to be the most important adenoviral species with respect to infantile dysentery and are shed in high concentrations by infected children (Sattar et al., 2002, Wadell, 1984).
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Figure 1.3.6.3.1: Adenovirus structure (http://signagen.com/).
Adenovirus type 35 (Ad35) strain, belonging to adenovirus subgroup B, was isolated in 1973 from the lungs and kidney of a 61-year old woman (Flomenberg et al., 1994) Subgroup C includes adenovirus type 2 and adenovirus type 5. These are considered to be endemic and account for over half of adenoviral infections. They mainly infect children and result in both gastrointestinal and respiratory disease. Adenovirus types from Species B (Ad3) and Species E (Ad 4) have been identified as causative agents of recreational waterborne outbreaks of conjunctivitis and pharyngoconjunctival fever (McMillan et al., 1992). Adenoviruses exhibit an increased stability in the environment compared to other viruses. They are generally considered to be emerging human pathogens (Yates et al., 2006).
Adenoviruses are nonenveloped, icosahedral particles consisting of a protein coat, or capsid, surrounding a DNA-protein core. They range in size from 70-100 nm (Strauss and Strauss, 2002). The protein coat contains several different types of proteins, mainly hexons which are located at each vertex of the virus’s icosahedral coat, creating a penton complex from which a fiber protein protrudes (Rux and Burnett, 1999) (figure 1.3.6.3.1). Each adenovirus particle has 12 molecules of fiber protein which are responsible for the attachment of viruses to their host cells. Adenovirus attaches to the coxsackie and adenovirus receptor (CAR) on the surface of host cells (Bergelson et al., 1997). According to Seth (1999a) adenovirus enters cells via receptor-mediated endocytosis, during which the portion of the cell membrane containing the CAR and bound virus become a membrane-bound vesicle. After binding to the cell surface, viral particles enter endosomes within the host cell. When the endosomes lyse, viral particles are released to the cytosol and travel along microtubules to the host cell’s nucleus, where adenoviral DNA is replicated by the host cell’s DNA replication machinery. Then, new viral Page 58 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki particles are formed and released from the cell. The virus travels from its attachment site on the surface of the host cell to the nucleus in about 30-min (Greber et al., 1993). Attachment of adenovirus to the cell surface involves mainly the CAR receptor and the fiber protein (Strauss and Strauss, 2002).
1.3.7 Pathogens and Infectious Dose
Infectious dose or infective dose (ID) is the amount of pathogens (measured in number of microorganisms) that are required to cause an infection in the host. ID varies dramatically across pathogen species, as well as according to consumer's age and overall health (Leggett et al., 2012).
Foodborne illness resulting from the consumption of any food is dependent upon a number of factors. The produce must first be contaminated with a pathogen and the pathogen must survive until the time of consumption at levels sufficient to cause illness. The infective dose (minimum numbers of organisms necessary to cause foodborne illness) is very low in many cases, which means that the microorganism needs only to contaminate the food and survive without reproducing. For example, pathogenic parasites and viruses are unable to multiply outside of a human or animal host and only need to survive in sufficient numbers to cause illness (FDA, 2011).
The infectious dose of a foodborne pathogen depends on many variables including the immune status of the host, the virulence and infectivity of the pathogen, the type and amount of contaminated food consumed, the concentration of the pathogen in the food and the number of repetitive challenges (EU, 1999). In general, infectious pathogens may enter the body and invade or colonize host tissues. This requires some time (e.g., usually greater than 8 h for onset of illness). Toxigenic pathogens create food “poisoning” situations by producing an enterotoxin in the food (Behling et al., 2010).
Risk assessment and impact of foodborne pathogens on the health of different populations remains one of the goals of every country. For certain pathogens, such as Listeria monocytogenes and Escherichia coli O157:H7, there are no feeding studies due to ethical reasons, and the results from outbreaks are normally used to estimate the infectious dose (Kothary and Babu, 2007).
The infectious dose for different serovars of Salmonella and strains of E. coli was quite large (>105 organisms), while the infectious dose for some Shigella spp. seemed to be as
Page 59 Literature Review low as less than 10 organisms (Kothary and Babu, 2007). The infectious dose of salmonellae can vary, depending on the bacterial strain ingested as well as on the immuno-competence of individuals. For serotypes not presenting particular adaptations to an animal host, experimental studies showed that between 105 to 107 bacteria were required to establish an infection. However, data from outbreaks of foodborne diseases indicate that infections can be caused by ingestion of as few as 10-45 cells (Lehmacher et al., 1995). It has repeatedly been reported that the infectious dose is lower when salmonellae are present in food with a high content of fat or protein, substances which protect bacterial cells against the low pH of gastric juices (Blaser and Newman, 1982). An oral dose of at least 105 Salmonella Typhi cells are needed to cause typhoid in 50% of human volunteers, whereas at least 109 S. Typhimurium cells (oral dose) are needed to cause symptoms of a toxic infection. Infants, immunosuppressed patients, and those affected with blood disease are more susceptible to Salmonella infection than healthy adults (Todar, 2012). The infectious dose of S. Enteritidis can be relatively small, 100 to 1,000 organisms are enough to cause the infection in some people. Food prepared from infected animals, insufficiently cooked and food contaminated prior to consumption are the principal causes of infection (CDC, 2005).
The infective dose of ETEC for adults has been estimated to be at least 108 cells. However, young and elderly people may be susceptible to lower levels. Because of its high infectious dose, analysis for ETEC is usually not performed unless high levels of E. coli have been found in a food. At least 106 EIEC organisms are required to cause illness in healthy adults. The infectious dose for O157:H7 is estimated to be 10-100 cells, but no information is available for other EHEC serotypes (FDA, 2011).
The infective dose for Listeria monocytogenes is uncertain, although it is generally considered to be high (105-107) (Farber et al., 1996) for healthy individuals, with food contamination rates of more than 1,000 cells/g being required. Due to the length of the incubation period, it can be difficult to determine the numbers of bacteria in foods at the time of consumption. An outbreak associated with frankfurters in the USA in 1998 is thought to have been caused by product containing less than 0.3 cells/g, although it is suspected that the strain involved may have been unusually virulent (Farber et al., 1996). The probability of exposure to a higher dose (> 1.000 CFU) was large enough to account for the observed rate of listeriosis.
Page 60 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki The infectious dose of S. aureus has been found to be at least 100,000 organisms in humans (PHAC, 2011). Therefore, if enterotoxinogenic staphylococci are able to grow in food to high numbers (more than 105 to 106 CFU/g or /ml) before they are killed there is still a risk for intoxication with consumption.
As virus infectivity dose is concerned, it has been estimated that NLVs and HAV have an infective dose of between 10 and 100 virus particles. Adenoviridae infectious dose is >150 plaque forming units when given intranasally (PHAC, 2011).
1.3.8 Methods for detection of foodborne pathogens
Generally, there are standard conventional methods for determination of the foodborne pathogens in food products including culture, microscopic, chemical and biological methods (immunological, molecular genetic methods, gel diffusion). Moreover, there exist more rapid methods including physical methods (biosensors, impedance, microcalorimetry, flow cytometry, biosys instrument) and bioassays (Yeni et al., 2014). Real-time PCR (rtPCR), microarrays, and biosensors have emerged as recent developments in the field of pathogen testing (Mattingly et al., 1988).
Among the culture-based conventional methods, standard plate count is the longest available detection and enumeration method. Culturing methods utilized to monitor for the presence of indicator organisms are time consuming and labor intensive (Shannon et al., 2007). As an alternative to standard plate count, incorporation of chromogenic and fluorogenic substrates into culture media exists for the biochemical identification of pathogenic microorganisms (Mattingly et al., 1988).
Emerging pathogens, that are not detectable by conventional microbiological methods, can be detected with nucleic acid (DNA and RNA)-based assays for the differentiation and identification of foodborne pathogens. These methods include polymerase chain reaction (PCR), pulsed-field gel electrophoresis, ribotyping, plasmid typing, randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) (Yeni et al., 2014). Most of them are available in commercial kits. Molecular- based detection methods, such as the polymerase chain reaction (PCR), amplifies specific target DNA of the desired organism being investigated, and have exhibited huge potential as routine and rapid analysis tools for the detection of specific micro-organisms (Shannon et al., 2007). However, the PCR technique is unable to distinguish between viable or dead cells, which could lead to false positive results (Moreno et al., 2011). Page 61 Literature Review
Immunology-based methods and several types of biosensors are the new developing techniques for detection of foodborne pathogens (Velusamy et al., 2010). For immunodetection, which is based on antigen-antibody binding selection, several types of antibodies can be used (conventional, heavy chain antibodies, polyclonal, monoclonal or recombinant antibodies). For instance, detection of L. monocytogenes can be performed via polyclonal antibodies (Jung, et al., 2003) and via monoclonal antibodies (Mattingly et al., 1988), but for Salmonella detection, monoclonal antibodies (Yeni et al., 2014) have been identified.
ELISA (enzyme-linked immunosorbent assay) is the most prominent method among other types of immunological pathogen detection methods. It integrates the specificity of antibodies and the sensitivity of simple enzyme assays by using antibodies or antigens connected to an enzyme (Lazcka et al., 2007, Yeni et al., 2014).
Another promising rapid detection method is the Matrix-assisted laser desorption/ ionisation time of flight mass spectrometry (MALDI-TOF MS) (Biswas and Rolain, 2013). The initial capital cost for purchasing the equipment is high, however, this technique seems cheap when utilised for the routine biotyping of bacteria (Biswas and Rolain, 2013, Clark et al., 2013).
The 3M™Molecular Detection system is a new technology being extensively used in food analysis for the detection of Listeria spp., Salmonella spp. and E. coli 0157:H7. The detection system makes use of a loop-mediated isothermal amplification (LAMP) method for the detection of foodborne pathogens. It then combines isothermal DNA amplification with bioluminescence detection, which allows for specificity and sensitivity in the testing for pathogens (Loff et al., 2014).
The culturing techniques that are commonly used for the detection and enumeration of pathogens in foods include selective chromogenic media for detecting each bacteria. A selective, chromogenic medium that can be used for the detection and enumeration of E. coli in foods is TBX Agar (Oxoid, CM 0595). TBX Medium is a tryptone Bile Agar which is based on a chromogenic agent -X-glucuronide- detecting glucuronidase activity. The released chromophore in TBX Medium is insoluble and accumulates within the cell. This ensures that colored target colonies are easy to be identified. Most E. coli strains can be differentiated from other coliforms by the presence of the enzyme glucuronidase. The chromogen in TBX Medium is 5-bromo-4-chloro-3-indolyl-beta-D-
Page 62 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki glucuronide (X-glucuronide), and is targeted by this enzyme. Escherichia coli cells are able to absorb this complex intact and intracellular glucuronidase splits the bond between the chromophore and the glucuronide. The released chromophore is colored and is accumulated within the cells, causing E. coli colonies to obtain a color of blue/green. The tryptone provides the essential growth nutrients (nitrogen, vitamins, amino acids) to the organisms whereas bile salts mixture inhibits Gram-positive organisms. Moreover, in order to detect E. coli O157:H7 in foods, an enrichment stage, is ofter required, since the number of cells is usually low, and injured cells are likely to be present. For this reason, enrichment in modified trypticase soy broth (mTSB), supplemented with either novobiocin or acriflavin, can be used (Taormina, 1998).
Baird Parker medium has been developed in order to detect S. aureus in foods. Baird- Parker (1962) developed this medium from the tellurite-glycine formulation. As a nitrogen source for the organism, casein peptone and beef extract are added to the medium. Yeast extract provides nitrogen as well as other important nutrients like B- complex vitamins. The selective agents glycine, lithium and potassium tellurite have been carefully balanced to suppress the growth of most bacteria present in foods, without inhibiting S. aureus. Egg yolk emulsion (Oxoid SR0047) makes the medium yellow and opaque. S. aureus reduces potassium tellurite to form grey-black shiny colonies and then produces clear zones around the colonies by proteolytic action. This clear zone with typical grey-black colony is characteristic for S. aureus detection. Most strains of S. aureus form opaque haloes around the colonies. This is probably due to the action of a lipase. However, not all strains of S. aureus produce both reactions.
A selective medium that can be used for the isolation and identification of Salmonella is Xylose-Lysine-Desoxycholate Agar (XLD Agar), which was originally formulated by Taylor (1965). It relies on xylose fermentation, lysine decarboxylation and production of hydrogen sulphide for the primary differentiation of shigellae and salmonellae from non-pathogenic bacteria. Salmonella spp. are differentiated from non-pathogenic xylose fermenters by the incorporation of lysine in the medium. Salmonellae exhaust the xylose and decarboxylate the lysine, thus altering the pH to alkaline and mimicking the Shigella reaction. However, the presence of Salmonella is differentiated from that of shigellae by a hydrogen sulphide indicator. Sodium Thiosulfate and Ferric Ammonium Citrate act as selective agents, allowing visualization of hydrogen sulfide production under alkaline conditions.The high acid level produced by fermentation of lactose and sucrose, prevents lysine-positive coliforms from reverting the pH to an Page 63 Literature Review alkaline value, and non-pathogenic hydrogen sulphide producers do not decarboxylate lysine. The acid level also prevents blackening by these micro-organisms until after the 18-24 hour examination for pathogens. Sodium desoxycholate is incorporated as an inhibitor in the medium. The sensitivity and selectivity of XLD. Agar exceeds that of the traditional plating media e.g. Eosin Methylene Blue, Salmonella-Shigella and Bismuth Sulphite agars, which tend to suppress the growth of shigellae (Dunn and Martin, 1971, Taylor and Schelhart, 1967).
Listeria Selective Medium (Oxford Formulation) is recommended for the detection of Listeria in foods. It is based on the formulation described by Curtis et al. (1989). The medium utilizes: (i) the selective inhibitory components lithium chloride, acriflavine, colistin sulphate, cefotetan, cycloheximide or amphotericin B and fosfomycin, (ii) the indicator system aesculin and ferrous iron for the isolation or differentiation of Listeria monocytogenes. Listeria monocytogenes hydrolyses aesculin, producing black zones around the colonies due to the formation of black iron phenolic compounds derived from the aglucon. Ferric Ammonium Citrate aids in the differentiation of Listeria spp. Since all Listeria spp. hydrolyze esculin, the addition of ferric ions to the medium will detect the reaction. A blackening of the colony and surrounding medium in cultures containing esculin-hydrolyzing bacteria results from the formation of 6,7-dihydroxycoumarin which reacts with the ferric ionsGram-negative bacteria are completely inhibited (Fraser and Sperber, 1988). Most unwanted Gram-positive species are suppressed, but some strains of enterococci grow poorly and exhibit a weak aesculin reaction, usually after 40 hours incubation. Typical Listeria colonies are almost always visible after 24 hours, but incubation should be continued for a further 24 hours to detect slow-growing strains.
1.4 Infection and Disinfection
Traditional thermal treatments are a basic pillar in the food industry providing required safety profiles and extensions of shelf-life of the product. However, attention must be given to losses of desired organoleptic properties and damage to temperature labile nutrients and vitamins. Consequently, the food industry is interested in alternative or combined approaches in order to achieve the objectives of disinfection. Thus, non- thermal technologies have been designed to meet the required food product safety
Page 64 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki standards or shelf-life demands while minimizing the effects on its nutritional and quality attributes.
1.4.1 The Bacterial Cell and Antimicrobial Interaction
The bacterial cell interacts with biocides through: the cell wall, the cytoplasmic membrane and the cytoplasm. However, a biocide can interact with one, two or three regions of the bacterial cell in order to have a successful antimicrobial effect. The cell wall is comprised of an open network of peptidoglycan (with a lipopolysaccharide overlayer for Gram-negative bacteria) that serves as an excellent target for antimicrobials. Thus, the presence of the outer envelope adds an additive resistance of Gram negative bacteria to antimicrobials when compared to Gram-positive bacteria (Denyer and Maillard, 2002) (figure 1.4.1.1). The peptidoglycan layer and its associated anionic polymers provide access to the membrane of microorganisms to molecules with molecular weights ranging from 30- 57 KDa. Antimicrobials such as hydrogen peroxide, phenols, alcohols, aldehydes, QACs and biguanides are small enough, thus their cross to the cell wall is easy (Lambert, 2002). Porins, which are large protein structures embedded within the outer membrane, not only allow the diffusion of cellular nutrients but can also serve as channels to hydrophilic biocides of molecular weight less than 600 Da (Denyer and Maillard, 2002). The cytoplasmic membrane is available to biocide attack as it acts as a rich matrix of balanced interactions between phospholipid and enzymatic/structural proteins where intracellular homeostasis and transport/metabolisms are maintained (Denyer and Stewart, 1998). The cytoplasm is considered to be the most common target for biocides due to the presence of catabolic and anabolic processes. Damage to the membrane can take place due to physical disruption, dissipation of the proton motive force and inhibition of membrane associated enzyme activity (Maillard, 2002). Hypochlorous acid reacts with a wide variety of biological molecules including proteins, DNA, cholesterol, lipids, free thiols and sulfides (Hawkins et al., 2003, Noguchi, et al., 2002). The bactericidal activity of hypochlorite has been attributed to the formation of secondary products (chloramines), which react with subcellular compounds such as ammonia ions and organic amines (Miche and Balandreau, 2001). However, chloramines are toxic compounds capable of diffusing through cell membranes and reaching intracellular components such as DNA. Consequently, hydrogen bonding is disrupted, resulting in the dissociation of DNA (Hawkins et al., 2003). Studies have demonstrated that radicals that result from the decomposition of chloramines on
Page 65 Literature Review hypochlorous acid treated proteins can cause damage to other substrates as well. The oxidation of proteins by hypochlorous acid can lead to side chain modification. Many of these reactions occur primarily with thiols, sulfides, amines, amides and aromatic rings
(Hawkins et al., 2003). The effectiveness of disinfectants like H2O2, and NaOCl, is dependent on concentration, pH, temperature, microbial strain, and the presence of organic materials.
Figure 1.4.1.1: Schematic illustration of cell wall structures of microbial pathogens. (A) Gram- positive bacteria, (B) Gram-negative bacteria (Yin et al., 2013).
1.4.2 The virus genome and infectivity
The virus must be able to attach to the host cell in order to inject its material into the host cell thus being able to replicate. When pasteurization is used as a disinfection method, the heat that is produced inhibits the bonding with the host cell. Thus, the virus in incapable of recognizing the host and is unable of attaching to it. In general terms chlorine and ozone affect the viral protein material, whereas UVC affects the genomic material more than the protein material (Nuanualsuwan and Cliver, 2003, Thurston- Enriquez et al., 2005, Toropova et al., 2008). When chlorine dioxide is used, the reactions take place in the genome and proteins, and byproducts that further react with aminoacids and as a concequence nucleotides are formed (Pecson et al., 2009). When
Page 66 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki radiation is used, chemical reactions are created that can destroy the genome, inhibiting virus replication. Radiation leads to direct photolysis of photolabile virus components, thus the virus protein capsid is broken. When the capsid is broken, there is no way for the virus to inject its material into the host cell. Finally, when chlorination is used for virus disinfection, the attack of chlorine to genome, prevents it from replication and destroys its injection into the host cell (figure 1.4.2.1).
Figure 1.4.2.1: Effects of capsid of virus when different situations occur (Wigginton et al., 2012).
1.4.3 Conventional Food Processing/Preservation Technologies
1.4.3.1 Chemical Methods
Chlorine compounds can be categorized to three different types: chlorine gas, chlorine hypochlorites (e.g., sodium, calcium, lithium and potassium) and chlorine releasing agents (trichloroisocyanuric acid, sodium dichloroisocyanurate, dichlorodimethylhydantoin, chloramines T) (McDonnel and Russell, 1999). Chlorine compounds are bactericidal and sporicidal (Russel, 1983). Their activity is mainly related to their solubility, amount of available chlorine present and pH of the solution. However, their activity is impaired by the presence of organic matter.
Hypochlorites are powerful oxidants and can induce lysis in Gram-negative bacteria by affecting the cell wall (Maillard, 2002). The manufacture of sodium hypochlorite is a
Page 67 Literature Review simple procedure that involves the reaction of chlorine with caustic soda in a batch or continuous process (Gordon et. al., 1995). The reaction that takes place follows:
2 NaOH + Cl2 → NaOCl + NaCl + H2O + Heat
Other commonly used terms include free available chlorine and available chlorine. Free available chlorine (FAC) refers to the concentration of molecular chlorine (Cl2), hypochlorous acid (HOCl), and hypochlorite ion (OCl-) in water expressed as available chlorine. Available chlorine is used to express the amount of chlorine in chlorine gas and hypochlorite salts. Commercial grade sodium hypochlorite can be produced by manufacturers at concentrations as high as 16% chlorine, with typical concentrations ranging between 5 and 15% chlorine. The stability of a sodium hypochlorite solution is affected by factors such as concentration, light, pH, temperature and heavy metals (Casson and Bess, 2003). Liquid hypochlorite typically has a pH between 11 and 13. In basic solution, hypochlorite ion (OCl-) decomposes to form chlorate ion (ClO3-) (a toxic by-product). This is a second order process and involves the reaction of OCl- with chlorite ion ClO2- (an intermediate ion). The reactions are the following (Bolyard and Fair, 1992):
2OCl- → ClO2- + Cl- (slow reaction)
OCl- + ClO2- → ClO3- + Cl- (fast reaction)
At pH higher than 9, the HOCl is almost completely dissociated to OCl- (HOCl ↔ H+ + OCL-), which results to a very poor disinfectant. Thus, lower pH is important for an adequate disinfection. The reaction that takes place when sodium hypochlorite is added to water is the following:
+ - NaOCl+H2O → HOCl + Na + OH
Chlorine is an economical disinfectant and has been used in water and food disinfection (EPA, 1999c). Chemicals that have been evaluated for use as disinfectant agents in food produces, are chlorinated water and chlorine dioxide (Park et al., 2008). However, there are also several disadvantages related to chlorine disinfection. Chlorine reacts with organic and inorganic compounds and produces undesirable trihalomethanes (THMs) and other carcinogenic disinfection by-products (DBPs), such as chloroform and bromodichloromethane. Thus the use of chlorine has been associated with the formation of carcinogenic compounds (EPA 1998a). Moreover, some foodborne pathogens have Page 68 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki been shown to be more resistant to the lethal action of chlorination (Furata et al., 2004). Furthermore, higher concentrations of chlorine can cause poor taste and odor in treated produces. To improve food safety and maintain the freshness, the products are subjected to appropriate washing systems containing chemical sanitizers. One of the most commonly used disinfectants in food industry is chlorine (50–200 ppm). However, it is reported that chlorine is not effective to inactivate internalized pathogens due to its limited penetration into the internal complex areas of foods (Sapers, 2001).
The USFDA recommends 50–200 mg/l total chlorine at pH 6.0–7.5 and contact times of 1–2 min for this purpose (Beuchat, 1998). The International Fresh-Cut Produce Association (IFPA) Model HACCP Plan for shredded lettuce suggests a chlorination of 100–150 mg /l total chlorine at pH 6.0–7.0 (Delaquis et al., 2004). Chlorinated water has been used to wash and disinfect vegetables and fruits shortly after harvesting and at various stages of handling and processing (Beuchat, 1998). The effectiveness of treatment with water containing up to 200 mg/ml chlorine in reducing numbers of naturally occurring microorganisms and pathogenic bacteria is minimal, often not exceeding 2 log10 on lettuce (Zhang and Farber, 1996) and tomatoes (Beuchat et al., 1998, Wei et al., 1995, Zhuang et al., 1995). However, Sapers and Simmons (1998) reported that the quality of some products may be degraded by browning induction (in mushrooms and lettuce) or bleaching of anthocyanins (in strawberries and raspberries).
In order to improve efficacy, chemical treatments are combined with other disinfection methods such as ultrasound (Seymour et al., 2002, Zhou et al., 2009), vacuum infiltration (Sapers, 2001), and adding detergents or enzymes to dissolve extracellular polymeric substances (Johansen et al., 1997).
1.4.3.1.2 Organic Acids
Organic acids, mainly citric, lactic and acetic acid, which are involved in GRAS (Generally Recognized As Safe) status, have been investigated because of their ability to inactivate foodborne pathogens (Akbas and Ölmez, 2007). Organic acids act rapidly and kill a broad spectrum of bacteria. Moreover, they are effective within a wide temperature range and are not affected by water hardness (Sagong et al., 2011). The antimicrobial action of organic acids, in general, is attributed to the pH reduction in the environment, and it changes widely among the organic acids (Ölmez and Kretzschmar, 2009).
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Antimicrobial properties of acetic acid have been shown to inactivate food-borne pathogenic bacteria (Bell et al., 1997). Many studies have been conducted related to the use of organic acids as disinfectants. It was reported that the fecal coliforms and the coliforms were reduced by 1.0 and 2.0 log10 CFU/g, respectively, on mixed salad vegetables treated with 1.0% lactic acid (Torriani et al, 1997). Francis and O’Beirne (2002) found that 1.0% citric acid solution reduced mesophilic population densities on lettuce by about 1.5 log10 CFU/g in 5 min. Dipping of fresh-cut iceberg lettuce in 0.5% citric acid or 0.5% lactic acid solutions for 2 min were found to have the same effect like 100 ppm chlorine, in reducing the natural microbial population (Akbas and Ölmez,
2007). Chang and Fang (2007) found that 5% acetic acid could reduce 3 log10 population of E. coli O157:H7 in iceberg lettuce, however, may alter the sensory quality by causing an unacceptable sour flavor. However, it is obvious that the antimicrobial activity of organic acids varies in a wide range depending on the type of organic acid. In general, the exposure times needed for a significant reduction in microbial load is between 5 and 15 min. Moreover, organic acids also have disadvantages such as high cost, odor, and corrosiveness. It must also be taken into consideration that the use of organic acids for disinfection purposes in fresh RTE industry would have an impact on the wastewater quality, characterized by high COD and BOD values in the wastewater (Ölmez et al., 2009). 1.4.3.1.3 Peroxyacetic Acid
Peroxyacetic acid (PA), which is also referred to as peracetic acid, is an aqueous quaternary equilibrium mixture of acetic acid and hydrogen peroxide (Dell’Erbaa et al., 2007). Like ozone and chlorine dioxide, peroxyacetic acid is an effective disinfectant able to kill pathogenic microorganisms in suspension at lower concentrations. Moreover, it is important to note that, the efficacy of PA is not affected by the organic compounds present in the process water, whereas the efficacy of chlorine is affected (Ruiz-Cruz et al., 2007). PA has become and interesting disinfectant among chlorine-alternative chemical disinfectants due to the fact that only harmless disinfection by-products have been formed from its spontaneous decomposition (i.e acetic acid, water, oxygen) (Dell’Erbaa et al., 2007). The US Code of Federal Regulations states that the use of peroxyacetic acid in fruits and vegetables is allowed up to 80 ppm in wash water. However, studies revealed that 80 ppm peroxyacetic acid in wash water is not sufficient to obtain a substantial reduction in the microbial load of the fresh RTE fruits and vegetables (Ruiz-Cruz et al., 2007, Vandekinderen et al., 2007).
Page 70 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.4.3.1.4 Hydrogen Peroxide
Hydrogen peroxide (H2O2), also referred to as hydrogen dioxide, has both a bacteriostatic and a bactericidal activity due to its strong oxidizing power and its generation of cytotoxic species (Juven and Pierson, 1996). Although it is involved in the GRAS status, its use in the food industry is limited only to some products (milk, dried egg, starch, tea and wine) as an antimicrobial or bleaching agent in the range of 0.04– 1.25%. The effectiveness of hydrogen peroxide as a disinfectant and antimicrobial agent has been established (Ölmez and Kretzschmar, 2009).
One of the main advantages of using H2O2 as a disinfecting agent is that it produces no residue as it is decomposed into water and oxygen by the enzyme catalase which is naturally found in plants. The main drawback is its phytotoxicity, which has been observed in some products like lettuce and berries. Studies revealed that the H2O2 treatment induces extensive browning on some products (lettuce, mushrooms) (Ölmez and Kretzschmar, 2009).
1.4.3.2 Physical Methods 1.4.3.2.1 Heat Processing
Foods are processed by various processing technologies to reduce or remove any potential pathogen or biological hazard that might be introduced while handling or food processing. Traditional food processing technologies, such as pasteurization and heat sterilization, use heat in order to kill or inactivate microbiological contaminants. Pasteurization is a mild heat treatment in which foodstuffs are heated to a temperature lower than 100°C (Fellows, 2000). This process is used to minimize potential health hazard through destruction of non-spore forming pathogenic microorganisms. Pasteurization can kill 99-99.9% of spoilage micro-organisms and inactivate enzymes, thus enhancing the shelf life of the product (Parikh, 2007).
The HTST pasteurization has been successfully implemented and has achieved a five- log10 reduction, killing 99.999% of the number of viable micro-organisms. This is considered adequate for destroying almost all yeasts, molds, and common spoilage bacteria and also to ensure adequate destruction of common pathogenic, heat-resistant organisms (Doyle et al., 2001).
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On the other hand, heat sterilization is a severe heat treatment in which foods are heated at sufficiently high temperatures for prolonged time-periods to destroy microbial and enzyme activity. As a result, sterilized foods are shelf-stable with more than six months of shelf life (Fellows, 2000). A sterilized product may contain viable spores that cannot grow due to environmental conditions, such as low pH, low water activity, etc. However, it can be concluded that the product treated with heat sterilization is considered as commercial sterile product (Parikh, 2007).
However, limitations exist when thermal disinfection technologies are used. Heat alters or destroys nutrient components of foods that are responsible for the special flavor, color, taste, or texture, and as a result they are perceived nowadays to have lower quality and value (Fellows, 2000). 1.4.3.2.2 Radio Frequency (RF) and Microwave Heating (MH)
RF and MW systems operate by the same principle,forcing polar molecules such as water, and ionic species to realign themselves by reversing and electric field around the food products. The promise of rapid and volumetric heating has called the attention of food industry for the potential use of dielectric systems. Industrial and experimental applications of MW for food preservation, among others include sterilization and pasteurization of ready to eat meals. RF systems could be particularly suitable for heat processing of whole meat products (Pereira and Vicente, 2010).
1.4.3.2.3 Ohmic Heating
An ohmic heater also known as a joule heater is an electrical heating device that uses a liquid’s own electrical resistance to generate the heat. Heat is produced directly within the fluid itself by Joule heating as alternating electric current (I) is passing through a conductive material of resistance (R), with the result energy generation causing temperature rise (Sakr and Liu, 2014). OH technology is distinguished from other electrical heating methods by the presence of electrodes contacting the foods (in microwave and inductive heating electrodes are absent), the frequency applied (unrestricted, except for the specially assigned radio or microwave frequency range), and the waveform (also unrestricted, although typically sinusoidal) (Pereira and Vicente, 2010).
Page 72 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1.4.4 Non-thermal Technologies (Alternative Technologies)
Consumers increasingly perceive fresh foods or minimally processed foods as healthier, compared to heat-treated foods. Thus, the industry is now developing alternative processing technologies. Novel non-thermal processing technologies have the ability to inactivate microorganisms at ambient or near ambient temperatures, thereby avoiding the process-induced changes that heat may have on the flavor, color and sensory, quality and nutritional characteristics of foods. Therefore, alternative processing technologies are now being used to effectively destroy any microbial threat, and to maintain at the same time the quality and storage-stability of foods (Ahvenainen, 1996).
Non-thermal technologies are processing technologies able to achieve microbial inactivation without exposing foods to adverse effects of heat. At the same time, an extension of product shelf life and retention of their fresh-like physical, nutritional, and sensory qualities is also achieved (Butz and Tauscher, 2002). These technologies include pulsed or radio frequency electric fields (PEF/RFEF), ultraviolet light (UV), pulsed light (PL), Near Ultraviolet Light (NUV Light), ultrasound (US), high pressure processing
(HPP), ionizing radiation, dense phase carbon dioxide (DPCO2) and ozone (Mohd. Adzahan and Benchamaporn, 2007).
Like any other process, non-thermal technologies can be combined with thermal or other non-thermal processes as a hurdle technology or as a compliment to other processes. The hurdle approach is widely used to produce minimally processed and microbiologically stable food. The microbial stability is achieved by combining different hurdles to increase the destruction of the microbial cytoplasmic membrane as well as preventing cell repair of survivors from treatments (Leistner, 2000).
It is true that combining non-thermal processes with conventional preservation methods, their antimicrobial effect is enhanced, and as a consequence lower process intensities as well as shorter treatment times can be used, which is an advantage to the food industries (Mohd. Adzahan and Benchamaporn, 2007, Ross et al., 2003). 1.4.4.1 Ultraviolet Light (UV)
Radiation from the UV region of the electromagnetic spectrum can be used for the disinfection of food produce. The wavelength for UV processing ranges from 100 to 400 nm (figure 1.4.4.1.1) (Sastry et al., 2000).
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Figure 1.4.4.1.1: Electromagnetic Spectrum (Guerrero- Beltrán and Barbosa-Cánovas, 2004).
The UV light is easy to use and has been proved lethal to microorganisms (Bintsis et al., 2000). The wavelength between 200 and 280 nm (UV-C) is considered germicidal against microorganisms such as bacteria, viruses, protozoa, moulds and yeasts, and algae (Bintsis et al., 2000), compared to other types of UV (table 1.4.4.1.2).
Table 1.4.4.1.1: Ranges, Wavelengths and Characteristics of different types of UV (Guerrero- Beltrán and Barbosa-Cánovas, 2004).
The highest germicidal effect is observed between 250 and 270 nm. More precisely, the wavelength of 254 nm (UV-C, generated by LPM lamps) is used for disinfection of surfaces, water, and food products (Bintsis et al., 2000).
Mode of Action of Continuous UV to Microorganisms
In general terms, because the UV-C bactericidal effect is mainly observed at the nucleic acid level, radiation absorbed by DNA can stop cell growth and lead to cell death (Liltved and Landfald, 2000, Wright et al., 2000). In detail, the UV-C light absorbed by DNA molecule, causes a physical shifting of electrons to render splitting of the DNA bonds, delay of reproduction or cell death. Then, cross-linking effects between Page 74 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki neighbouring thymine and cytosine (pyrimidine nucleoside bases) in the same DNA strand occur (figure 1.4.4.1.3). The produced DNA photoproducts are the cyclobutyl pyrimidine dimers. The cross-linking effects in the DNA are proportional to the amount of UV-C light exposure (dosage, time). The consequence is the cell death due to the blocked DNA transcription and replication as well as the blockage of many cellular functions (Guerrero- Beltrán and Barbosa-Cánovas, 2004).
UV-C irradiation is also responsible for the production of DNA mutations in the injured organism (Sastry et al., 2000). Photoreactivation can occur only when the UV-C injured cells are exposed to wavelengths higher than 330 nm (Liltved and Landfald, 2000). Protein factors (DNA repair genes) can repair the DNA damage (Yajima et al., 1995). Moreover, the activation of the enzyme photolyase that monomerises the dimer species (splitting of thymine and other pyridines) can photoreactivate the split nucleic acid (Stevens et al., 1998). However, a dark environment can avoid photoreactivation of irradiated products (Stevens et al., 1998) or restore cells exposed to UV-C light.
It must be mentioned, that the effect of UV radiation on microorganisms inoculated on food surfaces may vary from species to species. In the same species it depends on many factors such as strain, growth media, stage of culture (Chang et al., 1985, Wright et al., 2000), density of microorganisms and other characteristics, such as type and composition of the food. Generally, fungi and yeasts (large microorganisms) are more resistant during UV disinfection (Bachmann, 1975).
UV-C light is also applied to fresh fruits, vegetables and roots before being stored in order to reduce the initial count of microorganisms on the surface of the product and to induce host resistance to the microorganisms. The beneficial effect of UV-C light on fresh food products is called ‘hormesis’ and the agent (UV light) is called ‘hormetin’ or ‘hormetic effect’ (Stevens et al., 1997, Stevens et al., 1999). The hormetic effect of UV- C light may cause the production of phenylalanine ammonia-lyase (PAL) that induces the formation of phytoalexins (phenolic compounds). Phytoalexins’ role is the improvement of the resistance of fruits and vegetables to microorganisms (D’hallewin et al., 2000, Stevens et al., 1997, Stevens et al., 1998, Stevens et al., 1999).
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Figure 1.4.4.1.2: Structure of DNA before and after absorbing a photon of UV light (Koutchma, 2009).
UV intensity flux or irradiance is usually expressed in W/cm2, and the dose or radiant exposure is expressed as J/m2 (Bintsis et al., 2000). The UV-C dose (D) is defined as:
D=I254 nm*t “Equation 1”
2 2 Where: D is the dose (J/cm ), I254 is the intensity or dosage rate (W/cm ) and t is the retention time in seconds (Chang et al., 1985, Morgan, 1989, Stevens et al., 1999).
In a flow system, the retention time is obtained as: t= Volume of chamber / Flow Rate
Figure 1.4.4.1.3: UV chamber (inside photo)
Among the advantages, it can be concluded that the photoinactivating process by UV-C (figure 1.4.4.1.3) is a physical method which does not produce undesirable by-products (Chang et al., 1985) that could change the sensory characteristics (odor, taste and color) of the final product. Moreover, it does not generate chemical residues nor residual radioactivity (compared to gamma radiation) to the final product (Morgan, 1989). It can
Page 76 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki be concluded that it is a non-thermal process that can be simply and effectively used at low cost compared with other sterilization methods. It can be obtained by simply applying UV-C to the desired food surface at low intensity for long periods or high intensity for short periods of time (Morgan, 1989). 1.4.4.2 High Intensity Light Pulses (HILP)
High-intensity light pulses (HILP) (figure 1.4.4.2.1) is an emerging non-thermal technology which uses short (100–400 s) high-power, intense pulses of broad-spectrum light (200–1100 nm) and has been used𝜇𝜇 to inactivate bacteria (vegetative cells and spores), yeasts, moulds, and even viruses (Marquenie et al., 2003, Gomez-Lopez et al., 2007, Woodling and Moraru, 2007). The mode of action of HILP on microorganisms is based on the photochemical action of the UV-C part of the light spectrum that causes thymine dimerization in the DNA chain preventing transcription and replication and ultimately leading to cell death (Muňoz et al., 2012, Gomez-Lopez et al., 2007, Rajkovic et al., 2010). Microbial inactivation using HILP has gained attention in recent years due to lower energy consumption compared to conventional thermal processes (Barbosa- Cánovas et al., 1998). Depending on the energy delivered through each flash, the distance between the lamps and the contaminated matrix, the targeted microorganism, and even the nature of the contaminated matrix itself, HILP has been reported to result in a 0.5 to 8 log10 CFU/mL bacterial reduction (Hsu and Moraru, 2011). In addition, it has also been shown that both the visible and infrared regions of HILP in combination with its high peak power also contribute to the killing effect on microorganisms (Elmnasser et al., 2007). High-intensity light pulse (HILP) is an emerging technology that has been shown to be highly effective against a wide range of pathogenic microorganisms, including S. aureus, E. coli O157:H7, S. enterica, and Cryptosporidium parvum (Bialka and Demirci, 2007, Krishnamurthy et al., 2007, Lee et al., 2008). The germicidal action of HILP has been also attributed to the localized elevated temperature due to the simultaneous use of UVs and IR radiations leading to bacterial disruption (Dunn, 1995, Nicorescu et al., 2013, Takeshita et al., 2003, Uesugi and Moraru, 2009).
The use of HILP to food products such as apple juice, milk, minimally processed vegetables, berries, alfalfa seeds, hot dogs and salmon fillets have been studied with the intention to extending shelf-life and/or inactivating pathogens (Bialka and Demirci, 2007, Gomez et al., 2012, Huang and Chen, 2014, Oms-Oliu et al., 2010, Ramos- Villarroel et al., 2011). Moreover, HILP treatment does not result in the development of
Page 77 Literature Review volatile organic compounds or suspended airborne particulates. It is cost effective and generally does not change the “special” characteristics of food matrices (Luksiene et al., 2007).
However, there are some disadvantages that limit the HILP application to the fresh produce industry. One issue is that HILP treatment causes substantial heating of the samples, which might damage the quality of fresh produce. Another issue is that microorganisms on an opaque food surface must directly face the HILP-strobe in order to be inactivated due to the shallow penetration depth of HILP. In addition, samples positioned in different parts of the HILP chamber (figures 1.4.4.2.1, 1.4.4.2.2) might be exposed to different doses of HILP (Huang and Chen, 2014).
Figure 1.4.4.2.1: Equipment of HILP (www.xenoncorp.com)
Figure 1.4.4.2.2: Internal Part of pulsed Light with a Data Logger (www.xenoncorp.com). 1.4.4.3 Near UV-Vis Light (NUV-Vis)
Novel technologies utilizing visible wavelengths of light, in the violet/blue region of the electromagnetic spectrum induces a phenomenon called “photodynamic inactivation (PDI)”. Traditionally PDI has involved the use of dyes and other exogenous photosensitiser molecules coupled with light exposure to induce inactivation of
Page 78 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki microorganisms, but recently natural photosensitiser molecules, particularly porphyrins endogenous within microbial cells have been targeted (Murdoch et al., 2013).
NUV-vis light 395±5 nm (figure 1.4.4.3.1) is a safe, non-UV based disinfection technology which is thought to act by stimulating endogenous microbial porphyrin molecules to produce oxidizing reactive oxygen species (ROS), predominantly singlet 1 oxygen ( O2) that damages cells leading to microbial death (Elman and Lebzelter, 2004, Feuerstein et al. 2005, Lipovsky et al. 2010, Maclean et al. 2008b, Murdoch et al., 2012). Exposure of microorganisms to visible light particularly at wavelengths of 405 nm, has been shown to be effective in inactivating a range of bacteria, including Gram- positive and Gram-negative bacterial species and antibiotic-resistant microorganisms such as Methicillin-resistant Staphylococcus aureus, and its use has been suggested for a range of decontamination applications (Dai et al. 2012, Dai et al., 2013, Enwemeka et al. 2008, Guffey and Wilborn 2006, Maclean et al. 2008a, Maclean et al., 2009, Maclean et al., 2010, Murdoch et al. 2012).
Figure 1.4.4.3.1: High intensity near ultraviolet/visible (NUV–vis) 395±5 nm light unit (Haughton et al., 2012).
1.4.4.4 Ultrasound
Power ultrasound consists of pressure waves with a frequency from 20 kHz to 10 MHz (Brondum et al., 1998, Butz and Tauscher, 2002). These cyclic sound pressure waves have a frequency beyond the upper limit of human hearing. Ultrasound can be classified to: a) low intensity ultrasound with a frequency range of 5-10 MHz with sound intensities in the range of 0.1 to 1 W/cm2 (diagnostic ultrasound) and b) high intensity
Page 79 Literature Review ultrasound with a frequency range of 20-100 kHz and a sound intensity ranging from 10 to 1,000 W/cm2 (McClements, 1995). Higher power ultrasound at lower frequencies is referred as power ultrasound (Piyasena et al., 2003) which has been recognized as a promising non-thermal processing technology, which can replace or complement conventional thermal treatment in the food industry.
Ultrasonic apparatuses are divided to three categories. All of them have their advantages and disadvantages which are summarized in table 1.4.4.4.1 and are shown in figures 1.4.4.4.1, 1.4.4.4.2, 1.4.4.4.3.
Table 1.4.4.4.1: Advantages-Disadvantages of electrochemical ultrasonic apparatuses (Zhou, 2010)
Figure 1.4.4.4.1: Figure 1.4.4.4.2: Figure 1.4.4.4.3: Ultrasonic Cleaning Bath Ultrasonic Probe or horn Ultrasound cup-horn (Elma-ultrasonic.com) (www.hielscher.com) (www.hielscher.com)
Page 80 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki The mode of action of ultrasound is based on cavitation phenomenon. The general mode of action is based on bubbles which pass through the liquid solution and create a series of compression/rarefaction (expansion/collapse) cycles creating a negative pressure affecting the molecules of the liquid. When the distance between the molecules exceeds the minimum molecular distance, the liquid breaks down and a void is formed. In successive cycles, voids or cavities continuously grow with a small amount of vapor from the liquid (Bilek and Turantas, 2013). There are two types of cavitation: transient and stable cavitation.
Transient cavitation is based on bubbles which are nonstable and collapse quickly in a very short time period and then disintegrate into a mass of smaller bubbles. Moreover, they are produced when sound intensity exceeds 10 W/cm2. The radius of bubbles expands to at least twice of their initial size, and then the bubbles collapse violently on compression into smaller bubbles. No mass transfer through the bubble by diffusion of gas is produced due to the short lifetime of transient bubbles, whereas evaporation and condensation of liquid might take place. The generation of extremely high temperature (5,000ᵒC) and pressure (2,000 atm) within these bubbles is believed to play an important role, thus causing sonoluminescence. The subsequent release of pressure from bubble implosion creates shock waves which may be responsible for surface cleaning and disinfection. A powerfull inrush of liquid to fill the void occurs due to the sudden collapse of the bubble. This in turn produces shear forces in the surrounding bulk liquid (Bilek and Turantas, 2013, Lauternborn and Ohl, 1997, Lee et al., 2005, Mason et al., 2002).
Stable cavitation is based on bubbles which are non-linear, have some equilibrium size during pressure cycles, and form large bubble clouds. These bubbles happen at low sound intensities (1-3 W/cm2), containing mainly gas and some vapour, which oscillate, often nonlinearly, during many acoustic cycles. Mass and heat transfer of gas through the bubble by diffusion of gas occurs due to the longer lifetime. The stable bubbles like transient bubbles are also accompanied by evaporation and condensation of liquid. The stable bubbles can be transformed into transient bubbles, but the violence of their implosion will be less than that of the transient bubbles due to the cushion effect of gas. Stable bubbles can also continue to grow, float to the liquid-gas interface and be expelled to air, which is the process of ultrasonic degassing (Mason et al., 2002).
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The mechanism of microbial killing is mainly due to increase of permeability of membranes and lost selectivity, thinning of cell membranes, localized heating (Suslick, 1998), and production of free radicals (Bilek and Turantas, 2013, Butz and Tauscher, 2002, Fellows, 2000, Piyasena, 2003). Moreover, the chemical effect of a 20 kHz ultrasound unit is correlated to the increase of inactivation of microorganisms due to the antimicrobial mechanisms of hydroxyl radicals (Bilek and Turantas, 2013, Butz and Tauscher, 2002). Many studies have shown that the temperature increase which is localized inside a collapsing bubble, generates hydroxyl radicals (Bilek and Turantas, 2013, Suslick, 1989). According to researchers, the hydroxyl radical (OH−) is able to react with the sugar-phosphate backbone of the DNA chain and cause the secession of the phosphate-ester bonds and breaks in the double strand microbial DNA (Bilek and Turantas, 2013). The effectiveness of a power ultrasound treatment is influenced by many factors, including the frequency and intensity of ultrasound, the solvent, gas type and content in working medium, treatment temperature, geometry of the reactor, uniformity of the acoustic field in the treatment chamber, and externally applied pressure (Zhou, 2010). In general, increasing ultrasonic frequency results in a decrease in the intensity of cavitation in liquids. Recently, surface decontamination of fresh produce with ultrasound has gained attention of many researchers (Alexandre et al., 2011, Sagong et al., 2011, Seymoor et al., 2001). The advantages of ultrasound over heat pasteurisation include: reduction of flavour loss, greater homogeneity, and possible energy savings.
1.4.5 Other Methods
1.4.5.1 Ozone
Ozone (O3) is a triatomic form of oxygen and is characterized by a high oxidation potential that conveys bactericidal and viricidal properties (Kim et al., 1999). Moreover, ozone has also been effective against fungi and protozoa (Khadre et al., 2001). Ozone results from the rearrangement of atoms when oxygen (O2) molecules are subjected to high-voltage electric discharge. The product is a bluish gas with a characteristic pungent odour and strong oxidizing properties. It is an environmentally friendly antimicrobial agent which can be used in both gas and liquid phases in the food industry as it does not produce toxic byproducts (Kim et al., 1999). The inactivation of bacteria by ozone is
Page 82 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki performed through an oxidation reaction. Inactivation of bacteria by ozone is a complex process because ozone attacks numerous cellular constituents including proteins, unsaturated lipids and respiratory enzymes in cell membranes, peptidoglycans in cell envelopes, enzymes and nucleic acids in the cytoplasm, and proteins and peptidoglycan in spore coats and virus capsids (Cho et al., 2010). Ozone also reacts with amino acids and modifies purine and pyrimidine bases in nucleic acid (Scott and Lesher, 1963). Ozone produces single strand breaks in DNA which cause extensive breakdown of DNA, resulting in loss of cell viability. Ito et al. (2005) proposed that ozone caused DNA backbone cleavage, due to production of hydroxyradicals. Ozone has been approved as safe (GRAS) for treatment of bottled water and as a sanitizer for process trains in bottled water plants (FDA, 1995). In 2001, ozone was approved as an antimicrobial agent in foods in the USA (USFDA, 2001). Scott and Lesher (1963) found that treatment with ozone causes alteration in E. coli cell membrane permeability leading to leakage of cell contents. Thanomsub et al. (2002) also supported that bacteria were inactivated by ozone as their cell membrane was destructed.
1.4.5.2 Pulsed Electric Fields (PEF)
The basic theory of microbial inactivation caused by PEF is based on electroporation of cell membranes, causing reversible or irreversible pore formation depending on the electric field intensity. The application of high voltage electric field (5–80 kV/cm) in short electric pulses (1–100 μs) is known to disrupt the cell membrane by generating a potential difference across cell membranes high enough to cause the membranes to “break down” (Jeyamkondan et al., 1999). This phenomenon is based on the formation of pores (electroporation). This results in loss of the semipermeability properties of the cell membranes, altering homeostasis and causing cell death (Teopfl et al., 2006). PEF treatment is carried out in liquid media, usually in continuous mode. However, permeabilization takes place only if a certain level of the electrical energy is exceeded. In order to profit from the advantages of PEF as a non-thermal technology, the temperature at every location in the chamber should remain low enough not to damage the valuable nutritive and sensorial qualities (figure 1.4.4.6.1). Hence, a detailed knowledge of temperature as well as field strength distributions in the chamber is necessary for an efficient application of PEF (Cullen, 2012).
PEF has also been successfully combined with other non-thermal technologies such as UV irradiation to achieve bacterial inactivation in food beverages (Noci et al., 2009,
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Walkling-Ribeiro et al., 2008), or with US (Halpin et al., 2014). Many researchers have studied the effect of PEF on food (Jeyamkondan et al., 1999, Teopfl et al.,2006).
Figure 1.4.4.6.1: Schematic Representation of PEF equipment (www.foodengineeringmag.com, www.intechopen.com)
1.4.5.3 High Pressure Processing
High pressure processing is another non-thermal method where the food is subjected to elevated pressures (in the range of 100–1000 MPa) (figure 1.4.4.7.1) to achieve inactivation of microorganisms and enzymes, without the severe degradation effects associated with flavor and nutrients (Ramos et al., 2013). The factors that are responsible for the successful implementation of this method are pressure, treatment time, and types of enzymes and/ or microorganisms (Guerrero-Beltrán et al., 2005).
Moreover, high quality products with a fresher taste are produced, as there is little heat damage to nutrients or natural flavors and colors. This effect can be attributed to the use of ambient or even chill temperatures (Ramos et al., 2013). The type of composition of the food can play an important role on the response of microorganisms during pressure treatment. Carbohydrates, proteins, lipids and other food constituents can confer a protective effect (Garcia-Graells et al., 1999). This is probably due to the fact that, in contrast to heat, HPP does not denature covalent bonds, which in turn leaves primary protein structure largely unaffected (Murchie et al., 2005).
Many researchers have already involved HPP in fruit and vegetable products processing. This treatment provides high quality food with higher safety and extended shelf-life, while maintaining similar characteristics to fresh products (Guerrero-Beltrán et al., 2005). Currently, the food industry has used HPP for products such as guacamole, cooked RTE meats, tomato-based salsa, fruit juices, whole-shell oysters, and other
Page 84 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki shellfish (Patterson, 2005). HPP food products are available in the United States, Europe, and Japan. Furthermore, combining HPP with other microbial agents such as lacticin 3147, lactoperoxidase and nisin has shown a synergistic effect regarding bacteria inactivation. The combination of HPP with alternative non-thermal treatments for use as a combined hurdle technology has also possibilities of enhancing the synergistic effect (Considine et al., 2008).
Figure 1.4.4.7.1: High pressure processing Unit (www.ibebvi.be)
1.4.5.4 Electrochemical (Cold Plasma) Method
An emerging antimicrobial non-thermal technology for decontamination is the use of non-thermal ionized gases (cold gas plasma). Briefly, plasma is composed of gas molecules, which have been dissociated by an energy input. It is constituted by photons, electrons, positive and negative ions, atoms, free radicals and excited or non-excited molecules that, when combined together, have the ability to inactivate microorganisms (Fernández et al., 2012). The action is based on the use of electricity and a carrier gas, such as air, oxygen, nitrogen, or helium. The primary modes of action are due to UV light and reactive chemical products of the cold plasma ionization process. More precisely, an electrical current is applied between an anode and cathode in an electrolytic solution containing water and a solution of highly electronegative anions. A mixture of oxygen and ozone is produced at the anode. The advantages associated with this method are the use of low- voltage DC current, no feed gas preparation, reduced equipment size, possible generation of ozone at high concentration and generation in water. The degree of inactivation can be affected by the type of microorganisms, the inactivation medium, number of cells, operating gas mixture, gas flow, and physiological state of cells, among others (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Studies on produce had shown that cold plasma is highly effective on the removal of surface human pathogens, such as E. coli O157:H7 and Salmonella spp. (Fernández et al., 2013, Misra et al., 2011, Wang et al., 2012).
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The use of bacteria, viruses, and bioengineered compounds (e.g. enzymes, bacteriocins) can prevent infections in fresh produce (Castaneda-Ramirez et al., 2011). Non- pathogenic bacteria and natural microflora on fresh produce encourage and discourage the survival and proliferation of pathogenic bacteria (Cooley et al., 2006, Gragg and Brashears, 2010, Heaton and Jones, 2008). Natural microflora and plant pathology play an important role on encouragement or discouragement of pathogens. Moreover, symbiosis has a key role in pathogen increase (Allen et al., 2009). Finally, the use of bacteriophages is another biological control method that is of importance. Bacteriophages are viruses that attack bacteria, degrade extracellular polymeric substances (Warning and Datta, 2013), and undergo either a lytic or lysogenic cycle. E. coli phages have been shown to significantly reduce the bacterial loads on cantaloupes, lettuce (Sharma et al., 2009), tomatoes, spinach, broccoli, and ground beef (Abuladze et al., 2008).
1.5 Control of foodborne diseases
Although preventing bacterial attachment and growth on the surface of fresh produces is nearly impossible, contamination can be controlled and reversed. Removal of bacteria from produce without lowering product quality can be facilitated through: mechanical removal, chemical death, biological control, or alternative disinfection methods. Mechanically removing bacteria is facilitated through the scrub of the surface. However, this method is not very effective against internalized bacteria. Chemical death is a very common method of removal, however, its use is continuously reduced due to the production of by-products. Finally, alternative disinfection technologies are used nowadays due to their enhanced effect against microorganisms (Warning and Datta, 2013).
Foodborne diseases once they emerge, they spread very fast. Thus, ways of reducing and preventing foodborne diseases must be addressed. Improving on-farm sanitation and biosecurity, antimicrobial use, and other good agricultural practices (GAP) can be of paramount importance (Tauxe, 2002).
Spoilage microorganisms can be introduced to the crop on the seed itself, during crop growth in the field, during harvest and postharvest handling, or during storage and
Page 86 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki distribution. The soil-borne spoilage microbes that occur on produce are the same microorganisms that are present on harvesting equipment, on handling and packaging equipment, in the storage facilities, and on food contact surfaces throughout the food distribution chain. Therefore, through the use of good agricultural practices (GAP), early intervention measures can be taken during crop development and harvesting. Examples of GAPs include foliar fungicide application in the field, cross-contamination prevention measures in the packaging equipment and storage facilities, and use of postharvest fungicides. In 1998, FDA published the Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables, recommending GAPs that growers, packers, and shippers must implement in order to address the common microbiological hazards that may be associated with their operations (FDA, 1998). GAPs can be implemented in: water, manure and municipal biosolids, worker health and hygiene, sanitary facilities, field sanitation, packing facilities sanitation, transportation and traceability (Tauxe, 2002).
Furthermore, hazard analysis-critical control point (HACCP) strategies, and controlling contamination during transport and storage are important. Training and certification of foodhandlers, as well as regular handwashing could prevent many infections (Tauxe, 2002).
1.5.1 Public Health Surveillance
Public health surveillance is based on the implementation of disease prevention programs. Surveillance is defined as a systematic collection of reports of specific health events as they occur in a population (Tauxe, 2002). Surveillance defines the current magnitude and burden of a disease for which prevention measures are in place. It identifies unusual clusters or outbreaks of the disease, in order to take actions to control them. Surveillance also measures the impact of control and prevention efforts, and it serves to reassure the public that this critical part of public safety is in place (figure 1.5.1.1). Surveillance may ensure that immediate control measures are implemented, and it may also identify areas that need more applied research so that better control measures can be developed (Tauxe, 2002).
Page 87 Literature Review
Figure 1.5.1.1: Cycle of the public health prevention (Tauxe, 2002).
It is true that published data of foodborne diseases is reported much too late after the events have occurred. This is the reason for limited data existence (Soon et al., 2011). However, it is known that complete data from large countries with a number of levels of government are difficult to obtain, due to local/regional/county or state burocracy. Furthermore, resources to conduct full traceback investigation are often limited (Todd, 1990). There may be substantial under-reporting in mild and common illnesses as most individuals for example regard diarrhea as an inconvenience rather than a symptom of disease, and hence may not consult the doctor. In addition, the general practitioner must order a stool culture, the laboratory must identify the etiologic agent and report the positive results to the local or state public health institution (Soon et al., 2011). Taking these limitations into consideration, the actual number of cases that occur is likely to be greater than the number of cases that are reported, and a surveillance pyramid is created (figure 1.5.1.2).
Page 88 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 1.5.1.2: Surveillance pyramid. The number of illnesses reported to public health department is limited compared to the total number of illnesses (Soon et al., 2011).
It is well understood from the surveillance pyramid that for every case that is reported, it has been estimated that 38 cases of salmonellosis occur. Once the food is implicated in an outbreak, then a detailed review of its production process can reveal the points of possible contamination sources. As a consequence, a multi-disciplinary approach is needed by risk assessors, in order to identify as soon as possible the hazard, thus developing strategies for eliminating it (Soon et al., 2011).
1.5.2 Food Legislation
Fresh cut RTE fruits and vegetables must adhere to the food laws of the country where they are grown, harvested, processed, transported, and sold to consumers by caterers and retailers. The applicable food law is important when they are subjected to be traded internationally. The Codex Alimentarius Commission (CAC) is the international intergovernmental organization for food standards, guidelines, and recommended practices. Special attention must be given to good practices, especially to Good
Page 89 Literature Review
Agricultural Practices (GAPs) and Good Manufacturing Practices (GMPs), for the successive stages in the food production chain. Good Hygienic Practices (GHPs) follow the produce from the beginning to the end of the production chain. Systems based on the Hazard Analysis and Critical Control Point (HACCP) principles are also applicable throughout the production chain. Local Governments publish also guidance documents to explain their legislation (Martín-Belloso and Soliva-Fortuny, 2011).
Generally, RTE foodstuffs should not contain microorganisms, toxins or metabolites in quantities that present an unacceptable risk for human health. In order to contribute to the protection of public health, Commission European Regulation (EC) No 2073/2005 establishes harmonized microbiological criteria for microorganisms, their toxins or metabolites in certain foodstuffs and includes a number of implementing rules. Food business operators at all stages of the food chain, i.e. primary production, processing, manufacturing, distribution, retail and catering must comply with the relevant criteria. Generally, compliance with Regulation on the Hygiene of Foodstuffs EC No.852/2004 as well as with the Directive 2000/13/EC, is of paramount importance.
1.5.3 Guidelines for the microbiological quality of RTE foods in Greece
Due to the fact that RTE food is consumed in the same state as that in which it is produced, sold and distributed, it must be given special attention to the microbiological criteria. Enterobacteriaceae are useful indicators of hygiene and of post-processing contamination of foods. Their presence in high numbers in RTE foods indicates that an unacceptable level of contamination has occurred or there has been under processing (e.g. inadequate cooking). The presence of E. coli in RTE foods is undesirable because it indicates poor hygienic conditions which have led to contamination or inadequate heat treatment. E. coli and Enterobacteriaceae are used as indicators of faecal contamination of RTE foods (EC. No 2073/2005). Ideally E. coli should not be detected in RTE foods, however, levels of E. coli exceeding 1000 CFU/g, are unacceptable and indicates a level of severe contamination. Furthermore, contamination of RTE foods with coagulase- positive staphylococci is a result of human contact. Contamination should be minimized through good food handling practices and growth of the organism prevented through adequate temperature controls. The presence of coagulase-positive staphylococci is considered as potentially hazardous, as this level of contamination may result in food
Page 90 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki borne illness. RTE foods should be free of Salmonella and Listeria (absent in fruits and vegetables in a sample of 25g), as consumption of food containing these pathogens may result in food borne illness (EC. No 2073/2005).
1.5.4 Predictive Models-Risk Assessment Support Systems and Public Health
In a processing operation, the basic principles of GMPs, HACCP, sanitation and documented operating procedures are commonly employed to ensure the production of safe products (FDA, 2006). The complexity of food systems and the large number of “critical points” in food production chain impose the necessity of the development of mathematical models for the prediction of food safety as well as the prevention of contamination. New practices, predictive models, methods and valuable tools have been emerging as complements to decisions taken in Food Science problems (FDA, 2006).
Several types of models are used ranging from qualitative (e.g., tree structure) to quantitative (e.g., microbial growth models) (Wijtzes et al., 1998). Prototype dynamic models which describe the growth and inactivation of a microbial population as a function of time and temperature have already been presented by Baranyi et al. (1996) and Van Impe et al. (1992).
Quantitative microbiological risk assessment (QMRA), predictive modeling (PM) and Hazard Analysis Critical Control Points (HACCP) have gained increased attention in food microbiology recently. Structures and tools have been created in order to ensure food safety by evaluating the safety of foodstuffs and predicting the effects of intervention measures in food production processes. HACCP is typically linked to industrial processes, whereas QMRA is more often used for public health purposes, in order to elucidate ‘farm to table’ models. HACCP system and QMRA studies study the potential bacterial growth and incorporate predictive food microbiology models. Moreover, PM can quantify the increase or decrease of bacterial population sizes (Nauta, 2002).
QMRA is the quantitative estimation of the risks posed to public health when food and pathogen are combined (Oscar, 2011). QMRA can be a useful tool in the development of scientific-based strategies to manage risks and safeguard public health. QMRA is based on: 1) hazard identification, 2) exposure assessment, 3) hazard characterization and 4)
Page 91 Literature Review risk characterization (Codex, 1999). The development of QMRA models focusing on fresh produce is important because RTE vegetables are mostly eaten raw, without a definitive cooking step before consumption.
Modeling bacterial survival curves have become an important issue nowadays, due to the increasing use of mild heat treatments for food products which have to guarantee the safety of the products and due to the increasing use of risk analyses aiming to offer a better control of the foodborne diseases (Geeraerd et al., 2000). Most microbial survival curves have a non-log linear behavior. Availability of effective survival models is needed if unbiased estimates of the probability of cross contamination are to be estimated (Pérez-Rodríguez et al., 2013).
Predictive models are excellent tools for assessing and controlling food safety, particularly when dynamic conditions are used (Ding et al., 2010). In the study of Koseki and Isobe (2005), the Baranyi model (Baranyi and Roberts, 1994) and the Ratkowsky model (Ratkowsky et al., 1982) were used in order to estimate the E. coli O157:H7 growth on non-packaged iceberg lettuce, and to predict growth parameters such as maximum growth rate, latent phase and maximum density of population, as a function of temperature (5-25°C) (Posada-Izquierdo et al., 2013). Posada-Izquierdo et al. (2013) have proposed a model which permits predictions over a wide range of temperatures and also incorporates variability, thereby making it suitable for Quantitative Risk Assessment (QRA) studies.McKellar and Delaquis (2011) have developed a secondary death growth model based on data from different studies dealing with E. coli O157:H7 growth in leafy vegetables. Predictive models that consider the influence of stresses such as washing in chlorinated water of MAP would provide more accurate and realistic estimates of risk (Posada-Izquierdo et al., 2013).
Meanwhile, Decision Support Systems (DSS) in the field of Food Science require flexibility, autonomy, intelligence, reliability but above all should be trusted by people related to Food Science. To fulfil all these diverse and difficult requirements, food scientists investigate new models and techniques that will integrate and combine known advanced theories and new techniques that will be the core of these sophisticated systems (Halder et al., 2011). A Decision Support System (DSS) is defined as any interactive computer - based support system for making decisions in any complex system, when individuals or a team of people are trying to solve unstructured problems on an uncertain environment. DSS are especially valuable in situations in which the
Page 92 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki amount of “scientific data’’ is prohibitive for the “human decision maker’’ to precede in solving difficult problems (Groumpos, 2010). Advanced DSS can aid human cognitive deficiencies by integrating various methodologies and tools utilizing a number of different information sources in order to reach “acceptable decisions”. The benefits in using DSS are that they increase efficiency, productivity, competitiveness, and offer cost effectiveness and high reliability. This could give to a food science business a comparative advantage over other competitors (Groumpos and Stylios, 2000).
Fuzzy Cognitive Maps are a combination of methods of fuzzy logic (FL) and neural networks. Fuzzy logic develops multi-valued, non-numeric linguistic variables for modelling human reasoning in an imprecise environment. FL has been applied in solving problems in crop management, soil and water, food quality and safety, animal health and behaviour, agricultural vehicle control, precision agriculture, greenhouse control, agricultural machinery, food processing, air quality and pollution, agricultural facilities, agricultural robotics, chemical application, and others such as natural resources management and agricultural product design. Artificial Neural Networks (ANNs) provide a way to emulate biological neurons to solve complex problems in the same manner as the human brain. ANNs have the largest body of applications in agricultural and biological engineering when compared with other soft computing techniques. ANNs have been applied in solving problems in food quality and safety, crop, soil and water, precision agriculture, animal management, post-harvest, food processing, greenhouse control, agricultural vehicle control, agricultural machinery (Huang et al., 2010).
Page 93 Literature Review
Page 94 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki AIM OF THE STUDY
The Health promotion and foodborne disease prevention remain important issues in the 21st century. Many outbreaks are reported and contaminated product recalls continue to occur. It is, therefore, very important to eliminate pathogens from foods because of the high risk, fatality rate, and economic burden of diseases (e.g listeriosis, salmonellosis etc) caused by principal foodborne pathogens like E. coli, S. aureus, S. Enteritidis, L. monocytogenes and HAV virus. Consumers nowadays prefer RTE foods, due to many advantages that they offer. Moreover, Mediterranean diet based on consumption of fruits and vegetables on an everyday basis remains popular. However, fruits and vegetables are associated with a number of illness outbreaks of human pathogens. Outbreaks of E. coli, S. aureus, S. Enteritidis, L. monocytogenes infections have been found to be associated with consumption of RTE lettuce, strawberries and tomatoes.
The aim of the present study was to study the effect of different emerging, sustainable disinfection technologies on the decontamination of artificially inoculated RTE foods. The scope was to evaluate their effectiveness as promising technologies to be used by food industries with the final insight to assure public health.
In the first part of the dissertation, an in vitro initial experiment took place. Liquids inoculated with indicator microorganisms (E. coli and L. innocua), -one Gram positive and one Gram megative-, representing potentially foodborne pathogens, were treated with alternative, non-thermal technologies for different treatment times. Three light technologies (NUV-Vis, Continuous UV, HILP) were used. In addition the disinfection efficiencies of Gram negative and Gram positive microorganisms when different light methods, as well as different dosages and treatment times of each method were used.
In the second part, the disinfection efficiency of minimally processed RTE fruits and vegetables (lettuce, strawberry, cherry tomatoes) inoculated with the indicator pathogens (E. coli, S. aureus, S. Enteritidis, L. innocua and HAdV) were investigated. The main objective of this study was to study the effectiveness of different chemical sanitizers on their capacity to adequately disinfect RTE foods. For this reason, conventional and alternative disinfection technologies were used. Immersions in NaOCL solutions of a low and a high concentrations with additional physical hurdles (UV, US), as well as combinations of the above treatments were used. Moreover, different initial
Page 95 Aim of the Study concentrations of the above microorganisms were inoculated, in order to evaluate the disinfection efficiency of the above technologies. For adenovirus, culture assays were performed in order to confirm the results obtained with PCR assay. Finally, after the use of selected disinfection technologies, storage at 6°C of the above products followed for 15 days, and analysis of RTE produces were conducted at day 3rd, 7th and 15th.
In the third part of the phD thesis, the effect of the treatments on selected quality (color) and physicochemical characteristics (total antioxidant capacity, total phenolic content, ascorbic acid concentration) of the above RTE foods were investigated. The above characteristics were measured before and after the treatments in order to evaluate their possible change.
In the fourth part, a predictive model based on Decision Support Systems (DSS) in the field of Food Science was constructed, in order to take decisions in the complex system of a vertical lettuce company, where individuals or a team of people are trying to solve unstructured problems on an uncertain environment. The DSS is based on Fuzzy Cognitive Maps are a combination of methods of fuzzy logic (FL) and neural networks. The beneficial tool will be of importance as it is a way to enhance the efficiency, productivity, competitiveness of any production company as well as offer cost effectiveness and high reliability. This risk assessment model could be a valuable tool to a food science business as a comparative advantage over other competitors exists.
Finally, after gathering all the above results, and taking into account that preventing bacterial attachment and growth on the surface of fresh produces is nearly impossible, contamination can be controlled and reversed. Thus conclusions of assessment and evaluation of the proposed disinfection technologies, based on infective doses for each pathogen were taken into consideration, in order to assure public health.
Page 96 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Chapter 2. MATERIALS AND METHODS
During the first experimental approach, in vitro experiments with liquids (MRD solutions) inoculated with E. coli and L.innocua were conducted. Then, disinfection with three light technologies (NUV, UV, HILP) took place and microbiological analysis followed for enumeration of bacteria in the treated samples.
At the second experimental approach, RTE foods were used for the experiments. Romaine lettuce, strawberries and cherry tomatoes were artificially inoculated with a cocktail of bacteria (E. coli, S. aureus, S. Enteritidis, L. innocua) and virus (HAdV-35) and then conventional, alternative and combined disinfection technologies followed for different treatment times. Furthermore, the disinfection capacity was tested when different concentrations of microorganisms were used. For adenovirus, culture assay was used in order to confirm the results obtained with PCR. Moreover, the effect of storage of the above treated RTE foods on their microbial load was evaluated for a period of 15 days.
During the third experimental approach, quality and physicochemical characteristics of the treated RTE foods were evaluated. Color, TAC, TPC and AA content were tested before and after the disinfection treatments.
The computerized model that was then proposed, as a fourth approach, was based on a Decision Support System (DSS) that used the decisions of three Experts for the effect of critical control points on a final safe product. The DSS is based on Fuzzy Cognitive Maps, which are a combination of methods of fuzzy logic (FL) and neural networks.
All the results obtained throughout the study were evaluated for their significance with different tests using SPSS 21.0 program.
During the experimental approaches, lab coat and gloves were mandatory, as they assured that aseptic conditions were kept throughout the experiments. Moreover, the RTE foods were kept in refrigeration in protective bags before, during and after the experiments.
Page 97 Materials and Methods 2.1 In Vitro Experiments with 3 Light Technologies
2.1.1 Equipment
Equipment Origin (Model, Company) UV Chamber Baro Applied Technology, Ireland NUV-Vis Unit AP Technologies, Bath, UK HILP Unit Xenon, USA K-Type Thermocouple Grant Instruments, Cambridge, UK Grant Data Logger Grant Instruments, Cambridge, UK Incubator Grant Instruments, Cambridge, UK Colony Counter IUL Instruments, Spain Fridge Tricity Bendix, UK Freezer Tricity Bendix, UK Centrifuge Beckman J2-HS, USA Electronic Balance Denver S-2002, Germany Autoclave Rodwell SS14 3SD, Biosciences, UK Vortex Genie 2, Scientific Industries, INC, K
2.1.2 Disposables- Plasticwares
Disposables-Glass-Plasticwares Origin (Company) Bottle top Dispenser - Pipettes (10 μL, 100 μL, 200 μL, 1 mL) Eppendorf Micropipette plastic tips (1000 μL, 200 μL and 10 μL) Sarstedt Gloves VWR Centrifuge and microcentrifuge tubes/bottles (1.5 mL, 15 mL, 50 mL) Sarstedt Gosselin, Villeurbanne, Stomacher bags France Glass tubes - Petri dishes 90mm diameter Sarstedt Sterile glass spreaders - Beakers (600 mL, 1 mL) - Volumetric flasks (50 mL, 100 mL) - Conical flasks (500 mL, 1 L, 2 L) - Sterile Food Pincers - Pyrex Bottles (500 mL, 1 L) -
Page 98 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.1.3 Culture Media
2.1.3.1 Selective Medium for E. coli (Tryptone Bile X-Glucuronide Medium (TBX, Oxoid)
36.6 g of TBX Medium were suspended in 1 L of distilled water and boiled to dissolve the medium. Then the medium was sterilized by autoclaving at 121°C for 15-min. The medium was then cooled to 50°C until it was finally poured into sterile Petri dishes. The plates were stored at 4°C.
2.1.3.2 Selective Medium for L. innocua (Listeria Selective Agar (Oxford Formulation, Oxoid)
27.75 g of the Listeria Selective Agar Base (Oxford Formulation) were suspended in 500 ml of distilled water. The solution was dissolved gently to the boil point. It was then sterilized by autoclaving it at 121°C for 15-min. It was then cooled to 50°C and aseptically the content of one vial of Listeria Selective Supplement (Oxford Formulation) was added, after diluting it in 50% ethanol:water solution. It was mixed well and poured into sterile Petri dishes. The plates were stored at 4°C.
2.1.4 Solutions for microbiological analysis
2.1.4.1 Tryptone Soya Broth (TSB, Merck)
30 g were added to 1 L of water (purified), mixed well and distributed into final containers. The medium was then sterilized by autoclaving at 121°C for 15 min. The broth was stored at 4°C.
2.1.4.2 MRD (Maximum Recovery Diluent, Oxoid)
9.5 g of MRD were dissolved in 1 L of distilled water. The solution was dispensed into the final containers and sterilized by autoclaving at 121°C for 15 min.
2.1.4.3 Microorganisms and Culture Preparation
Experiments were conducted using E. coli K12 (DSM 1607) and L. innocua (NCTC 11288). For inoculation of the model solutions, cultures of E. coli or L. innocua grown overnight at 37°C in Tryptone Soya Broth, TSB (Oxoid) were used. The 24 h cultures were then centrifuged for 10 min at 10,000 x g and the resulting pellets were washed and
Page 99 Materials and Methods centrifuged twice in Maximum Recovery Diluent (MRD, Oxoid) before being mixed together by resuspending in a final volume of 10 mL MRD. This resulted in mixed culture cell suspensions of ~108 colony forming units per milliliter (CFU/mL). The suspensions containing both E. coli and L.innocua inoculates were assessed for susceptibility to three light technologies in a liquid matrix (MRD). Samples (10 ml) were then placed into Petri dishes (50 mm diameter). After removal of covers, Petri dishes containing the MRD solutions were subjected to different light equipments.
2.1.5 Disinfection Light Treatments
2.1.5.1 High intensity NUV–vis light unit
The NUV–vis light was produced by a light-emitting diode (LED) array (OD-2049) (Opto Diode Corp) with a central wavelength of 395±5 nm, a bandwidth of 12 nm full- width at half maximum (FWHM) and a half intensity beam angle of 30°. The irradiance (J * cm-2) of light emitted from the LED unit was measured using a UV–VIS Radiometer (model no. RM12, Dr. Gröbel UV Electronik, GmbH, Ettlington, Germany) fitted with a RM UV-A sensor (part no. 811030, Dr. Gröbel UV Electronik) (figure 2.1.5.1.1). Distances of 3, 12 and 23 cm from the light source were chosen for treatments. The corresponding energy intensities and time needed to achieve them are presented in table 2.1.5.3.1. Sample temperatures were measured during the treatment using a K-type thermocouple attached to a Grant Data Logger to ensure that the maximum temperature reached was non-lethal to the bacteria under the treatment times investigated (<50°C).
Figure 2.1.5.1.1: Schematic representation of NUV Vis Equipment
2.1.5.2 Continuous UV Equipment
The UV unit was a custom-made unit with intimal dimensions (length × width × height) of 790 × 390 × 345 mm and consisting of four 95-W bulbs (Baro) 500 mm in length (figures 2.1.5.2.1, 2.1.5.2.2). The UV dosages (J/cm2) were varied by altering the Page 100 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki distance of the sample (6.5, 17, and 28.5 cm) from the light source and by changing the treatment time (table 2.1.5.3.1). Sample temperatures were measured during the treatment using a K-type thermocouple attached to a Grant Data Logger to ensure that the maximum temperature reached was nonlethal to the bacteria under the treatment times investigated (<0˚C).
Figure 2.1.5.2.1: Layout of UV treatment unit; 2, safety interlock; 3, treatment chamber with dimensions (length, width, and height) of 790 by 390 by 345 mm; 4, UV lights (95 W) 500 mm in length.
Figure 2.1.5.2.2: Custom made UV treatment unit (outside and inside the chamber)
2.1.5.3 HILP (High Intensity Light Pulses) Unit
The HILP unit was a benchtop SteriPulse-XL system (Xenon, USA) (figure 2.1.5.3.1). The system comprised a high-energy pulsed ultraviolet-visible flash lamp (Type C, 190 nm spectral cut-off point) delivering a maximum of 1.27 J/cm2. The pulse width produced was 360 μs at a fixed pulse rate of 3 Hz. The pulse energy delivered to the sample varied depending on its distance from the quartz window within the HILP chamber. Distances of 2.5, 8, 11.5 and 14 cm were selected for treatments, in order to achieve a wide spectrum of dosages varying between 0.18-106.2 J/cm2. The corresponding dosages and time needed to achieve them are presented in table 2.1.5.3.1.
Page 101 Materials and Methods
During HILP treatment, samples were placed in an iced bath to minimize heating. Sample temperatures were measured during the treatment using a K-type thermocouple attached to a Grant Data Logger to ensure that the maximum temperature reached was nonlethal to the bacteria under the treatment times investigated (<50˚C).
Figure 2.1.5.3.1: Representative Scheme of HILP Unit
Dose per treatment (J/cm2)
0.18 0.36 0.7 1.44 2.832 6 17.7 27 36 54 106.2
3 6 11 22 45 88 186 548 836 111 * *
VIS -
12 28 55 110 221 435 * * * * * * NUV
23 149 298 595 119 2341 * * * * * *
6.5 30 60 120 240 472 * * * * * *
17 UV 36 72 144 288 566 * * * * * *
28.5 45 90 180 360 708 * * * * * *
2.5 NT NT NT NT 0.8 NT 5 NT NT NT 30 Distance from light source (cm)
8 0.1 0.2 0.4 0.8 NT NT NT NT NT 30 NT
HILP
11.5 NT 0.3 0.6 NT NT 5 NT NT 30 NT NT
14 0.2 0.4 0.8 NT NT NT NT 30 NT NT NT
Table 2.1.5.3.1: Calculated exposure time (sec) of non-thermal light technologies at selected distances from the light source.(NUV-Vis: NUV–vis light; UV: Ultraviolet Light; HILP: High Intensity Light Pulses, *Samples that were not analyzed due to high temperature, NT: Not tested samples, due to inconsistency of correspondence of time and dose).
Page 102 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.1.6 Microbiological analysis
After treatment of liquid samples, the contents of each Petri dish were transferred to sterile containers. Ten-fold dilution series were prepared in MRD and 0.1 mL of each dilution was pour plated in duplicate using TBX (Oxoid) for E. coli and Listeria Selective Agar (Oxford formulation, Oxoid) for L. innocua. The plates were incubated at 44˚C and 37˚C for 24 and 48 h respectively. Mean counts for each treatment were calculated and converted to log10 CFU/mL values with results for surviving numbers of microorganisms in MRD expressed per mL (CFU/mL). The plates were then used to enumerate viable cells in untreated controls and in samples following processing. The survival of bacterial cells following illumination was monitored by counting their viable number after exposure of the suspended bacteria to light. Bacterial cultures grown under the same conditions but without light exposure served as controls. The results were expressed as the logarithmic reduction (log N/N0), where N0 is the initial microbial load and N the number remaining after treatment. All experiments were repeated at least three times.
Page 103 Materials and Methods 2.2 Food Disinfection
2.2.1 Equipment
Equipment Origin (Model, Company) Ultrasound Bath Elmasonic P60, Elma Ultraviolet Light 4 95-W lamps (Baero Hellas) Ultraviolet Light Cabinet 4 8-W lamps (Osram Germicidal G5) Autoclave Yamato Autoclave SM52 BagMixer, Interscience, St Nom la Bretêche, Stomacher France Bag Rack Interscience pH Meter Consort (C830) Refrigerator Frigorex Electronic Balance GF 3000-EC A&D Instruments LTD Incubators WTC Binder, Memmert Real-time PCR platform Stratagene MX 3005 Waterbath GFL D3006 DNA/RNA UV-Cleaner UVC/T-M-AR Colonies Counter WTC BZG 30 Scientific GE industries Bohemia n.y.11716 Vortex U.S.A Model K-550-GE Environmental Shaker-Incubator ES-29 Rocking platform Biosan
Incubator with CO2 Heal Force HF 90 Smart Cell Dynal MPC-S Magnetic Particle Concentrator A13346, Invitrogen Freezer Telstar Igloo Block Heater Stuart Scientific Bibby Refrigerated centrifuge(s) and rotor(s) Technolab Sigma 3K 30 Biosafety cabinet class II Cytair 155, FluFrance Centrifuge HermLe 2383 K Epifluorescence microscope ZEISS 2.2.2 Disposables- Plasticwares
Disposables-Glass-Plasticwares Origin (Company) Automatic Pipet Pipetboy Plus, Technomara Pipettes 10 μL, 100 μL, 200 μL, 1 mL Eppendorf Micropipette plastic tips (1000 μL, 200 μL and 10 μL) Sarstedt Gloves VWR Centrifuge and microcentrifuge tubes/bottles (1.5 mL, 15 mL, 50 mL) Sarstedt Tubes (1.5 mL) with screw caps Sarstedt Stomacher bags Gosselin, Villeurbanne, France Glass tubes - Petri dishes 90 mm diameter Sarstedt Sterile glass spreaders - Beakers (600 mL, 1 L) -
Page 104 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Disposables-Glass-Plasticwares Origin (Company) Volumetric flasks (50 mL, 100 mL) - Conical flasks (500 mL, 1 L, 2 L) - Sterile Food Pincers - 96-well polypropylene plates Agilent Technologies Optical adhesive covers Agilent Technologies TC-12-well Plates with Lid Cellstar, Greiner Bio-one Pyrex Bottles (500 mL, 1 L) - Flasks CellstarGreiner Bio-one 2.2.3 Culture Media
2.2.3.1 Selective Medium for E. coli (Tryptone Bile X-Glucuronide Medium (TBX), Merck)
36.6 g of TBX Medium were suspended in 1 L of distilled water and boiled to dissolve the medium. Then the medium was sterilized by autoclaving at 121°C for 15-min. The medium was then cooled to 50°C until it was finally poured into sterile Petri dishes. The plates were stored at 4°C.
2.2.3.2 Selective Medium for S. aureus (Baird-Parker Agar, Oxoid)
63 g were suspended in 1 L of distilled water and boiled to dissolve the medium. Then, the medium was sterilized by autoclaving at 121°C for 15-min. Then the medium was cooled to 50°C and were aseptically added 50 mL of Egg Yolk Tellurite Emulsion (SR0054, Oxoid). The medium was finally mixed well before pouring into sterile Petri Dishes. The plates were stored at 4°C.
2.2.3.3 Selective Medium for S. Enteritidis (Xylose-Lysine-Desoxycholate Agar (XLD) Agar, Oxoid)
53 g were suspended in 1 L of distilled water. The medium was heated with frequent agitation until boil. Then, it was transferred immediately to a water bath at 50°C. Finally, it was poured into sterile Petri dishes as soon as the medium was cooled. The plates were stored at 4°C.
2.2.3.4 Selective Medium for L. innocua (Listeria Selective Agar (Oxford Formulation, Oxoid)
27.75 g of the Listeria Selective Agar Base (Oxford Formulation) were suspended in 500 mL of distilled water. The solution was dissolved gently to the boil point. It was then
Page 105 Materials and Methods sterilized by autoclaving it at 121°C for 15-min. It was then cooled to 50°C and aseptically the content of one vial of Listeria Selective Supplement (Oxford Formulation) was added, after diluting it in 50% ethanol:water solution. It was mixed well and poured into sterile Petri dishes. The plates were stored at 4°C.
2.2.4 Solutions for microbiological analysis
2.2.4.1 Tryptone Soya Broth (TSB, Merck)
30 g TSB were added to 1 L of water (purified), mixed well and distributed into final containers. The medium was then sterilized by autoclaving at 121°C for 15-min. The broth was stored at 4°C.
2.2.4.2 0.1% Peptone Saline Solution (Bacteriological Peptone (Oxoid)
8.5 g sodium chloride and 1 g bacteriological peptone were suspended in 1 L distilled water. Then the pH was adjusted to 7.0±0.2. It was heated to dissolve the medium completely. It was finally sterilized by autoclaving 121°C for 15-min. The solution was stored at 4°C.
2.2.4.3 Buffered Peptone Water (Merck)
20.0 g of Buffered Peptone Water (ISO) were added to 1 L of distilled water. The solution was mixed well and was distributed into final containers. It was sterilized by autoclaving at 121°C for 15-min.
2.2.4.4 Sodium Hypochlorite Solutions
A low (50 ppm) and a high (200 ppm) concentration of sodium hypochlorite solution was prepared, by using a stock NaOCl solution of 14%. To prepare a 50 ppm solution, 0.357 mL was added to 1 L of sterile water and the pH was adjusted to 6.5. For a 200 ppm solution 1.43 mL of stock solution was added to 1 L of sterile water and the pH was adjusted to 6.5.
Page 106 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.2.5 Solutions for virus concentration
2.2.5.1 5X PEG/NaCl solution (50% (w/v) PEG 8000, 1.5M NaCl)
500 g PEG 8000 (Biochemica, AppliChem), 87 g NaCl (Sigma-Aldrich) and 450 mL molecular grade water were added to a bottle. The solution was mixed with gentle shaking/stirring until the solids were dissolved. Finally the final volume was adjusted to 1 L.
2.2.5.2 Chloroform:Butanol solution
Equal volumes of chloroform and butanol were added in a pyrex bottle. It was then shaked to mix.
2.2.5.3 Tris Glycine 1% Beef Extract (TGBE) Buffer
12.1 g Tris base (Tris-hydroxymethylaminomethan, Merck), 3.8 g glycine (Glycine Molecular biology Grade, AppliChem), 10 g beef extract powder (BBL, BD) and 1 L molecular grade water were added to a bottle. They were mixed with stirring until the solids are dissolved. The pH was adjusted to 9.5. It was then sterilized by autoclaving at 121°C for 15-min.
2.2.5.4 Phosphate Buffered Saline (PBS)
2 PBS tablets (Gibco) were added in 1 L deionised water. Using a magnetic stirrer, the PBS tablets were dissolved in the deionised water. Finally, PBS solution was sterilized in an autoclave at 121°C for 15-min.
2.2.6 Bacterial Strains
Bacterial strains used were Escherichia coli NCTC 9001, Staphylococcus aureus NCTC 6571, Salmonella Enteritidis NCTC 6676 and Listeria innocua NCTC 11288 (HPA, Colingdale, U.K). Lenticules with the microorganisms were rehydrated in 9 mL of peptone saline (0.1%) (Oxoid) and after 20-min, working cultures were streaked into Tryptic Soy Agar (Oxoid), incubated at 37°C for 24 h, and stored at 4°C. Bacterial strains were maintained as frozen stocks at -70ºC in the form of microorganism protective beads (Mast DIagnostixa GmbH).
Page 107 Materials and Methods
2.2.7 Cell lines and virus Adeno-35 stock
Human adenovirus serotype 35stocks were cultivated in human lung carcinoma cell line A549 cells. A549 cells were then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, NY, US), containing 4,5 g/L D-Glucose, L-glutamine and pyruvate supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, US). A549 cells were cultured confluent (80-90%) in 175 cm3 flasks at 37°C and 5% CO2, and infected with Adenoviruses serotype 35.Viruses were released from cells by freezing and thawing the culturing flasks for 3 times. A centrifugation step at 3000 × g for 20-min was applied to eliminate cell debris. The obtained supernatant was ultracentrifuged for 1h at 34,500 × g and finally resuspended in PBS, quantified and stored in 10 mL aliquots at −80 °C until used. The initial concentration of HAdV stock suspensions were quantified by Real-Time PCR and were calculated as 108-109 genome copies/mL.
2.2.8 Bacterial Preparation
Each bacterial type was cultured in 40 mL Tryptone Soya Broth (TSB) at 37°C for 18-20 h, harvested then by centrifugation at 4000 × g for 20-min at 4°C and washed three times with buffered peptone water (BPW). The final pellets were resuspended in BPW, corresponding to concentrations of approximately 107-108 CFU/mL, depending on different microbe.
2.2.9 Sample Selection
A leafy green vegetable such as romaine lettuce (Lactuca sativa L. var. longifolia), cherry tomatoes (Solanum lycopersicum var. cerasiforme) and strawberries (Fragaria x ananassa) were selected as fresh ready to eat produces in order to study the level of decontamination. These fresh produces were purchased from a local supermarket (Patras, Greece) the day of the experiment and stored under refrigerated conditions (4ºC) until the time of the experiment. As cherry tomatoes are concerned, a careful selection was made in order to secure a uniform maturity stage and size of samples with a color of light-red. Fruits with bruises, sign of infection or those different from the group were discarded from the samples. Uniform, unblemished tomatoes having similar size and color were finally selected.
Page 108 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.2.10 Sample preparation
All samples were rinsed with sterile water to remove some of the natural flora or any other matter before treatment. For lettuce, two to three outer leaves were discarded and the intact internal leaves were removed and weighted to give samples of 10 g. Likewise pieces of whole strawberries without calyx were weighted to give final weight of 10 g. Finally pieces of whole cherry tomatoes were weighted to give final weight of approximately 10-11 g.
2.2.11 Bacterial Cocktail
Bacterial cocktails (E. coli, S. aureus, S. Enteritidis, L. innocua) were prepared by mixing equal volumes of each bacterial type of high concentration in a 50 mL tube.
2.2.12 Sample Inoculation
A spot-inoculation method was used to inoculate the pathogenic bacteria on lettuce leaves and strawberry pieces (Mahmoud, 2010). Briefly, 100 μL (10 drops) of bacterial cocktail corresponding to 107-108 of each bacteria type was spotted with a micropipette on the surface of each produce. The bacteria cocktail was evenly applied throughout the skin surface of the strawberry, approximately midway between the calyx and cap (Bialka et al., 2008). In lettuce, the cocktail was placed to the centre (abaxial) outer surface of the lettuce, in order to simulate real conditions that can occur when contaminated compost and irrigation water can be transferred to lettuce leaves (Oliveira et al., 2011). 100 μL (10 drops) of deionized sterile water was spotted on the surface of control samples. To allow bacterial attachment, the samples were air dried on sterile aluminum foil in a class II biosafety cabinet for 2 hours in 25°C prior to treatments. The fact that the inoculums were attached to the vegetable surfaces was verified by comparing the results with a control sample containing food only in BPW for 60-min. Whereas, a dipping inoculation method was used to inoculate the pathogenic bacteria on cherry tomatoes (Hadjok et al. 2008). Briefly, batches (10-11 g) of whole cherry tomatoes were submerged in a beaker containing 300 mL of bacterial suspensions diluted in PBW corresponding to 106–107 of each bacteria type and were agitated in a shaker for 1h. The samples were then air-dried for 1h to allow bacterial attachment.
Page 109 Materials and Methods
2.2.13 Virus inoculation
The spot-inoculation method was used to inoculate the adenoviruses on the ready to eat produces. Briefly, 100 μL (10 drops) of Adeno-35 corresponding to concentration of 108–109 infectious units/mL was spotted with a micropipette on 10 different areas of the surface of each produce. After spiking, to allow viral attachment, the samples with inocula were dried in a class II biosafety cabinet for 20-min at 22±2°C prior to treatments.
2.2.14 Disinfection Treatments
2.2.14.1 Chlorine Treatment
For the chlorine treatment, the inoculated samples were immerged in a beaker containing 100 mL of chlorinated water (NaOCl solution) of 2 concentrations (50 ppm and 200 ppm) and were handed agitated for 3-min at 22±2°C. After that, the samples were transferred to a beaker with sterile water and were left for another 3 minute period. Finally, they were left on a sterile paper for drying. The sodium hypochlorite treatments used are included in table 2.2.14.1.1.
Treatments Sodium Hypochlorite (ppm) Time (minutes) 1 50 1 2 50 3 3 50 5 4 200 1 5 200 3 6 200 5
Table 2.2.14.1.1: Sodium Hypochlorite Treatments selected throught the experiments.
2.2.14.2 Continuous UV Treatment
A UV cabinet with four UV-C (Osram Germicidal G5) lamps was used. The peak emission of the lamps was 254 nm. The inoculated samples were placed in sterile petri dishes and were placed at 8 cm distance from the lamps and treated for 1, 3, 5, 10, 20, 30, 45 and 60-min. The treatment was conducted at an intensity of 2 mW/cm2 at dosages 0.12, 0.36, 0.6, 1.2, 2.4, 3.6, 5.4 and 7.2 J/cm2. Throughout the experiments, the UV-C
Page 110 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki light intensity was kept constant, and the applied doses varied by altering the exposure distance and time.
The UV dose (D) was calculated by using the equation 1 (1.4.4). The Intensity measured in the UV chamber was 2 mW/cm2 in a 8 cm distance from the lamp. The Energy (mJ/cm2) delivered was calculated according to the time (table 2.2.14.2.1).
Treatments Time (minutes) Intensity (mW/cm2) Energy (J/cm2) 1 1 2 0,12 2 3 2 0.36 3 5 2 0.6 4 10 2 1.2 5 20 2 2.4 6 30 2 3.6 7 45 2 5.4 8 60 2 7.2
Table 2.2.14.2.1: Corresponding Energy-Intensity- Time for different UV treatments selected for the experiments.
2.2.14.3 Ultrasound Treatment
For the Ultrasound treatment, a 5.75 L ultrasound tank (Elmasonic, Germany) was filled with 3 L of distilled water and used at an operating frequency of 37 kHz and a power up to 30 W/L. A glass beaker (600 mL) was placed in the US tank and filled with 9-fold dilution of distilled water (figure 2.2.14.3.1). The ratio fruit (strawberry) or vegetable (lettuce or cherry tomatoes) to distilled water for the ultrasound treatments was 1 part of food (10 g) to 9-fold dilution of liquid. Inoculated lettuce leaves, strawberries and cherry tomatoes were immersed in the glass beaker and processed with US at a constant frequency of 37 kHz for 1, 3, 5, 10, 20, 30, 45 and 60-min. At least three replicates of each treatment were performed (table 2.2.14.3.1).
Treatments Ultrasound Frequency (kHz) Time (minutes) 1 37 1 2 37 3 3 37 5 4 37 10 5 37 20 6 37 30 7 37 45 8 37 60
Table 2.2.14.3.1: Various ultrasound treatments for various treatment times. Page 111 Materials and Methods
Figure 2.2.14.3.1: Ultrasound equipment outside and inside (Elmasonic P, Elma, Germany).
2.2.14.4 Combined Treatments
The combined treatments consisted of combinations of alternative technologies as well as of combinations of alternative and conventional disinfection technologies (table 2.2.14.4.1).
Combined Disinfection Treatments Treatments Time(minutes) Alternative+Conventional UV+NaOCl 1+3 Alternative+Conventional UV+NaOCl 3+3 Alternative+Conventional UV+NaOCl 5+3 Alternative+Conventional UV+NaOCl 10+3 Alternative+Conventional UV+NaOCl 20+3 Alternative+Conventional UV+NaOCl 30+3 Alternative+Conventional US+NaOCl 1+3 Alternative+Conventional US+NaOCl 3+3 Alternative+Conventional US+NaOCl 5+3 Alternative+Conventional US+NaOCl 10+3 Alternative+Conventional US+NaOCl 20+3 Alternative+Conventional US+NaOCl 30+3 Alternative+Alternative UV+US 5+5 Alternative+Alternative UV+US 10+10 Alternative+Alternative UV+US 10+20 Alternative+Alternative UV+US 20+10
Table 2.2.14.4.1: Combined Disinfection Treatments for various treatment times.
2.2.15 Storage conditions
After each treatment, samples were stored under refrigerated conditions (4±1 °C) in refrigerator. The temperature of the fridge was monitored using a calibrated thermometer. Samples were collected throughout different storage time intervals (3rd
Page 112 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki day, 7th day, 15th day) and analyzed in terms of bacterial populations for a total of 15 days storage.
Selected treatments (7 in total) were examined after 3 days of storage (3rd, 7th, 15th) which were: UV (30-min), US (30-min), NaOCl 200 ppm (3-min), UV 30-min followed by NaOCl 200 ppm 3 -min, US 30-min followed by NaOCl 200 ppm 3-min, UV 10-min followed by US 20-min and UV 20-min followed by US 10-min.
2.2.16 Microbiological Analysis
For enumeration of bacteria, 10 g of treated lettuce, strawberry or cherry tomato sample was transferred into a sterile stomacher bag containing 90 mL of Peptone Buffer water (PBW) and homogenized in a stomacher for 2-min. One mL of the homogenized sample was then 10-fold serially diluted in 9 mL of sterile PBW, and appropriate dilutions were pour or spread-plated into appropriate selective media. All samples were analyzed according to ISO standard methods (table 2.2.16.1).
Selective Culture Microorganisms IncubationTemperature ISO Method Media E. coli TBX 44 ± 1 °C 16649-1:2001 S. aureus Baird Parker 37 ± 1 °C 6888-1:1999 S. Enteritidis XLD 37 ± 1 °C 6579:2002 L. innocua Oxford Listeria Agar 37 ± 1 °C 11290:1996
Table 2.2.16.1: ISO Methods implemented throughout the experiments for E. coli, S. aureus, S. Enteritidis and L. innocua enumeration.
2.2.17 Bacteria Enumeration
Reductions of bacteria were calculated on a per Gram of fruit and vegetable basis. Mean counts for each treatment were calculated and converted to log10 CFU/g values. The results were then expressed as the logarithmic reduction (log N/N0), where N0 is the initial microbial load and N the number remaining after treatment. All experiments were repeated at least three times. Negative and Positive Controls were included.
Page 113 Materials and Methods
2.2.18 Analysis for Detection of Viruses
2.2.18.1 Virus concentration from fresh produce surfaces
The sample was processed by the method of Dubois et al. (2006) as described by Kokkinos et al. (2012) with slight modifications.
• Approximately 25 g of vegetable and fruit pieces were weighed and transferred to a sterile beaker. • 40 mL of TGBE buffer were added to each sample. • The samples were agitated at room temperature for 20-min by rocking at 60 rpm. • The eluate was decanted from the beaker through a strainer into one 50 mL or two smaller centrifuge tubes. • The sample was centrifuged at 10,000 × g for 30-min at 4ºC. Then the supernatant was decanted into a single clean tube. • The pH of the sample was adjusted to 7.2 with Hydrochloric acid (1 N and 0.1 N). • 0.25 volumes of 5 × PEG/ NaCl solution were added and mixed by inversion, then incubated with gentle rocking at 4ºC for 60-min. • The tube was then centrifuged at 10,000 × g for 30-min at 4ºC. • The supernatant was discarded. • It was centrifuged at 10,000 × g for 5-min at 4ºC to compact pellet. • Finally the pellet was resuspended in 500 μL PBS and 500 μL chloroform:butanol solution (1:1) and mixed by vortexing.
• It was allowed to stand for 5-min.
• Finally it was centrifuged at 10,000 × g for 15-min at 4ºC and the aqueous phase was transferred to a clean tube and stored at -20ºC.
2.2.18.2 Virus concentration from fruits
The procedure was the same with the vegetables. The only difference was indicated at the initial phases. After 10-min of agitation at room temperature by rocking at 60 rpm the pH of the eluate was checked. If the pH fall below 9.0 it was adjusted to 9.4 with sodium hydroxide (4% w/v). The period of agitation was extended by 10-min for every time the pH was adjusted. Then, the following procedure was the same and finally the aqueous phase was transferred to a clean tube and stored at -20ºC.
Page 114 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.2.18.3 DNAseTreatment
An enzymatic digestion treatment was applied to reduce false positive results by detection of free DNA when using qPCR analysis (Nuanualsuwan and Cliver, 2002). Each analyzed sample was treated with DNase I (free – RNase) (DNase I, Molecular Grade, Invitrogen), before DNA extraction to degrade DNA released from damaged viral capsids, according to manufacturer's instructions, before the nucleic acid extraction.
Briefly, 400 μL of concentrated sample was added to 2.5 μL of DNase I and 97.5 μL Reaction Buffer and incubated for 2 h at 37°C.
2.2.18.4 Nucleic Acid Extraction The principle of the protocol used for Nucleic Acid Extraction is based on the Nuclisens miniMAG (Biomerieux, Paris) using Boom technology and magnetic silica (figure 2.2.18.4.1). The steps followed for nucleic extraction are: • 500 μL of the extract from soft fruits or vegetables were added into a clean centrifuge tube. • 4.5 mL of Nuclisens lysis buffer were added to the tube, and were mixed by vortexing briefly. • The sample was incubated for 10-min at room temperature. • Centrifugation for 2-min at 1,500 × g was followed to ensure that entire sample was brought down into the tube. • 50 μL of well-mixed magnetic silica solution was added to the tube and mixed by vortexing briefly. • Incubation for 10-min at room temperature followed. • Centrifugation for 2-min at 1,500 × g and then the supernatant was carefully discarded. • 400 μL wash buffer 1 was added and resuspension of the pellet by pipetting/vortexing. • Transfer of suspension to a 1.5 mL screw-cap tube. (It was very important to avoid creating foam at this stage. Very gentle pipetting should be done when using wash-buffer 1, because of the GuSCN incorporated into it. This avoided any loss of nucleic acids). • It was washed for 30 sec using the automated wash steps of the miniMAG extraction systems.
Page 115 Materials and Methods
• After washing silica was allowed to settle using magnet of the magnetic rack. • The supernatant was discarded. • Separation of the tubes from magnet followed. • 400 μL of wash buffer 1 was added. • Pellet was resuspended and washed for 30 sec. • After second washing, silica was allowed to settle using magnet. • The supernatant was discarded. • Separation of the tubes from magnet. • 500 μL wash buffer 2 were added. • Pellet was resuspended and washed for 30 sec. • Silica allowed tosettle using magnet. • The supernatant was discarded. • After this washing with Buffer 1, the total sample was transferred to a clean 1.5 mL tube to eliminate GTC residues. • Washing with Buffer 2 was repeated. • The tubes were separated from magnet. • 500 μL wash Buffer 3 were added. • Washing for 15 sec was followed to allow silica to settle using magnet. • Supernatant was discarded. • 50 μL elution buffer were added, and the tubes were transferred to thermoshaker • Incubation for 5-min at 60ºC was followed. • Tubes were placed in magnetic rack and allow silica to settle. • Transfer of the eluate to a clean tube. • Repetition of the step with elution Buffer (total volume of the eluate 100 μL). • The samples were retained at -80ºC for up to one week.
Page 116 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 2.2.18.4.1: Schematic Presentation of the procedure of Nucleic Acid Extraction (Kokkinos et al., 2012).
2.2.18.5 PCR Quantification
The mix was prepared in DNA/RNA UV-Cleaner room according to the tables (tables 2.2.18.5.1 and 2.2.18.5.2). Firstly, the stock volumes of primers and probes were prepared (table 2.2.18.5.1).
Final Stock volume H2O volume Molarity
Primers (AdF, AdR) 225 μL 275 μL 500 μL 45 μΜ
Probe AdP1 56.25 μL 443.75 μL 500 μL 11.25 μΜ
Table 2.2.18.5.1: Working solutions of primers and probe.
Page 117 Materials and Methods
Then, the PCR mix was prepared according to the table 2.2.18.5.2.
Working Final Volume Reagent Concentration Concentration (μL)
Mix 2x 1x 12,5
Primer AdF 45 900 nM 0,5
Primer AdR 45 900 nM 0,5
Probe AdP1 11,25 225 nM 0,5
H2O 1
Total volume of PCR mix 15
Table 2.2.18.5.2: Volumes of reagents for PCR mix.
Once the mix has been prepared aliquots of 15 μL were added into each well. The total volume for one reaction after addition of target was 25 μL (15 μL mix + 10 μL sample). The samples were then added in duplicate in a separate area. DNA standard was added as positive control (PAC) in duplicate. Finally, 10 μL of nuclease-free dd-water were added in the Non-template control (NTC) wells. The assay included a NTC to prove mix does not produce fluorescence. Whereas the PAC must be added to verify that the reaction has worked and has not failed. The wells were then closed with adhesive cover. The QPCR was performed in a real-time PCR platform, selecting the appropriate parameters (considering the use of adhesive cover and the total volume in each well, etc). Following activation of the UNG (2-min, 50°C) and activation of the AmpliTaq Gold for 10-min at 95°C, 45 cycles (15 s at 95°C and 1 minute at 60°C) were performed. Once the reaction is completed, the results are stored. The amount of DNA was calculated as GC/mL and was converted to log10.
2.2.19 Evaluation of disinfection with different initial bacteria cocktail
The RTE foods were inoculated with different initial concentrations of bacteria and virus. For bacteria cocktail experiments, serial dilutions of high cocktail inocula were done, and then the RTE foods were inoculated with different initial concentrations. Then, selected disinfection treatments followed and RTE foods were subsequently microbiologically analyzed.
Page 118 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.2.20 Culture Assay for HAdV35
Briefly, A549 monolayers were incubated overnight in 12-well plates at 37 °C in 5%
CO2 until they reached 90–100% of confluence. Thirty microliters (30 μL) of direct and diluted samples were inoculated into each well and incubated for 90-min at 37 °C on a shaking incubator. After that, the media with the inoculum was discarded and 1% FBS- supplemented with DPH (DMEM supplemented with hepes and pest). The flasks were incubated for 3-4 days at 37 °C in 5% CO2. Finally, cells were observed under an epifluorescence microscope for cytopathic effect. The theoretical detection limit of this technique is 10 viral particles/mL and even viral particles that have lost their ability to develop cytopathic effect may be still detected. The final result of each sample analyzed was expressed as the geometric mean of the most probable number of cytopathic units (MPNCU) per milliliter calculated for two independent replicates. All assays were performed in triplicate and negative and positive controls were included.
Page 119 Materials and Methods 2.3 Food Quality parameters
2.3.1 Color Measurement
A colorimeter (CIE colorimeter) was used for color measurements. All the treated samples were surface dried; they were positioned then in a plastic bag and held on ice until all experiments have been completed. A lettuce, a strawberry piece and cherry tomatoes (10 g) were then placed directly on the colorimeter sensor and measured in at least three different points of the product. Three measurements were performed per treatment and results were averaged. The L* parameter shows lightness to darkness and ranges from 0 (black) to 100 (white). The a* parameter measures the degree of redness (+a*) or greenness (-a*). The b* parameter indicates the degree of yellowness (+b*) or blueness (-b*). The net color difference (ΔE*) and chroma or saturation index (C*) were determined using L*, a* and b* values and were compared with the values of unprocessed samples (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Finally, the Tomato Color Index (TCI) and the Whiteness Index (WI) were calculated for cherry tomatoes and lettuce respectively as described by Clément et al. (2008) and Obande et al. (2011). The above calculations were calculated according to the following equations:
C*= (a*2 + b *2) ½
WI= 100 – [(100 - L*) 2 + a*2 + b*2]0.5
Page 120 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.3.2 Physicochemical Parameters
2.3.2.1 Equipment
Equipment Origin (Model, Company) Ultrasound Bath Elmasonic P60, Elma Absorption Spectrophotometer Hitachi U-1900 Colorimeter CIE Colorimeter Electronic Balance AND HR-300 pH meter Consort (C830) Waterbath Edelstahl, RostFrei Stirrer Heidolph Ruhren
2.3.2.2 Disposables
Disposables, Glass-Plasticwares Origin (Model, Company) Burette - Spectrophotometer cuvettes - Plastic Tubes (15 mL, 50 mL) Sarstedt Conical Flasks - Filter papers - Pipettes 10 μL, 100 μL, 200 μL, 1 mL Eppendorf Micropipette plastic tips (1000 μL, 200 μL and 10 μL) Sarstedt
2.3.2.3 Chemicals
Chemicals, Reagents Origin (Company) Acetone Sigma Sodium Acetate Sigma Acetic Acid Glacial 100% Merck TPTZ (2,4,6 Tris 2-Pyridyl-s-triazine) Sigma-Aldrich FeCl3• 6H2O (Iron (III) chloride Hexahydrate) AnalaR BDH Hydrochloric Acid Fuming 37% Merck Folin-Ciocalteau Reagent Merck Sodium Hydrogen Carbonate AnalaR Gallic Acid monohydrate Sigma-Aldrich 2,6 Dichlorophenolindophenol Sigma-Aldrich Sodium salt hydrate Meta-Phosphoric Acid Sigma-Aldrich L-Ascorbic Acid AnalaR BDH
Page 121 Materials and Methods
2.3.2.4 Methods
2.3.2.4.1 Samples extraction
The samples were extracted using 60% acetone (v/v) and distilled water. Each sample of 2 g was weighed into conical flask and 50 mL 60% acetone solution was added. The samples were then placed in an ultrasound bath at temperature 30˚C for 2 hours. The “extracts” were then filtered to obtain a clean solution. The extracts were finally left in a dark place to cool.
2.3.2.4.2 Total antioxidant capacity
The total antioxidant capacity of samples was determined according to FRAP (Ferric Reducing Antioxidant Power) of Benzie and Strain (1996), as introduced in section 1.2.1.1.1. This method is based on the reduction of Fe+3-TPTZ yellow complex to the ferrous blue form (Fe+2) by the antioxidants of sample.
2.3.2.4.2.1 FRAP reagents
- Acetate buffer solution 0.3 Μ, pH=3.6.
In order to prepare this solution, 3.1 g of sodium acetate were mixed with 16 mL of acetic acid and were diluted in 1 L of buffer solution with an adjusted pH. This solution was prepared once and used throughout the experiments.
- Solution 40 mM HCl
1.6 mL of HCl Fuming 37% were added to 500 mL of water.
- Solution TPTZ 10 mM.
This solution was a solution of 10 mM TPTZ in 40 mM of HCL. This solution was prepared every 2 days and was maintained in the fridge.
- Solution FeCl3•6H20 20 mM
This solution was prepared by dilution of 1.3525g FeCl3•6H20 in 250 mL of distilled water. This solution was prepared once and was used throughout the experiments.
- FRAP reagent
Page 122 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki It was prepared by mixing 25 mL of acetate buffer solution, 2.5 mL of TPTZ solution and 2.5 mL FeCl3 solution. This solution was prepared on a daily basis, before the experiments were carrying out. It was maintained in 37°C in a water bath. After the end of experiments the solution FRAP was thrown away.
2.3.2.4.2.2 Total antioxidant capacity (TAC) determination
Briefly, 1 mL of sample was added to 5 mL FRAP solution and was left in a dark place for 30-min. The absorbance was recorded after 30-min incubation at 595 nm. Control samples were also measured. The results were based on a standard curve of 2+ FeSO4•7H20. The results were finally expressed in μmol Fe equivalents per g of fresh weight of food sample.
2.3.2.4.3 Total Phenolic Content (TPC) Determination
The determination of total phenolic content was measured according to the method of Spanos and Wrolstad (1990) using Folin-Ciocalteau reagent. Briefly, 3 mL of each filtered sample, plus 1 mL Folin-Ciocalteau reagent, plus 1mL sodium carbonate (7.5%) were added. After incubation at room temperature in a dark place for 60-min, the absorbance of the reaction mixture was measured at 765 nm against aquatic methanol blank on a spectrophotometer. Standards were prepared from gallic acid in water. From the standard curve, the total phenolic contents of samples were expressed as mg gallic acid/g of fresh weight of food sample.
2.3.2.4.4 Determination of Vitamin C
The determination of ascorbic acid was made by titration against 2.6- dichlorophenolindophenol. The key point was to carry out the titration in an acid environment and rapidly.
2.3.2.4.4.1 Reagents needed for the determination of Vitamin C:
- Ascorbic acid solution
Preparation of ascorbic acid solution 1 mg/mL in metaphosphoric acid solution.
-Metaphosphoric acid solution
Page 123 Materials and Methods
15 g HPO3 were dissolved in 40 mL CH3COOH and 200 mL deionised water. Dilution was made in 500 mL deionised water. HPO3 was converted gradually to phosphoric acid. This solution was unstable and was kept in fridge. It was thrown away after 1 week in order to avoid the conversion into orthophosphoric acid.
- Standard solution 2.6 dichlorophenolindophenol
50 mg of meta sodium salt of 2.6 dichlorophenolindophenol were dissolved in 50 mL of deionised water which had 42 mg NaHCO3 content. The solution was diluted to 200 mL water, then it was filtered from folded filter and it was kept in a dark bottle.
2.3.2.4.4.2 Ascorbic Acid determination
Food extracts (2 mL) were inserted in a conical flask. The titration was made rapidly with standard solution of 2.6 dichlorophenolindophenol until a slight pink color appeared. This pink color must be conserved for at least 5 seconds. The determination was carried out in triplicates.
In the same time, the standard solution of ascorbic acid was titrated in order to find out its content in ascorbic acid, more precisely in order to correspond the amount (mg) of ascorbic acid to amount (1 mL) of pigment solution.
The Ascorbic Acid determination was made according to the following equation:
mg ascorbic acid/mL = (X – B) × (F/E) ×(V/Y)
X - average volume for test solution titration (mL)
B - average volume for test blank titration (mL)
F - mg ascorbic acid equivalent to 1.0 mL indophenol standard solution,
E - number of g assayed
V - volume of initial test solution
Y - volume of test solution titrated
Page 124 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain
The purpose of the mathematical model was to focus on the construction and the use of FCM in modelling a fit-for-purpose Decision Support System which diagnoses the possibility of the cross-contamination of lettuce from production to the point of sale in a vertical production company of vegetables.
2.4.1 Selection of critical points
Nine critical points were selected from three (3) experts, with different scientific background, as they play a crucial role throughout the lettuce production procedure (figure 2.4.1.1). The selection of critical points was based on background information questionnaires, based on HACCP audit principles, which were completed for the premise during the European FP7 project VITAL (Integrated Monitoring and Control of Foodborne Viruses in European Food Supply Chains) (http://www.eurovital.org) aimed to gather data on virus contamination with the aim of providing a basis for subsequent quantitative viral risk assessment and recommendation of control measures. These critical points were selected among others as the most important ones to be taken seriously into consideration, in order to be able to estimate the infection risk for humans through consumption of the leafy vegetables.
1. Labour, Manpower
Vegetable production is very labour intensive work which requires both dedication and skill to effectively undertake it. Basic training in agronomic principles or experience in the same field is very crucial. Moreover, hygiene training is of great importance. Lettuce/leafy greens may be harvested mechanically or by hand and are almost always consumed uncooked or raw. Because lettuce/leafy greens may be hand-harvested and hand-sorted for quality, there are numerous “touch points” early in the supply chain and a similar number of “touch points” later in the supply chain as the products are used in foodservice or retail operations. Each of these “touch points” represents a potential opportunity for cross-contamination (FDA, 2006). Emphasis in hand washing where there is risk of contamination (e.g. before starting work, after using the bathroom, etc.) must be given. Workers with any notifiable infectious disease must be excluded from
Page 125 Materials and Methods work. Harvesting equipment (knifes) should be cleaned and/or sanitised daily. Suitable protective clothing has to be worn by food workers, except for disposable gloves.
2. Quality and Safety of Food Systems
The existence of Good Agricultural Practices (Global, GAP) is a necessity in a vegetable company. Internal and external auditing must be in place. Quality systems, i.e. ISO 22000, Sanitation Standard Operating Procedures (SSOPs) must be present throughout the food supply chain. HACCP guidelines are also important so as to ensure the safe production and handling of lettuce/leafy greens products from field to fork (FDA, 2006). The Recommended International Code of Practice General Principles of Food Hygiene (CAC, 2003) indicates that “Prior to application of HACCP to any sector of the food chain, that sector should have in place prerequisite proGrams such as good hygienic practices according to the Codex General Principles of Food Hygiene, the appropriate Codes of Practice, and appropriate food safety requirements” (Garayoa, 2011).
3. Location-Surroundings of the growing field
Caution must be given with domestic animals which in primary production must not have access or presence on the premises. Moreover, emphasis must be given to the place of storage of raw manure and to the existence or not of any industrial, and/or farming activity adjacent to the field. Fields that contain animal manure are more likely to be contaminated with enteric pathogens because of their ability to survive in soils for months or years (Doyle and Erickson, 2008). Faeces may naturally contain between 102 and 105 CFU/g E. coli and between 102 and 107 CFU/g Salmonella spp. (Himathongkham et al., 1999, Olaimat and Holley, 2012).
4. Lettuce Nursery
The quality of lettuce nursery is of great importance. Melotto et al. (2006) reported that phytopathogen infections, which occur frequently during field cultivation, could affect the interaction between human pathogens and plants (Ge et al., 2013). Microbial and chemical testing of products can be carried out in accredited laboratories on a scheduled base and the results must be satisfactory. A labelling and traceability system as well as a recall system must be in place.
Page 126 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 5. Produce Land
Lettuce grows best in fields that are level and well drained. Lettuce is highly sensitive to salinity. High salinity causes one of the most widespread types of abiotic stress worldwide, severely limiting crop productivity. Thus, the choice of soil with an appropriate electrical conductivity is highly desirable. Land used for lettuce is often pre- irrigated before land preparation is completed to facilitate salt leaching. The variety and seed selection as well as the seed handling are very important. The irrigation and the cultivation procedure are key elements in the production of lettuce (Zhu, 2001).
6. Harvesting the crop
The handling technique that will be followed is important during the harvesting. Because most lettuce undergoes little processing, great emphasis is placed on producing a high quality product. It is essential that the product be free of pest damage and contamination at harvest. Lettuce is open to contamination from a wide variety of sources that includes manure amended soil, irrigation water, insects and wild animals (Warriner et al., 2009). A wash step is applied in fresh-cut processing in an attempt to remove field acquired contamination or at least prevent cross-contamination between batches (Barrera et al., 2012, Gil et al., 2009, Nou et al., 2011). Lettuce trimming and coring-in-field (CIF) are relatively recent industry developments designed to increase processing plant production. This process significantly reduces shipping and waste disposal costs while maintaining the market quality of lettuce (Brown and Rizzo, 2001). However, core removal requires additional human handling per head in the field and exposes the internal leaf tissues, increasing the risk of direct contamination, which is already high in field environments (FAO/WHO, 2008). Cut leaf tissues, such as those resulting from coring, provide a moist, nutrient rich environment especially conducive to direct and rapid infiltration, and pathogen attachment, growth and survival (Takeuchi et al., 2000, Yang et al., 2012).
7. Postharvest Processing
Field packaged lettuce can be packed "naked" in the carton, film-wrapped in perforated or non-perforated cellophane; or bagged in perforated plastic bags. After harvest, the lettuce is transported to a cooling shed and distribution centre where it is stored at low temperatures and it must be shipped with 48 hours. Cross contamination combined with
Page 127 Materials and Methods the growth of pathogens during storage are fundamental risk factors for listeriosis (Ding et al., 2013, Hoelzer et al., 2012).
8. Transportation
The transport of fresh fruit and vegetables is a complicated topic. The equipment should be maintained in good condition, and the cleaning frequency must be documented and verified. The storage/carriage conditions afforded the produce should be such that excessive water loss does not occur. Optimum transit temperature is around 0°C, container temperature 1-2°C, and relative humidity 90-95%. Fast transportation with minimum damage during shipment is vey important in successful marketing of perishable. Although optimal storage temperature (0-2°C) is very useful for prolonging the shelf-life of vegetables, the recommended temperature is not always maintained during postharvest storage and normal temperature is usually found during the transportation of lettuce from farm to retails since some of the transportation equipment are open air vehicles (Ding et al., 2013, Yang et al., 2012).
9. Point of Sale
The lettuce packaging should be designed to preserve the content as fresh and safe as possible. Its second function is to make the product look attractive to customers, using colourful prints. In addition, cross contamination might be occurred during the period of transport or storage in the market, restaurant and home (Ding et al., 2013). Moreover, the final packing containers must be properly handled in order to prevent cross- contamination and be kept covered. The premises must be regularly cleaned according to a documented cleaning plan.
C1. Labor, Manpower
C2. Quality and Safety of Food Systems C9. Point of Sale
C3. Location-Surroundings of the growing field
C8. Transportation C4. Lettuce Nursery
C7. Postharvest Processing C5. Produce Land
C6. Harvesting the crop
Figure 2.4.1.1: Flow Chart of Lettuce/ Leafy Greens Production including 9 concepts (critical points) Page 128 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 2.4.2 Decision Making Support System in lettuce’s safety using fuzzy cognitive maps
The concepts that were selected to be tested during the lettuce production procedure were extracted from questionnaires that were filled from experts. The methodology described extracts the knowledge from experts and exploits their experience of the process. Each expert based on his/her experience knows the main factors that contribute to the decision. Experts describe the existing relationship firstly as “negative” or “positive” and secondly, as a degree of influence using a linguistic variable, such as “low”, “medium”, “high” etc.
More specifically, the causal interrelationships among concepts are declared using the variable influence which is interpreted as a linguistic variable taking values in the universe of discourse U = [-1, 1]. Its term set T (influence) is suggested to be comprised of nine variables. Using nine linguistic variables, an expert can describe the influence of one concept on another in detail and can discern it between different degrees. The nine variables used here are: T (influence) = {negatively very strong, negatively strong, negatively medium, negatively weak, zero, positively weak, positively medium, positively strong, and positively very strong}. With this method the purpose is to diagnose and predict the effect of different factors during the lettuce production chain in their contribution to a final safe fresh lettuce.
Page 129 Materials and Methods STATISTICS
All experiments were carried out in triplicate. During each experiment two samples were taken at any time to conduct microbial counts. The microbiological data were analyzed in terms of log10 ( / 0), where is the microorganism load at a given time, and 0 corresponds to the initial𝑁𝑁 𝑁𝑁 microbial𝑁𝑁 load of untreated samples. 𝑁𝑁
N is calculated from two successive dilutions using the following equation:
N=Σα / V (n1+0,1n2) d
Σα: is the sum of the CFU counted on all the dishes retained from two successive dilutions.
n1: is the number of dishes retained at the first dilution.
n2: is the number of dishes retained at the second dilution.
V: is the volume of inoculum, in millilitres, applied to each dish.
D: is the dilution factor corresponding to the first dilution retained
All the data were analyzed for statistical significance using SPSS 21.0 (SPSS Inc., Chicago, USA).
An assessment of the Normality of the data was done with Shapiro-Wilk test. Results were then compared by an analysis of variance (ANOVA) followed by Tukey’s pairwise comparison of the means with significance defined at the < 0.05 level.
𝑃𝑃 Tukey's HSD test is a post-hoc test, meaning that it is performed after an analysis of variance (ANOVA) test. The purpose of Tukey's HSD test is to determine which groups in the sample differ. Thus, in the present study, Tukey Test determined the differences between different groups of microorganisms, different groups of RTE foods and different groups of disinfection technologies, as well as differences between physicochemical and color values.
Moreover, Pearson coefficient was used for measuring correlation between values. Correlation between different sets of data is a measure of how well they are related.
Page 130 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Pearson Correlation in the present study was used in order to correlate different microorganisms as far as their disinfection efficiency is concerned. The Pearson product- moment correlation coefficient is a measure of the strength of the linear relationship between two variables. It is referred to as Pearson's correlation or simply as the correlation coefficient. If the relationship between the variables is not linear, then the correlation coefficient does not adequately represent the strength of the relationship between the variables. Pearson's r can range from -1 to 1. An r of -1 indicates a perfect negative linear relationship between variables, an r of 0 indicates no linear relationship between variables, and an r of 1 indicates a perfect positive linear relationship between variables. Figure 1 shows a scatter plot for which r = 1.
Finally, pairwise t-tests concern the comparison of the same group of individuals, or matched pairs, being measured twice, before and after an “intervention”. In the present study, the “intervention” was all the selected disinfection treatments for treatment times ranging from 1 to 60-min.
Page 131 Materials and Methods
Page 132 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Chapter 3. RESULTS
In this study four experimental approaches were conducted in order to evaluate food safety and public health. The final aim at the fifth part was to assess the efficiency of different disinfection technologies on their ability to reduce or totally disinfect pathogens which may be present in ready to eat fresh produces, thus ensuring public health.
The aim of the first experimental approach was to evaluate the effectiveness of three non-thermal light technologies (NUV-Vis, continuous UV and HILP) on their ability to inactivate two pathogens on a liquid matrix. The liquid matrix used for the disinfection experiments was a liquid matrix (MRD solution). The indicator microorganisms that were selected were Escherichia coli K12 and Listeria innocua. E. coli K12 was selected as a representative microorganism for the enterohaemorrhagic foodborne pathogen E. coli O157:H7 and L. innocua as a surrogate microorganism for the common foodborne pathogen Listeria monocytogenes, respectively.
The second experimental approach involved the use of non-thermal technologies (UV and US) as well as conventional sodium hypochlorite (NaOCl) solutions, in order to evaluate the disinfection efficiency of three ready-to-eat produces (romaine lettuce, strawberry and cherry tomatoes). The series of these disinfection technologies included also combinations of the above technologies. More precisely, UV+US, UV+NaOCl and US+NaOCl combined technologies were used. The disinfection efficiency was tested against Gram positive and Gram negative microorganisms (E. coli, S. aureus, S. Enteritidis, L. innocua) as well as adenovirus-35 which have been artificially inoculated on the above fresh produces. Moreover, infectivity assays were conducted based on different initial concentration inocula of RTE produces. For this reason, the RTE produces were inoculated with different concentrations of the above bacteria and virus and their disinfection efficiency with selected disinfection technologies was tested. Furthermore, with the aim of investigating how the above pathogens survive during refrigerated storage, the three fresh produces inoculated with the cocktail of the above four microorganisms were treated with selected disinfection technologies and were kept in refrigerated conditions for 15 days. The microbial load of romaine lettuce, strawberry and cherry tomatoes was recorded after 3, 7 and 15 days of storage at 6˚C.
Page 133 Results
In the third experimental approach, the quality and the physicochemical characteristics of the above fresh ready-to-eat produces were tested before and after the use of disinfection technologies. More precisely, color was recorded as a quality indicator and TAC, TPC and AA were studied as valuable physicochemical characteristics of the fresh produces.
The fourth approach was a computerized model, which was proposed, in order to evaluate and explore problems that can arise during the food production chain and predict the possibility of cross-contamination of fresh produce from production to the point of sale in a vertical production company of vegetables. The final aim was to obtain a risk assessment software tool to ensure food safety and public health.
Finally, conclusions based on infectivity doses for each pathogen and the results obtained from the present study, were exported.
Page 134 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 3.1 In Vitro Experiments with 3 Light Technologies
The first light equipment that was used was NUV-vis 395 ± 5 nm. The inactivation rate of E. coli (figure on the left) and L. innocua (figure on the right) was dose and time dependent. Generally, it was observed that as the distance from the lamp was increased, the time needed for inactivation for both microorganisms was longer (figure 3.1.1).
Figure 3.1.1: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 3 cm (∆), 12 cm (☐), 23 cm (○) and L. innocua placed at: 3 cm (▲), 12 cm (■) and 23 cm (●) from the high intensity near ultraviolet/ visible (NUV–vis) 395±5 nm light source (Results expressed as mean log10 CFU/mL).
Moreover, when higher dosages were achieved (36 J/cm2), the inactivation rates of L. innocua remained significantly higher with a maximum average log10 CFU/mL reduction of 2.74 achieved after 1115 sec of treatment, compared to that of E. coli where the maximum average log10 reduction after the same time was 1.37 log10 CFU/mL (p < 0.05, n=12). The higher susceptibility of L. innocua was even observed at 23 cm distance from the light source, giving a log reduction at the highest dose (2.832 J/cm2) of
1.10 log10 CFU/mL. It was significantly greater (p<0.05) in comparison with the corresponding reduction that was observed for E. coli which was 0.52 log10 CFU/mL (p>0.05).
Subsequently, experiments with continuous UV light source followed. The inactivation rate of both E. coli and L. innocua were also dependent on treatment time and dose.
Page 135 Results
Figure 3.1.2: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 6.5 cm (∆), 17 cm (☐), 28.5 cm (○) and L. innocua placed at: 6.5 cm (▲), 17 cm (■) and
28.5 cm (●) from continuous UV light source (Results expressed as mean log10 CFU/mL).
A positive correlation was observed between log10 reduction and duration of UV light exposure. The highest reductions were achieved at the shortest distance from the lamp
(6.5 cm) and at an exposure time of 472 sec. More precisely, reductions of 2.66 log10
CFU/mL and 3.04 log10 CFU/mL were achieved for E. coli and L. innocua, respectively. It must be stated that the susceptibility of the two microorganisms when this light technology was used, was not significantly different (p = 0.749).
The third light equipment used in this first approach of disinfection experiments was the High Intensity Light Pulsed (HILP) source. The results observed with this disinfection equipment are quite promising and are illustrated in figure 3.1.3 for E. coli (on the left) and L. innocua (on the right) respectively.
Figure 3.1.3: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed at: 2.5 cm (∆), 8 cm (☐), 11.5 cm (○), 14 cm (◊) and L. innocua placed at: 2.5 cm (▲), 8 cm (■), 11.5 cm (●) and 14 cm (♦) from high Intensity pulsed light source (Results expressed as mean log10 CFU/mL).
Page 136 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki In general, increased treatment times resulted in greater reductions for both E. coli and L. innocua. The least susceptible microorganism was E. coli. A dosage of 17.7 J/cm2 resulted in log reductions of E. coli and L. innocua populations (3.07 and 3.77 log10 CFU/mL, respectively). When a dosage of 54 J/cm2 (30 sec) was implemented, reductions of 4.81 and 5.56 log10 CFU/mL were achieved or E. coli and L. innocua, respectively. At a dosage of 36 J/cm2, a degree of variation was observed between the two tested microorganisms. For example, E. coli was reduced by 3.85 log10 CFU/mL, whereas L. innocua was reduced by 5.30 log10 CFU/mL (p < 0.05, n=12). At 2.5 cm distance, at longest exposure time (30 sec), both microorganisms were below the limit of detection (<0.22 log10 CFU/mL). The susceptibility of two microorganisms regarding this light technology was also significantly different (p < 0.05, n=192).
In figure 3.1.4 comparisons between three light technologies for E. coli are illustrated. The dosages that were used had a range between 0.18-106.2 J/cm2. Where no measurement took place (e.g due to temperature increase) no bar exists.
Figure 3.1.4: Mean Log CFU/mL E. coli on MRD after treatment at the same dosages at shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High Intensity Light Pulses (■).
It can be observed that High Intensity Light Pulses was the most powerful technology as far as the disinfection capacity on E. coli is concerned. Moreover, dosages greater than 2.832 J/cm2 were not appropriate for Continuous UV technology, due to the increase in temperature (>30°C) that was observed.
Page 137 Results
When low dosages were implemented (0.18, 0.36, 0.72, and 1.44 J/cm2), the observed inactivation rates were similar for both E. coli and L. innocua (p > 0.05, n=396) (figures 3.1.4, 3.15). However, when a higher dose of 2.832 J/cm2 was delivered, L. innocua exhibited a higher log reduction (1.25 log10 CFU/mL) compared to E. coli (0.68 log10 CFU/mL) after 88 sec of treatment (p < 0.05, n=84), which is around 2 times the inactivation log of the more resistant bacterium of E. coli.
In figure 3.1.5 comparisons between three light technologies for L. innocua inactivation are illustrated. The dosages that were used had a range between 0.18-106.2 J/cm2. Where no measurement took place (e.g due to excessive temperature increase) no bar exists.
Figure 3.1.5: Mean log CFU/mL L. innocua on MRD after treatment at the same dosages at shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High Intensity Light Pulses (■).
At low dosages (0.18, 0.36, and 0.72 J/cm2) the difference between the two microorganisms, when the three light technologies were used, were all significant (p < 2 0.05, n=354). When 1.44 J/cm was used, the log10 (CFU/mL) reduction at NUV-vis light and continuous UV light for both microorganisms was significant (p <0.05, n=72), whereas when comparisons with HILP light were performed, the differences between the susceptibility of the tested microorganisms did not differ (p >0.05, n=192). When 2.832 J/cm2 was implemented in both continuous UV light technology and HILP, the disinfection efficiency of E. coli and L. innocua did not differ significantly (p = 0.306
Page 138 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki and p = 0.116, respectively). Finally, at higher dosages, the correlations between log10 (CFU/mL) reduction of NUV-vis and HILP, as the two organisms are concerned, were all significant (p < 0.05). Generally, it was observed that in all light technologies (NUV- vis, continuous UV, and HILP) a significant correlation (p < 0.05, n=600) between doses and microorganisms log reduction existed.
The temperature increase for three light equipments and at different distances from the lamp are shown in figures 3.1.6, 3.1.7, 3.1.8.
Figure 3.1.6: Mean Temperature increase Figure 3.1.7: Mean Temperature increase (ΔΤ ᵒC) for NUV-Vis light technology at (ΔΤ ᵒC) for UV light technology at distances: distances: 3 cm (▲), 12 cm (■) and 23 cm (●) 6.5 cm (▲), 17 cm (■) and 28.5 cm (●)
Figure 3.1.8: Mean Temperature increase (ΔΤ ᵒC) for HILP light technology at distances: 2.5 cm (▲), 8 cm (■), 11.5 cm (●) and 14 cm (♦).
Page 139 Results
Temperatures remained below 50°C for all disinfection treatments used in the study. However, it must be stated that HILP exhibited the greater temperature increases, followed by NUV-Vis and Continuous UV light. For this reason, during HILP treatments, samples were placed in an iced bath to minimize heating caused by the infrared portion of the HILP light unit.
Page 140 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 3.2 Food Disinfection 3.2.1 Bacteria Disinfection
3.2.1.1 Lettuce
The first vegetable selected for food disinfection was lettuce. Its disinfection efficiency against four bacteria was tested, after immersing whole lettuce leaves in 50 ppm and 200 ppm NaOCl solutions.
Figure 3.2.1.1.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce. The inactivation effect of NaOCl against E. coli, S. aureus, S. Enteritidis and L. innocua increased with increasing treatment time and concentration. Significant reductions (p<0.05, n=48) were observed at 3-min treatment time when pairwise t-test was used, with both NaOCl 50 ppm solution and NaOCl 200 ppm solution. For instance, treatment time of 3-min and 200 ppm NaOCl solution reduced microorganisms significantly when tested with pairwise t-test (p<0.05, n=48) by 1.92, 1.82, 1.96 and 2.01 log10, for E. coli, S. aureus, S. Enteritidis and L. innocua respectively. Lower concentration of NaOCl (50
Page 141 Results ppm) reduced the above populations by 1.64±0.11 1.45±0.27, 0.92±0.21 and 1.63±0.14 log10, at the same treatment time. However, no significant differences (p>0.05, n=48) were observed with pairwise t-test, for microbe populations when the treatment time increased from 3 to 5-min. On the other hand, when the treatment time was increased from 1 to 3-min, significant differences when pairwise t-test was used (p<0.05, n=48) were observed for all the microorganisms, with the exception (p=0.199) of S. Enteritidis which was treated with NaOCl 50 ppm.
Lettuce was then treated with two non-thermal, alternative disinfection technologies Ultrasound and UV for treatment times ranging from 1 to 60-min and the results are illustrated in figure 3.2.1.1.2.
Figure 3.2.1.1.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce. Treatment with US significantly reduced the numbers of all microorganisms inoculated on lettuce. The reduction was found to be significant with anova test after 30-min of treatment (p< 0.05, n=240). The maximum reductions of E. coli, S. aureus, S. Enteritidis
Page 142 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki and L. innocua observed on lettuce after US treatment, were 2.30 ± 0.34, 1.71 ± 0.20,
5.72 ± 0.05 and 2.95 ± 0.57 log10 CFU/g.
Treatments with UV reduced significantly the concentrations of all types of microorganisms (p<0.05, n=240). The reduction of four bacterial types depended on the different time intervals as well as on the bacterial type concerned. Significant reduction of all bacteria was achieved after 20-min (p<0.05). The reduction was finally about 1–
1.7 log10 after 45-min treatment with UV in all different types of microorganisms. Both Salmonella and E. coli in lettuce showed similar reductions when treated for the same period with UV.
Combined technologies including alternative and conventional technologies in different treatment times were also examined. The treatment times for alternative technologies were in the range of 1-30-min. All the treatments were followed by 3 minute immersion of lettuce in NaOCl solutions of 50 and 200 ppm concentration. The results for four bacteria are illustrated in figure 3.2.1.1.3.
Figure 3.2.1.1.3: Disinfection Efficiency of combined alternative and conventional disinfection technologies (US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm) on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce.
Page 143 Results
All the combined disinfection treatments were effective after 1 minute treatment time with non-thermal technology followed by 3-min treatment with NaOCl. For instance, E. coli, S. aureus and L. innocua populations were significantly reduced (p<0.05), after 1 minute treatment of Ultrasound technology followed by 3-min of NaOCl 50 ppm, whereas S. Enteritidis was not significantly reduced (p=0.029) after the same time, but 20-min treatment time needed for significant inactivation rate (p=0.04). However, when 200 ppm NaOCl immersion followed, the disinfection efficiency was significant (p<0.05, n=336) at all treatment times, when tested with pairwise t-test.
Generally, US+NaOCl was more effective than UV+NaOCl. L. innocua was reduced by
1.83±0.32, 2.41±0.34, 1.78±0.08 and 1.95±0.11 log10, when US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm and UV+NaOCl 200 ppm were used respectively. US+NaOCl 200ppm was efficient and reduced by 2.71, 2.33, 2.94 and 3.34 log10 CFU/g E. coli, S. aureus, S. Enteritidis and L. innocua respectively, after 30-min treatment, plus 3 minute treatment with conventional technology.
Finally, combinations of alternative non-thermal technologies were tested. The results are observed in figure 3.2.1.1.4. The maximum treatment time for the combined treatments tested was 30-min.
Figure 3.2.1.1.4: Disinfection Efficiency of combined alternative disinfection technologies on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce.
Page 144 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Combinations of alternative disinfection technologies did not show an additive effect. However, when tested with pairwise t-test, they resulted in significant log reductions (p<0.05, n=120) of all microorganisms. The greater log reductions were obvious after 10-min UV treatment followed by 20-min of US where 0.67±0.07, 0.35±0.01, 0.87±0.29 and 1.22±0.29 log10 reduction for E. coli, S. aureus, S. Enteritidis and L. innocua were found respectively. The lowest log reduction was presented for S. aureus, regardless the combined disinfection method that was used.
Page 145 Results
3.2.1.2 Strawberry The fruit that dominates the Mediterranean diet, prior inoculated with bacteria cocktail was tested with a series of conventional and alternative disinfection technologies. Firstly, the results of the immersion in NaOCl 50 ppm and NaOCl 200 ppm are observed in figure 3.2.1.2.1.
Figure 3.2.1.2.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries. The disinfection efficiency of NaOCl was dependent on the concentration and the treatment time. Longer treatment times, resulted in greater log reduction of all microorganisms.
After 1 minute immersion in both NaOCl 50 ppm and NaOCl 200 ppm, no significant reduction with pairwise t-test (p>0.05, n=48) was observed for all microorganisms. However, at 3-min treatment time with NaOCl 200 ppm, significant population reductions for E. coli (p=0.017), S. aureus (p=0.015), S. Enteritidis (p=0.008) and L. innocua (p=0.07) were recorded. NaOCl 200 ppm and 3-min treatment time resulted in
Page 146 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
1.58±0.35, 1.29±0.28, 1.66±0.26 and 1.48±0.21 log10 reductions of E. coli, S. aureus, S. Enteritidis and L. innocua respectively. Whereas after 5-min treatment of strawberries with 200 ppm NaOCl resulted in 1.91±0.30, 1.44±0.12, 1.75±0.26 and 1.52±0.21 log10 of the above microorganisms respectively.
Significant differences were recorded with the use of pairwise t-test, when the treatment time was enhanced from 1 to 3-min (p<0.05, n=48). Whereas, when the time was increased from 3 to 5-min, the disinfection remained constant and the differences were not significant (p>0.05, n=48). Both Ultrasound and UV disinfection technologies were used for strawberry disinfection and the results are recorded in figure 3.2.1.2.2.
Figure 3.2.1.2.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries. In strawberries, the most significant reduction occurred after 10-min treatment with UV (1.2 J/cm2) depending on the microorganism (p<0.05, n=48), according to pairwise t- test. E. coli and S. Enteritidis were reduced significantly after 10-min (p=0.05 and
Page 147 Results p=0.037 respectively). S. aureus reduced significantly (p=0.037) after 5-min treatment with UV and L. innocua after 20-min UV treatment (p=0.038).
Treatment with US significantly reduced the numbers of all microorganisms on strawberries (p<0.05, n=240). The maximum reductions of E. coli, S. aureus, S. Enteritidis and L. innocua on strawberries were 3.04 ± 0.72, 2.41 ± 0.59, 5.52 ± 0.13 and 6.12 ±0.04 log10, respectively and were observed after the longest exposure times to US technology. L. innocua was reduced significantly (p=0.05) after 5-min treatment with US. However, S. aureus and S. Enteritidis needed 20-min to be reduced significantly (p=0.037). It must be stated that no viable (<0.22 log10 CFU/g) Salmonella or Listeria cells were observed after 30-min treatment time with US.
Combinations of alternative (US and UV) with immersion in NaOCl 200 ppm for 3-min were also selected for strawberries disinfection. The results are shown in figure 3.2.1.2.3.
Figure 3.2.1.2.3: Disinfection Efficiency of combined alternative and conventional technologies (US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm) on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries.
Page 148 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Combined alternative and conventional treatments were used for strawberry disinfection. All microorganisms inoculated in strawberry were reduced when combined technologies were used. E. coli was reduced significantly (p<0.05, n=168) by 1.91±0.11, 2.36±0.22,
1.96±0.26 and 2.08±0.35 log10 when the longest combined treatments US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, were used. The highest reductions among all microorganisms observed by S. Enteritidis where it was reduced significantly (p<0.05, n=168) by 2.93±0.35, 3.50±0.39, 2.20±0.25 and
2.40±0.21 log10 after longest exposure to combined treatments of US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm.
In general terms, combinations of UV followed by NaOCl resulted in less disinfection efficiency compared to US followed by NaOCl. However, all microbe populations were significantly reduced (p<0.05, n=672), with all the combined treatments that were selected.
Finally, combinations of alternative technologies were used for testing the disinfection efficiency of microbial populations in strawberries and are shown in figure 3.2.1.2.4.
Figure 3.2.1.2.4: Disinfection Efficiency of combined alternative technologies on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries.
E. coli is more resistant to combined alternative technologies showing decreased log10 reductions from 0.09±0.05 to 0.58±0.10. However, L. innocua population decreased by
0.64±0.03, 1.37±0.11, 1.83±0.15 and 1.47±0.07 log10 when the above combined alternative technologies were used. Moreover, S. aureus and S. Enteritidis were reduced by 0.64±0.08, 0.57±0.07, 1.18±0.06, 0.90±0.06 log10 and 0.62±0.06, 0.74±0.03,
Page 149 Results
1.10±0.2, 0.82±0.04 log10, respectively. All the populations reductions that were achieved were statistically significant (p<0.05, n=120) when tested with pairwise t-test.
3.2.1.3 Cherry tomatoes
Cherry tomatoes are very common vegetables consumed raw in an everyday basis in a Mediterranean diet. The disinfection methods used for cherry tomatoes are shown in figures 3.2.1.3.1 – 3.2.1.3.4.
Figure 3.2.1.3.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes.
Treatment with NaOCl, when tested with pairwise t-test, significantly (p<0.05, n=48), reduced all microorganisms from the first minute of treatment. Moreover, it is obvious Page 150 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki that the degree of disinfection was not enhanced significantly after 3-min treatment. However, a greater reduction (p<0.05, n=48) was observed at 3-min treatment with 200 ppm NaOCl compared to 3-min treatment of NaOCL 50 ppm. The reduction from 3 to 5- min was not significant (p>0.05, n=48).
For instance, 50 ppm NaOCl reduced E. coli, S. aureus, S. Enteritidis and L. innocua by
2.87±0.35, 2.18±2.25, 2.86±2.67 and 2.03±2.33 log10 respectively, whereas 200ppm NaOCl reduced the above microorganisms by 3.68±0.56, 2.98±2.67, 3.07±2.83 and
2.29±2.53 log10, respectively.
Then, US and UV were tested as fas as their disinfection efficiency is concerned and the results are shown in figure 3.2.1.3.2
Figure 3.2.1.3.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes.
Treatments with US led to reductions of 0.64 - 3.16 log10 CFU/g in the population of E. coli, 1.06-2.62 log10 CFU/g in the population of S. aureus, 1.23-3.29 log10 CFU/g in the
Page 151 Results population of S. Enteritidis and 0.76-3.16 log10 CFU/g in the population of L. innocua (p<0.05, n=240). The effectiveness of the 37 kHz ultrasound bath treatement was increased as the treatment time increased from 1-60-min. Significant reduction of all bacteria was achieved after 10-min (p<0.05). The reduction was finally about 3.29 log10 for S. Enteritidis followed by E. coli after the longest exposure time, with an average log reduction of 3.16 log10 (p<0.05). All microorganisms were significantly reduced (p<0.05) from the first minute of treatment with US, except E. coli which needed 10-min treatment (p=0.009), in order to be reduced significantly.
Treatments with UV, reduced the populations of all microorganisms, artificially inoculated in cherry tomatoes. More specifically, UV treatments evaluated in this study promoted the reduction of 0.82 - 2.39 log10 CFU/g in the population of E. coli, 0.98-2.05 log10 CFU/g in the population of S. aureus, 1.11-2.62 log10 CFU/g in the population of
S. Enteritidis and 0.84-2.56 log10 CFU/g in the population of L. innocua (p<0.05). The reduction of four bacterial types depended not only on the different time intervals (1-60- min) but also on the bacterial type. At 10-min treatment time significant reduction of all bacteria was achieved (p< 0.05). The increase in contact time from 10 to 60-min of treatment reduced contamination even further (p=0.04). S. Enteritidis achieved the greatest reduction among all bacteria with an average of 2.62 log10 CFU/g at 60-min treatment time (p<0.05). All microorganisms were significantly reduced (p<0.05) from the first minute of treatment with UV, except S. Enteritidis which needed 10-min (p=0.024), in order to be reduced significantly.
Combined technologies of US and UV followed by immersion in NaOCl 50ppm and NaOCl 200ppm are illustrated in figure 3.2.1.3.3.
Page 152 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.2.1.3.3: Disinfection Efficiency of combined alternative and conventional technologies (US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm) on E. coli, S. aureus, S. Enteritidis, L.innocua inoculated on fresh cherry tomatoes.
The combined alternative followed by conventional treatments, reduced significantly (p<0.05, n=672) all microorganisms. When US followed by 3-min immersion of cherry tomatoes in NaOCl 50 ppm solution was used, reductions of 3.63±0.28, 3.26±0.25,
3.63±0.03 and 3.39±0.05 log10 were achieved for E. coli, S. aureus, S. Enteritidis and L. innocua respectively. However, when US was combined with 3-min immersion in
NaOCl 200 ppm 0.50-1 log10 further significant reductions (p<0.05) were observed for all microorganisms. More precisely, the aforementioned microorganisms were reduced by 4.78±1, 3.63±0.13, 4.27±0.41 and 3.84±0.28 log10 respectively.
Furthermore, when UV was combined with NaOCl 50 ppm, the above microorganisms were reduced by 3±0.08, 2.50±0.19, 3.39±0.36 and 2.86±0.03 log10 respectively. Whereas, when it was combined with NaOCl 200 ppm, the above microorganisms were reduced significantly (p<0.05) by 3.94±0.22, 2.89±0.04, 3.9±0.55 and 3.28 ±0.29 log10 respectively.
Combinations of alternative technologies were tested for treatment times of 10-30-min.
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Figure 3.2.1.3.4: Disinfection Efficiency of combined alternative technologies on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes.
Treatment with UV for 10min followed by US 20-min significantly reduced, when tested with pairwise t-test, the numbers of all microorganisms (p<0.05, n=48) on cherry tomatoes. The reductions achieved were 3.06±0.18, 2.63±0.20, 3.70±0.05 and 2.73±0.02 log10 for E. coli, S. aureus, S. Enteritidis and L. innocua respectively.
Finally a more generalized statistical analysis was carried out. For instance, Tukey HSD test was used in conjunction with an ANOVA to find disinfection technologies that are significantly different from each other.
For lettuce, no significant differences were found between alternative disinfection methods (p>0.05, n=480). However, when compared with conventional as well as with combined alternative and conventional, the differences were significant (p<0.05, n=192 and n=672 respectively). The above was observed for all bacteria in lettuce except S. Enteritidis. At the case of S. Enteritidis, a significant difference (p<0.05) between Ultrasound and UV was found.
For strawberry, the results obtained for S. Enteritidis and L. innocua did not differ significantly (p>0.05) between different disinfection methods. The only difference for the aforementioned microorganisms (p=0.046, p=0.001 respectively) was found between Ultrasound and UV technology. For E. coli and S. aureus there was a difference between US and UV (p<0.05, n=240 for both microorganisms), as well as between alternative
Page 154 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki disinfection methods and combinations of alternative followed by conventional disinfection methods (p<0.05).
For cherry tomatoes, a greater difference between disinfection methods for microorganisms was observed. E. coli and S. Enteritidis exhibited the same inactivation pattern (p>0.05, n=240) when treated with US and UV, whereas they differed significantly (p<0.05, n=432) when treated with the rest of disinfection methods. Gram positive microorganisms susceptibility was the same for UV and US as well as for UV and combinations of alternative disinfection technologies (p>0.05), whereas differed for the other disinfection technologies.
Finally, for each disinfection methods used, Tukey HSD tests were performed to determine difference between microorganisms. Generally, a variation was observed among bacteria’s susceptibility to different disinfection methods. For lettuce, the four bacteria did not exhibit any difference (p>0.05, n=180) when conventional treatments were used. For US treatment S. Enteritidis differed significantly from the other three bacteria (p<0.05). Moreover, for UV treatment, gram negative bacteria susceptibility was the same (p=0.993) whereas a difference was observed for gram positive bacteria (p=0.004).
For strawberry, the four bacteria exhibited the same behavior (p>0.05) when strawberries were treated with conventional treatments, US and US followed by NaOCl immersion. S. aureus in UV followed by NaOCl 50 ppm immersion treatments exhibited a different behavior compared to the other three bacteria. When UV treatments were conducted, the only difference observed was between E. coli and S. Enteritidis (p=0.004), whereas the rest of microorganisms had a similar behavior (p>0.05).
Finally, for cherry tomatoes, a similar reduction of gram negative (p=0.878) and gram positive (p=0.903) bacteria is observed when US treatment was followed by NaOCl treatments. However, when UV followed by NaOCl treatments, the microorganisms behave differently (p<0.05) between each other. When cherry tomatoes were treated with combined alternative treatments, S. Enteritidis reduction was significantly (p<0.05) different from the other three bacteria.
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3.2.2 Adenovirus Disinfection
Figure 3.2.2.1: Standard Curve based on the entire hexon region of Ad35 cloned into pBR322
The construction of the standard curve is based on the use of the entire hexon region of Ad41 cloned into pBR322 (kindly donated by A. Allard, University of Umeå). High efficiency E. coli JM109 competent cells (Promega, L2001) were added to 1µl of pBR322 plasmid containing the entire Ad41 hexon gene sequence following the supplier instructions. The efficiency of these cells has been reported to be 2.2x108 CFU/µg. 100 µl of transformed E. coli JM109 were plated into a LB agar plate containing ampicilline 100µg/ml. Consequently, 500µl of transformed bacteria were aliquoted into a 1.5 ml tube and centrifuge 10-min at 5000xg. The supernatant was discarded, pellet was resuspended in 1ml of LB 15% glycerol and kept frozen at -80ºC for further production of standard. The other 400µl of transformed cells were kept at 4ºC for further production of larger amounts of DNA. Afterwards, some of the colonies growing were checked on LB plates by conventional PCR (using primers AdR and AdF at 55ºC of annealing temperature) to contain the target DNA. Then colonies were inoculated directly into PCR tubes by using sterile toothpicks. The target DNA was obtained by using the QIAGEN Plasmid Midi kit (Cat. No. 12143) and the transformed cells kept at 4ºC by following manufacturer’s instructions. Then dilution of the obtained Page 156 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki DNA into Tris-EDTA pH 8 (10 mM Tris and 0.1 mM for EDTA) and quantification by spectrophotometry several replicates of the DNA followed. Approximately 10μg of DNA was linearized with BamHI restriction enzyme. Then the DNA was purified with the QIAGEN QIAquick PCR Purification kit (Cat. No. 28104). The purity of linearised plasmid was checked in an agarose gel 1%. If a band corresponding to undigested plasmid was present more enzyme was added and the digestion was let to take place for an additional 2 hours. Several replicates of the DNA were quantified by spectrophotometry, then the DNA was serially diluted in order to obtain dilutions where 102 to 108 molecules per 10 µl are present.
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Figure 3.2.2.2: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and single step conventional Disinfection Treatments.
In the experimental conditions evaluated, HAdV was reduced when the samples were immersed in sodium hypochlorite solutions and significant inactivation (p<0.05, n=20) was observed as the treatment time of immersion to sodium hypochlorite 200 ppm was enhanced (from 3 to 10-min). It must be stated that after the longest exposure time, lettuce and strawberry achieved 4.95 and 5.02 log10 GC/g reduction, whereas cherry tomatoes achieved 3.76 log10 GC/g reduction. Moreover, the treatment time of 3-min was sufficient for inactivating HAdV as far as cherry tomatoes are concerned. An additional 0.79 log10 reduction was achieved for another 2-min immersion in sodium hypochlorite solution. For strawberry and lettuce the treatment time plays an important role in inactivation efficiency. The difference between 3 and 5-min was not found to be statistically significant (p>0.05). However, when 10-min treatment time was implemented, the inactivation rate was significantly enhanced (p<0.05). For instance, in strawberries a double inactivation log10 GC/g of HAdV was achieved. Whereas, in lettuce an additional 1.23 log10 reduction GC/g of HAdV was reported when the treatment time was increased from 3 to 10-min.
Page 158 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.2.2.3: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and single step Alternative Disinfection Treatments
In all foods, as expected, higher UV doses resulted in a greater decrease of viral growth in ‘romaine’ lettuce, in strawberry pieces and in cherry tomatoes. UV was less effective at reducing viral populations in lettuce. It was observed, that when the time was doubled (from 30 to 60-min), the mean reduction of HAdV was also doubled for strawberry
(from -1.26 to -3.98 log10 GC/g) and cherry tomatoes (from -0.92 to -2.22 log10 GC/g). Treatment with US was less effective (p>0.05, n=44) compared to UV. After the longest exposure time, lettuce exhibited the greatest reduction (-1.79 log10 GC/g) compared to other fresh produces. However, the treatment time also played an important role, as far as virus reduction is concerned.
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Figure 3.2.2.4: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and Cherry tomatoes (light grey bars) and combined Disinfection Treatments.
A synergistic effect was observed when UV and US were followed by immersion in sodium hypochlorite solutions, however, no additive effect was observed. The synergy was enhanced further, when UV was followed by sodium hypochlorite (p<0.05, n=40), rather than when US followed by sodium hypochlorite. Moreover, the sequential treatment of alternative methods exhibited more promising results compared to the combination of an alternative and a conventional treatment, in strawberries and cherry tomatoes. In all cases the sequential application of two alternative technologies depended on the time used for each method.
Page 160 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 3.2.3 High and Low Initial Load Disinfection Treatments
LETTUCE / DISINFECTION TREATMENTS MICROORGANISMS
Initial UV UV UV+NaOCL US US US+NaOCl NaOCl Concentration (30min) (60min) (33 min) (30min) (60min) (33 min) (3 min) E.coli 2,16E+08 3,86+07 1,81E+06 2,83E+05 1,50E+07 1,45E+06 7,36E+04 6,85E+05 1,68E+06 8,64E+05 3,41E+03 1,00E+04 4,41E+05 1,98E+04 2,73E+02 5,45E+03 1,55E+04 8,98E+03 1,33E+03 2,73E+02 9,23E+03 4,00E+02 0,00E+00 3,64E+01 6,36E+02 1,64E+02 7,27E+01 0,00E+00 3,64E+01 9,09E+00 0,00E+00 0,00E+00 S.aureus 1,35E+07 1,86E+06 7,64E+05 4,36E+05 1,20E+06 1,91E+05 7,27E+04 5,45E+05 1,32E+05 6,36E+04 9,73E+04 1,73E+03 2,73E+04 1,18E+04 8,18E+02 2,73E+02 5,55E+03 8,18E+02 1,82E+02 9,09E+02 1,09E+03 9,09E+01 5,45E+02 1,82E+02 8,18E+02 4,55E+02 9,09E+01 9,09E+01 9,09E+01 0,00E+00 0,00E+00 0,00E+00 S .enteritidis 3,25E+06 7,09E+05 7,27E+04 6,36E+04 2,00E+04 6,36E+02 2,27E+04 1,35E+05 2,88E+05 1,10E+04 6,27E+03 5,45E+03 1,18E+03 9,09E+01 9,09E+02 5,00E+03 4,55E+03 1,18E+03 7,27E+02 9,09E+02 4,55E+02 9,09E+01 9,09E+01 8,18E+02 2,73E+02 9,09E+01 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 L.innocua 4,49E+07 7,27E+05 1,73E+05 1,09E+04 5,09E+05 3,64E+04 6,36E+03 3,00E+04 1,35E+05 2,00E+04 4,55E+04 2,73E+02 2,73E+03 9,09E+02 3,64E+02 9,09E+01 2,00E+04 6,64E+03 2,00E+03 9,09E+01 3,64E+02 9,09E+01 0,00E+00 0,00E+00 2,73E+02 1,82E+02 0,00E+00 0,00E+00 9,09E+01 0,00E+00 0,00E+00 0,00E+00
Table 3.2.3.1: High and Low Inocula (Log10 CFU/g) on lettuce and disinfection with selected treatments.
STRAWBERRY / DISINFECTION TREATMENTS MICROORGANISMS Initial UV UV+NaOCL US US US+NaOCl NaOCl Concentration UV (30min) (60min) (33min) (30min) (60min) (33 min) (3min) E.coli 9,64E+07 1,96E+06 1,10E+06 1,48E+05 1,91E+04 3,64E+03 8,64E+04 1,35E+05 1,30E+05 3,64E+04 2,21E+04 1,00E+03 2,27E+03 1,82E+02 4,55E+02 2,45E+03 9,45E+02 2,73E+01 9,09E+00 0,00E+00 1,82E+01 0,00E+00 0,00E+00 0,00E+00 S.aureus 3,73E+07 8,18E+06 2,45E+06 7,09E+05 5,22E+06 8,91E+05 4,36E+05 1,31E+06 2,80E+05 2,25E+04 1,73E+04 1,09E+03 1,70E+04 1,55E+03 3,64E+02 1,82E+03 3,73E+03 9,09E+01 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 S.enteritidis 3,46E+06 7,79E+04 1,19E+04 1,73E+03 8,00E+03 9,09E+02 7,27E+02 4,91E+03 2,55E+04 2,36E+03 8,18E+02 5,45E+02 6,36E+02 3,64E+02 1,82E+02 8,18E+02 8,18E+02 4,55E+02 0,00E+00 0,00E+00 9,09E+01 0,00E+00 0,00E+00 0,00E+00 L.innocua 1,11E+07 1,22E+05 9,85E+04 3,10E+05 1,85E+04 3,09E+03 3,19E+04 1,01E+05 1,53E+05 3,18E+03 6,36E+02 9,09E+02 4,00E+03 1,82E+02 2,73E+02 2,09E+03 1,64E+03 2,73E+02 0,00E+00 0,00E+00 9,09E+01 0,00E+00 0,00E+00 0,00E+00
Table 3.2.3.2: High and Low Inocula (Log10 CFU/g) on strawberries and disinfection with selected treatments.
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TOMATOES / 5LSLNC9/TLON TR9ATa9NTS MICROORGANISMS Initial UV UV UV+NaOCL US US US+NaOCl NaOCl Concentration (30min) (60min) (33 min) (30min) (60min) (33 min) (3min) E.coli 1,28E+07 1,57E+05 6,91E+04 2,45E+03 3,00E+05 1,45E+03 7,27E+01 3,45E+03 4,01E+05 3,01E+04 7,82E+02 1,18E+02 1,34E+03 2,09E+02 1,82E+01 1,73E+02 1,55E+03 7,27E+01 0,00E+00 0,00E+00 9,09E+00 0,00E+00 0,00E+00 0,00E+00 S.aureus 6,43E+06 1,72E+04 9,09E+03 3,18E+03 1,50E+04 1,82E+03 1,45E+03 1,30E+04 4,91E+04 6,64E+03 7,27E+02 1,82E+02 1,91E+03 4,55E+02 9,09E+01 8,18E+02 5,45E+02 9,09E+01 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 S.enteritidis 1,87E+07 9,64E+04 4,57E+04 2,62E+04 4,25E+04 2,09E+03 2,18E+03 5,30E+04 1,39E+05 3,09E+03 2,09E+03 1,91E+03 1,73E+03 7,27E+02 5,45E+02 9,09E+02 9,09E+02 2,73E+02 0,00E+00 0,00E+00 1,82E+02 0,00E+00 0,00E+00 0,00E+00 L.innocua 1,36E+06 1,07E+05 1,50E+04 3,27E+03 5,77E+04 4,45E+03 8,18E+02 2,52E+04 6,27E+04 8,91E+03 1,91E+03 4,55E+02 5,45E+03 9,09E+02 9,09E+01 7,27E+02 8,18E+02 1,82E+02 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00 0,00E+00
Table 3.2.3.3: High and Low Inocula (Log10 CFU/g) on cherry tomatoes and disinfection with selected treatments.
Adenovirus Tissue Culture Infectivity Assay and Lettuce
Culture Assay Treatments PCR (GC/mL) (PFU/mL) Control 5,04E+08 109 UV 30-min 6,28E+05 106 UV 60-min 1,42E+02 103 UV+NaOCl 33-min 6,12E+03 103 US 30-min 1,31E+07 107 US 60-min 1,03E+06 107 US+NaOCl 33-min 3,02E+05 106 NaOCl 3-min 5,74E+02 103
Page 162 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.2.2.5: The results of Real Time PCR were also evaluated with cell cultures observed under an Epifluorescence microscope.
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3.2.4 Storage Conditions
The three fruits and vegetables prior inoculated with the cocktail of bacteria, and treated with selected conventional and alternative treatments were stored for 15 days at 6°C.
Lettuce
Figure 3.2.4.1: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on romaine lettuce before and after selected disinfection treatments during storage for 15 days at
6°C. (control), (UV treated), (US treated), (NaOCl treated),
(UV+NaOCl treated), (US+NaOCl treated), (UV10+US20 treated), (UV20+US10 treated).
Page 164 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Populations of E. coli, S. aureus, S. Enteritidis and L. innocua inoculated on control samples, increased during the storage period in romaine lettuce. More precisely, they were increased by 0.95, 0.10, 1.11 and 1.73 log10 respectively.
E. coli and S. Enteritidis in treated lettuces showed a similar behavior during storage period and were steadily increased from day 0 to day 15. E. coli in UV- and US- treated lettuces increased 0.74 and 1.62 log10 respectively. Whereas, S. Enteritidis in the aforementioned treated lettuces, increased 2.72 and 3.04 log10.
Populations of S. aureus and L. innocua in treated lettuces were generally decreased from day 0 to day 3 and from day 3 onwards an increase was recorded. In UV- and US- treated lettuces after storage of 15 days a decrease of 0.15 and 0.72 log10 was recorded for S. aureus but an increase of 1.46 and 1.82 log10 was observed for L. innocua respectively.
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Strawberry
Figure 3.2.4.2: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on strawberries before and after selected disinfection treatments during storage for 15 days at 6°C.
(control), (UV treated), (US treated), (NaOCl treated),
(UV+NaOCl treated), (US+NaOCl treated), (UV10+US20 treated), (UV20+US10 treated).
From day 0 and throughout storage an increase on growth of E. coli and S. aureus was observed (p<0.05) after 15 days of storage. They were increased from 7.20 to 8.34 log10 and from 7.44 to 8.16 log10 respectively. However, S. Enteritidis and L. innocua reduced by 1.44 and 3.14 log10 respectively.
Page 166 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki All treated samples collected at storage day 3, exhibited a reduction in their populations. However, at storage day 7 and 15, an increase was observed in some treated samples. For example, E. coli, S. aureus and S. Enteritidis in US-treated samples showed an increase of 0.64, 0.74 and 1.12 log10 respectively after storage for 15 days. Moreover, an increase of the same microorganisms (1.01, 0.67 and 1.33) in combined US+NaOCL treated samples was observed after 15 days of storage.
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Cherry Tomatoes
Figure 3.2.4.3: Populations of E. coli, S. aureus, S. Enteritidis and L. innocua inoculated on cherry tomatoes before and after selected disinfection treatments during storage for 15 days at
6°C. (control), (UV treated), (US treated), (NaOCl treated),
(UV+NaOCl treated), (US+NaOCl treated), (UV10+US20 treated), (UV20+US10 treated).
At refrigerated conditions, populations of all microorganisms increased steadily until day 15, reaching control samples final populations of 8.05, 7.46, 6.65 and 7.75 log10 CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua respectively. Moreover, all treated cherry tomatoes samples significantly (p<0.05) increased their populations.
Page 168 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
3.3 Food Quality parameters
3.3.1 Color
Lettuce
Physical Quality Control NaOCl 50 ppm NaOCl 200 ppm Parameters Time (min) 0 1 3 5 1 3 5 L* 59.51±0.72 57.75±1.33 56.21±3.19 51.84±2.43 53.73±1.18 52.73±0.44* 52.83±0.98 a* -19.32±1.40 -18.31±0.80 -19.09±1.12 -18.47±1.42 -19.30±0.62 -19.38±0.19 -18.29±0.17 b* 31.25±0.21 27.48±1.39 26.03±1.68 24.42±1.14 28.74±1.47 27.63±1.05* 26.24±0.57 ΔΕ* 4.74±1.33 7.03±0.44 10.69±1.88 6.75±1.26 7.99±0.48 8.69±2.58 C* 36.75±0.57 33.02±2.52 32.29±1.81 30.62±2.76 34.62±1.52 33.75±0.97* 31.98±1.14 WI 45.46±0.63 46.37±1.79 45.58±3.40 42.89±1.77 42.19±0.45 41.92±0.86* 42.97±1.47
Page 169 Results
Parameters Control Ultrasound Treatment Time (min) 0 1 3 5 10 20 30 45 60 L* 59.71±0.72 58.82±0.65 57.82±0 57.94±0.65 57.57±0.49* 54.47±0.27* 54.86±0.52* 51.35±0.49* 47.54±2.92*
a* -19.32±1.40 -16.47±22.63 -19.35±0 -18.50±2.88 -20.31±0.62 -21.49±0.60 -20.67±0.51 -19.51±2.28 -20.75±1.99 b* 31.11±0.22 30.46±0.29 30.07±0.58 30.61±1.92 28.79±0.74 28.89±1.12 28.97±0.94* 27.21±0.81* 27.79±1.07* ΔΕ* 1.71±0.37 2.48±0.58 2.80±2.21 3.53±0.60 6.24±0.77 5.56±1.13 9.29±1.17 12.86±3.01 C* 36.64±0.64 36.21±0.17 35.76±0.49 35.81±2.80 35.24±0.45 36±1.12 35.60±0.50 33.50±1.91 34.70±1.98 WI 45.54±0.76 45.16±0.57 44.70±0.32 44.72±1.36 44.84±0.31 41.95±0.77* 42.51±0.27* 40.91±0.82* 37.11±3.51*
Control Ultraviolet Light Treatment
Time (min) 0 1 3 5 10 20 30 45 60 L* 60.50±0.98 60.07±0.60 59.23±0.18 58.21±0.29 57.93±0.41 58.26±0.12 56.28±1* 48.95±0.96* 45.91±1.60* a* -19.18±0.70 -21.05±0.65 -20.23±0.02 -20.65±0.13 -19.09±1.01 -19.71±1.64 -20.60±2.15 -21.34±2 -21.42±1.65 b* 32.71±0.95 30.98±0.05 29.13±1.09 28.61±1.59 31.24±0.23 29.63±0.27 27.13±1.75 22.79±1.61 26.82±3.14 ΔΕ* 3.13±0.82 4.33±1.52 5.41±2.14 3.94±1.53 4.13±2.73 7.54±4.13 15.67±1.56 16.59±2.09 C* 37.92±1.17 37.46±0.32 35.47±0.91 35.31±1.22 36.61±0.60 35.60±0.75 34.14±0.28* 31.29±0.25* 34.40±2.15* WI 45.19±0.11 45.24±0.22 45.94±0.72 45.28±0.57 44.22±0.33 45.14±0.44 44.53±0.96 40.13±0.90* 35.89±2.50*
Page 170 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Parameters Control US+NaOCl 50ppm
Time (min) 0 1 3 5 10 20 30 L* 59.71±0.72 54.61±0.04 53.73±0.38 53.16±1.95* 53.68±1.64* 51.81±1.08* 48.46±3.83* a* -19.32±1.40 -18.76±0.04 -18.51±0.70 -19.58±0.67 -19.14±1.12 -18.17±0.81 -17.95±0,.58 b* 31.12±0.21 27.43±0.55 26.46±1.51 26.81±2.38 26.43±1.88 25.07±1.33* 24.25±2.50* ΔΕ* 6.46±0.20 7.72±1.37 8.10±1.56 8.03±0.93 10.15±1.60 13.32±4.58 C* 36.65±0.64 33.23±0.46 32.29±1.64 33.22±1.95 32.64±1.86 30.96±1.51 30.19±2.35 WI 45.53±0.75 43.75±0.25 43.56±0.64 42.55±2.12 43.32±2.01 42,.70±0.60* 40.18±2.20*
US+NaOCl 200ppm Time (min) 0 1 3 5 10 20 30 L* 59.71±0.72 54.01±0.53 54.37±0.37 54.82±0.14* 54.16±0.92* 53.31±2.19* 52.80±2.33* a* -19.32±1.40 -19.77±0.29 -20.29±0.90 -20.78±0.82 -19.64±0.49 -20.05±0.98 -20.02±1.05 b* 31.12±0.21 27.45±0.87 29.61±1.18 29.75±0.73 26.85±0.44* 27.62±0.58* 26.79±1.32* ΔΕ* 6.97±0.22 5.74±0.87 5.37±0.69 7.07±0.49 7.35±1.64 8.36±1.05 C* 36.65±0.64 33.83±0.78 35.90±1.48 36.29±1.00 33.27±0.30* 34.14±0.62* 33.45±1.57* WI 45.53±0.75 42.91±0.81 41.93±0.65 42.04±0.53 43.35±0.60 42.14±1.63* 42.15±2.80*
Page 171 Results
Physical Quality Parameters Control UV+NaOCl 50ppm
Time (min) 0 1 3 5 10 20 30 L* 59.71±0.72 58.83±0.84 57.42±0.86* 55.27±1.27* 53.48±1.54* 52.34±4.01* 49.59±0.57* a* -19.32±1.40 -20.36±0.69 -19.61±0.73 -19.91±1.06 -22.33±3.07 -21.78±0.66 -21.88±0.58 b* 31.12±0.21 30.73±0.46 29.83±0.43 29.32±1.01 29.99±0.57 27.02±3.34 27.67±0.11* ΔΕ* 1.46±0.36 3.21±0.86 5.28±1.34 7.11±2.05 8.75±5,.44 10.92±0.46 C* 36,.65±0.64 36.86±0.68 35.71±0.59 35.44±1.37 37.43±2.32 34.75±2.70 35.28±0.32 WI 45.53±0.75 44.74±1.05 44.42±0.58 42.91±0.15 40.27±2.29 40.90±1.81 38.47±0.55*
UV+NaOCl 200ppm Time (min) 0 1 3 5 10 20 30 L* 59.71±0.72 57.20±1.49 54.86±0.25* 53.75±0.38* 50.98±0.73* 46.07±2.56* 43.91±2.08* a* -19.32±1.40 -19.49±1.30 -19.38±1.17 -20.17±1.86 -20.96±2.19 -20.30±0.40 -20.86±2.24 b* 31.12±0.21 28.44±1.73 27.43±0.26* 27.14±0.81* 23.38±1.31* 22.50±0.98* 23.18±0.58* ΔΕ* 4.23±0.26 6.13±0.43 7.25±0,.78 11.84±1.00 16.22±2.10 17.88±1.75 C* 36.65±0.64 34.47±2.14 33.59±0.84 33.83±1.58 31.44±1,.52 30.31±0.76* 31.23±1.07* WI 45.53±0.75 45.04±2.50 43.73±0.42 42.69±1.25 41.75±0.90 38.12±2.08 35.80±2.29
Page 172 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Parameters Time (min) Control UV5+US5 UV10+US10 UV10+US20 UV20+US10
L* 59.08±0.45 54.62±0.48* 53.44±0.47* 52.87±0.15* 54.10±1.29* a* -19.85±1.45 -19.01±0.73 -19.49±0.55 -20.54±0.79 -21.11±0.69 b* 31.12±0.21 29.56±1.81 26.32±0.99* 26.95±0.65* 27.59±0.85* ΔΕ* 5.13±0,.92 7.50±0.65 7.56±0.27 6.29±1.07 C* 36.93±0.59 35.15±1.78 32.76±0.48* 33.89±0.73 34.75±0.32 WI 44.88±0.48 42.59±0.92 43.07±0.58 41.95±0.42* 42.42±0.84*
Table 3.3.1.1: Values are average ± standard deviation of at least three experiments and represent the color parameters of romaine lettuce after each processing time with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200ppm, UV+US. *: Asterisks within different treatment methods indicate significant differences (p<0.05).
Page 173 Results
Strawberries
Physical Quality Control NaOCl 50 ppm NaOCl 200 ppm Parameters Time (min) 0 1 3 5 1 3 5
L* 42.02±0.55 39.29±0.51 37.95±2.11 37.85±2.47 39.32±0.46 38.52±1.61 37.85±2.39 a* 29.19±2.15 27.09±0.26 26.90±0.71 26.68±1.71 27.52±1.15 27.67±0.64 26.83±1.51 b* 21.52±1.39 18.34±0.40 16.67±2.38* 15.21±2.73* 18.17±0.62 16.71±2.32* 15.57±3.70* ΔΕ* 5.21±1.12 7.08±3.50 8.10±4.19 5.01±1.69 6.65±3.29 8.09±4.89 C* 36.32±1.08 32.72±0.01 31.68±1.82 30.76±2.53 32.98±1.30 32.36±1.44 31.10±2.92
Page 174 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Control Ultrasound Treatment Parameters Time (min) 0 1 3 5 10 20 30 45 60 L* 41.06±0.56 40.86±0.38 40.47±0.25 40.19±0.43 39.38±0.99 38.83±0.77 38.97±1.18 36.74±0.94 35.98±0.94* a* 27.17±0.37 26.94±0.63 27.07±0.54 26.58±0.55 26.85±0.64 26.30±0.77 25.82±0,57 25.87±0.60 25.74±0.83 b* 20.99±2.16 20.72±2.46 20.10±2.68 19.65±2.60 19.92±4.65 19.33±4.01 18.48±2.61 14.54±1.02 15.21±1.05* ΔΕ* 0.58±0.18 1.27±0.67 1.82±0.70 2.66±1.84 3.17±1.35 3.58±1.09 8.14±2.02 8.06±2.09 C* 34.37±1,15 34.03±1,45 33.75±1,76 33.11±1,34 20.41±4.66* 19.82±4.01* 18.98±2.61* 15.03±1.02* 15.70±1.05* Control Ultraviolet Light Treatment Time (min) 0 1 3 5 10 20 30 45 60 L* 41.36±0.68 40.65±0.53 40.63±0.32 40.57±0.29 40.37±0.01 40.30±0.12 40.27±0.27 38.31±0.13* 37.79±0.15* a* 28.09±0.96 23.29±5.23 23.93±5.24 22.82±4.34 21.36±5.15* 19.48±3.00* 18.89±2.26* 15.27±0.32* 15.52±0.40* b* 19.90±1.47 19.02±1.69 17.72±0.49 18.77±1.14 19.88±1.76 19.33±1.61 19.49±1.72 18.33±2.07 18.30±1.87 ΔΕ* 4.98±4.35 5.10±3.92 5.48±3.47 6.84±5.02 8.71±2.90 9.28±1.66 13.31±0.59 13.18±1.32 C* 34.44±1.19 19.51±1.69 18.21±0.49 19.26±1.14 20.21±2.05 19.82±1.61 19.98±1.72 18.82±2.07 18.80±1.87
Page 175 Results
Physical Quality Control US+NaOCl 50ppm Parameters Time (min) 0 1 3 5 10 20 30 L* 43.66±0.50 42.94±0.72 40.67±2.76 39.16±1 39.55±0.82 39.96±0.18 39.94±1.03 a* 30.64±0.42 30.27±1.05 28.43±1.54 28.27±1.15 26.92±0.33 26.77±3.05 28,.84±0.70 b* 23.30±0.47 22.49±1.52 20.03±2.38 20.73±0.64 20.53±1.19 19.74±0.46 20.09±0.58 ΔΕ* 1.86±1.61 5.44±2.58 5.85±0.44 6.28±0.64 6.75±1.87 5.36±0.60 C* 38.49±0.49 22.98±1.52 20.52±2.38* 21.23±0.64* 21.03±1.19* 20.24±0.46* 20.58±0.58* US+NaOCl 200ppm Time (min) 0 1 3 5 10 20 30 L* 44.02±1.31 43.05±1.01 41.45±1.31 40.32±0.56 40.42±0.32 40.45±0.37 40.21±0.52 a* 24.64±5.88 22.73±3.95* 22.59±3.37* 23.17±4.85* 23.42±4.51* 21.80±4.31* 20.87±4.47* b* 24.88±2.03 21.31±1.18 20.05±0.80 20.20±1.40 20.75±0.70 19.67±0.39 19.13±0.48 ΔΕ* 5.08±1.45 6.56±1.13 6.36±2.32 5.91±2.11 7.28±1.37 8.06±0.75 C* 35.30±3.03 21.81±1.18* 19.87±1.01* 20.70±1.40* 21.25±0.70* 20.16±0.39* 19.62±0.48*
Page 176 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Control UV+NaOCl 50ppm Parameters Time (min) 0 1 3 5 10 20 30 L* 41.86±1.33 41.09±0.37 40.41±1.28 39.78±1.25 39.31±0.45 38.92±0.23 38.06±1.52 a* 28.34±1.93 27.01±2.47 27.33±3.09 27.49±2,86 27.81±2.57 27.20±2.09 25.40±1.70 b* 21.67±2.40 20.62±0.84 19.02±0.90 19.15±0.96 18.37±0.88 16.85±2.17* 16.38±1.42* ΔΕ* 2.52±1.91 3.90±2.02 4.00±0.57 4.48±1.81 6.60±1.55 7.93±1.62 C* 35.72±2.42 21.12±0.84* 19.52±0.90* 19.64±0.96* 18.87±0.88* 17.35±2.17* 16.88±1.42* UV+NaOCl 200ppm
Time (min) 0 1 3 5 10 20 30 L* 41.86±1.33 41.74±1.52 40.73±1.52 38.62±2.27 38.16±1.93 39.55±0.44 40.19±1.59 a* 28.34±1.93 26.74±1.50 26.22±1,.20 25.86±0.28 24.49±2.19 23.88±3.01 25.13±2.69 b* 21.67±2.40 19.76±1.07 19.36±0.78 17.96±1.09 18.24±3.46 18.95±2.05 19.00±2.78 ΔΕ* 2.95±1.69 3.85±3.00 6.14±2.77 6.61±1.02 5.82±1.31 5.06±0.69 C* 35.72±2.42 20.26±1.07* 19.85±0.78* 18.46±1.09* 18.73±3.46* 19.45±2.05* 19.49±2.78*
Page 177 Results
Physical Quality Parameters Time (min) control UV5+US5 UV10+US10 UV10+US20 UV20+US10 L* 38.23±0.29 35.55±0.12 33.95±0.86* 34.86±0.04* 36.12±0.76* a* 26.91±0.84 25.74±0.15 22.76±0.76 25.21±1.78 25.20±1.33 b* 19.60±1.51 19.60±0.26* 15.01±0.29* 13.02±0.44* 17.07±1.16* ΔΕ* 4.61±0.52 7.84±0.75 7.98±0.89 7.60 ±1.45 C* 33.32±0.40 26.05±7.72 23.15±6.33 24.08±8.93 26.38±8.78
Table 3.3.1.2: Values are average ± standard deviation of at least three experiments and represent the color parameters of strawberries after each processing time with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, UV+US. *: Asterisks within different treatment methods indicate significant differences (p<0.05).
Page 178 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Cherry Tomatoes
Physical Quality Control NaOCl 50 ppm NaOCl 200 ppm Parameters Time (min) 0 1 3 5 1 3 5 L* 41.47±1.08 39.12±0.90 38.34±0.15 38.11±0.41 39.65±0.26 39.90±0.10 38.68±0.09 a* 19.34±2.97 16.96±3.10 14.54±0.60 14.64±1.03 16.02±0.50 15.70±0.61 15.29±0.15 b* 18.86±1.20 17.07±0.53 15.65±0.74 15.42±1.15 19.20±0.43 19.13±0.62 17.71±0.08 ΔΕ* 4.13±1.70 7.04±0.77 7.11±0.52 4.31±2.10 4.33±1.89 5.58±1.90 C* 23.54±4.82 21.12±2.70 19.54±2.33 19.74±2.94 25.01±0.23 24.75±0.48 23.40±0.12 TCI 220.97±25.20 223.68±14 219.90±4.74 228.23±0.50 203.47±5.44 200.82±6.88 210.12±1.39
Page 179 Results
Physical Quality Control Ultrasound Treatment Parameters Time (min) 0 1 3 5 10 20 30 45 60 L* 42.27±0.31 41.27±0.02 41.26±0.01 41.21±0.09 41.31±0.45 37.87±0.41* 36.31±0.27* 35.48±0.38* 35.41±0.66* a* 19.33±2.96 15.69±0.11 15.67±0.08 15.79±0.06 15.18±0.25 12.00±0.15* 10.72±0.19* 9.76±0.08* 9.09±1.51* b* 21.07±2.63 19.70±0.44 19.48±0.55 19.59±0.45 19.51±0.44 15.01±0.36 16.01±0.41 16.54±0.96 16±1.16 ΔΕ* 4.12±3.45 4.19±3.54 4.08±3.50 4.71±3.60 10.50±3.64 11.77±3.76 12.72±3.92 13.51±3.13 C* 28.59±3.94 25.19±0.39 25.00±0.41 25.16±0.38 24.72±0.50 19.22±0.37 19.27±0.45 19.21±0.83 18.46±0.35 TCI 207.69±2.24 193.98±2.21 195.15±3.65 195.52±2.09 191.09±0.58 202.96±0.35 184.74±0.38 170.89±7.76* 165.85±8.04*
Control Ultraviolet Light Treatment
Time (min) 0 1 3 5 10 20 30 45 60 L* 42.27±0.31 41.56±0.28 40.45±0.11 40.67±0.69* 40.42±0.09* 40.34±0.10* 41.27±0.27* 38.31±0.13* 37.79±0.15* a* 19.33±2.96 17.30±0.62 16.52±0.13 16.74±0.03 17.36±0.23 15.87±0.09 15.89±0.84 15.27±0.32 15.52±0.40 b* 21.07±2.63 19.59±1.31 18.25±1.49 17.59±1.59 20.61±0.51 20.20±0.26 18.82±0.91 17.99±1.29 17.64±1.90* ΔΕ* 2.83±2.72 4.52±2.79 4.85±2.72 3.45±2.58 4.37±3.32 4.26±2.67 6.45±1.90 6.66±2.87 C* 28.59±3.94 26.14±1.36 24.63±1.18 24.30±1.17 26.94±0.52 25.69±0.15 25.64±1.23 23.61±1.19 23.52±1.18 TCI 207.69±2.24 205.47±3.96 211.19±8.70 216.39±9.69 202.70±9.69 194.56±1.94 200.84±0.82 209.28±6.44 215.14±6.37
Page 180 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Control US+NaOCl 50ppm Parameters Time (min) 0 1 3 5 10 20 30 L* 41.82±0.20 40.33±0.04 39.77±0.26 37.97±0,.03* 37.29±0.13* 36.11±0.02* 35.77±0.46* a* 19.34±0.34 17.21±0,.08 16.22±0.34 15.32±2.18 14.84±2.98 14.18±1.70 12.84±0.57 b* 20.97±0.26 18.96±0.35 16.66±0.05 14.77±1.01 14.59±1.44 14.44±0.54 13,.91±0.69 ΔΕ* 3.49±3.57 5.82±4.15 8.68±5.28 9.48±6.28 10.30±4.62 11.42±4.71 C* 28.53±3.99 25.61±0.30 23.25±0.22 21.30±2.04 20.83±3.11 20.26±1.48 18.93±0.86 TCI 209,49±3.14 211.68±1.73 221.15±1.96 232.71±13.96 232.00±13.55 232.55±11.39 226.77±3.47
US+NaOCl 200ppm Time (min) 0 1 3 5 10 20 30 L* 41.82±0.20 40.21±0.06 37.51±1.24 36.77±0.70* 36.42±0.16* 36.85±1.07* 35.74±3.47* a* 19.34±0.34 14.85±2.27 13.86±2.70 13.20±2.76 12.23±2.65 12.66±2.16 12.24±4.22 b* 20.97±0.26 17.58±0.52 15.22±0.55 15.08±0.19 14.08±1.96 14.05±2.45 13.79±4.83 ΔΕ* 5.98±5.35 9.23±4.19 10.33±4.84 11.55±5.97 11.11±6.82 12.05±6.34 C* 28.53±3.99 23.04±1.88 20.61±2.62 20.13±1.89 18.71±2.86 18.95±3.53 18.48±3.53 TCI 209.49±3.14 202.49±14.05 218.30±16.64 214.41±25.65 215.19±23.75 220.03±17.88 222.05±21.59
Page 181 Results
Physical Quality Control UV+NaOCl 50ppm Parameters Time (min) 0 1 3 5 10 20 30 L* 41.82±0.20 40.41±0.47 40.02±0.44 40.45±0.32 40.35±0.42* 40.29±0.12* 39.00±1.07* a* 19.34±0.34 17.40±0.69 16.63±0.17 16.39±0.79 15.99±0.50 16.22±1.00 16.37±1.06 b* 20.97±0.26 18.90±0.82 18.41±0.47 18.51±0,.40 18.60±0.30 18.33±0.53 18.07±0.34 ΔΕ* 3.55±3.10 4.36±4.33 4.29±3.38 4.53±4.12 4.47±4.89 5.27±4.75 C* 28.53±3.99 25.69±1.07 24.80±0.46 24.73±0.58 24.53±0.53 24.48±1.04 24.38±0.84 TCI 209.49±3.14 213.16±1.70 211.96±2.86 208.38±6.07 205.23±1.93 208.70±4.22 214.80±4.56
UV+NaOCl 200ppm Time (min) 0 1 3 5 10 20 30 L* 41.82±0.20 40.95±0.02 39.78±0.51 39.73±1.04 39.59±1.21* 39.22±1.13* 38.91±0.06* a* 19.34±0.34 18.12±0.60 14.25±0.21 14.71±0.64 14.82±0.59 14.57±0.48 14.70±0.65 b* 20.97±0,.26 18.80±0.02 18.69±0.77* 18.93±0,.65* 19.25±0.42* 18.65±0.61* 18.56±0.13* ΔΕ* 3.13±3.37 6.08±3.46 5.79±3.46 5.64±3.46 6.05±2.85 6.17±3.31 C* 28.53±3.99 26.11±0.42 23.50±0.71 23.99±0.29 24.30±0.11 23.67±0.76 23.68±0.30 TCI 209.49±3.14 216.80±3.66 192.34±3.28 194.77±11.45 193.97±9.64 196.71±4.26 198.95±6.31
Page 182 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Physical Quality Parameters Time (min) control UV5+US5 UV10+US10 UV10+US20 UV20+US10 L* 41.82±0.20 37.22±0.17* 37.32±0.58* 37.80±0.52* 36.92±0.11* a* 19.34±0.34 11.59±0.34 10.72±0.66 12.96±0.15 11.40±0.35 b* 18.86±1.20 12.08±0.32* 11.57±1.05* 14.86±1.08* 13.55±1.18* ΔΕ* 12.92±3.39 13.75±2.98 10.11±4.27 12.26±4.26 C* 23.54±4..82 16.74±0.44 15.78±1.19 19.72±0.85 17.73±0,.67 TCI 209.49±3.14 227.00±2.00 222.70±4.47 213.90±7.23 212.06±14.88
Table 3.3.1.3: Values are average ± standard deviation of at least three experiments and represent the color parameters of cherry tomatoes after each processing time with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, UV+US. *: Asterisks within different treatment methods indicate significant differences (p<0.05).
Page 183 Results
Color was expressed in terms of L*, a* and b* values. The chromameter describes color in three coordinates. More specifically, L* value indicated luminosity from 0 (black, level of darkness) to 100 (white, level of light), a* indicated chromaticity on a green (- 60, negative number) to red (+60, positive number), and b* value was responsible for chromaticity on a blue (-60, negative) to yellow (+60, positive) scale respectively.
When lettuce samples were treated with US for 45 and 60-min, some parts of the leafy structure of lettuce lost their green color giving final values of ΔΕ* 9.29±1.17 and 12.86±3.01 respectively. Treatment with NaOCl 50 ppm for 7-min resulted in a ΔΕ*=10.69±1.88. For UV treatments of 45 and 60-min the ΔΕ* observed were 15.67±1.56 and 16.59±2.09 respectively. Combined treatment of US 30min followed by immersion in NaOCl 50 ppm for 3-min resulted in ΔΕ*: 13.32± 4.58. Whereas, combined treatment of UV 20 and 30-min followed by immersion in 200 ppm NaOCl has as a result an increase in ΔΕ* of 16.22±2.10 and 17.88±1.75 respectively.
As far as strawberry modifications are concerned, the highest net change of color was observed for treatment with UV for 45 and 60-min respectively where an increase of 13.31±0.59 and 13.18±1.32 were observed.
Comparing the color of treated cherry tomatoes, the highest net change of color (ΔΕ*) for lettuce was observed when the samples were treated with US at longest time intervals (after 20-min). Combined treatments of US followed by NaOCl 50ppm and 200 ppm exhibited a change (ΔΕ*>10) after 20-min and 10-min treatment respectively.
Page 184 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 3.3.2 Physicochemical Parameters
Lettuce
Figure 3.3.2.1: TAC of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies.
No significant differences (p>0.05, n=44) were observed as far as Total Antioxidant Capacity (TAC) is concerned when conventional treatments at different treatment times were used. However, when alternative disinfection treatments were used, an increase in TAC concentration was obvious from the first-min of treatment, however the increase was significant after 45-min with US (p=0.049) and at 60-min treatment with UV (p=0.004). The increases after longest treatment times with UV and US reached 731 and 273 μmol Fe2+/g respectively. When combined alternative technologies were used, a significant increase of 463 and 366 μmol Fe2+/g was detected at 20-min UV followed by 10-min US (p=0.005) and at 10-min UV followed by 20-min US (p=0.013), respectively. Finally, significant increases (p<0.05, n=156) were observed when combinations of alternative and conventional treatments occurred. Page 185 Results
Figure 3.3.2.2: TPC of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies.
Total Phenolic Content (TPC) concentration remained constant or was slightly decreased when lettuce was immersed in NaOCL solutions. However, TPC increased by 3.76 and 3.01 mg gallic acid / g lettuce when UV and US alternative disinfection technologies were used. However, the differences among different treatment times, were statistically significant, after 45 min US treatment (p=0.033) and 30 min UV treatment (p=0.025). The highest concentrations of TPC were detected in UV20+US10-, US+NaOCL50 -, UV+NaOCl200 - and UV+NaOCl 50 - treated lettuces and were 22.18, 22, 22.25 and 22.65 mg gallic acid / lettuce respectively.
Page 186 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.3.2.3: AA of Romaine Lettuce before and after conventional, alternative and combined disinfection technologies.
The Ascorbic Acid (AA) content of lettuces did not exhibit any significant changes during different treatments. However, AA was slightly decreased (p>0.05, n=132) when treatments of more than 30-min for US, UV and combinations of UV+US occurred.
Page 187 Results
Strawberries
Figure 3.3.2.4: TAC of Strawberries before and after conventional, alternative and combined disinfection technologies.
When the strawberries are immersed in sodium hypochlorite solutions a slight but not statistically significant increase is observed (p>0.05, n=44). However, the mean observed increases of UV and US treatment are after the longest treatment times, 309 and 223 μmol Fe2+/g respectively. US led to a significant (p<0.05) increase from 5 minute treatment, whereas UV led to a significant increase (p<0.05) from 3 minute treatment. Moreover, the combined alternative treatments also led to an increase (p<0.05, n=36) in TAC of strawberries. The higher content of total antioxidants is obvious after the first-min of treatments.
Page 188 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.3.2.5: TPC of Strawberries before and after conventional, alternative and combined disinfection technologies.
The values obtained in this study demonstrate the positive effect of alternative and combined alternative technologies on the TPC of strawberries. However, the results obtained by conventional treatments showed the negative or no effect of sodium hypochlorite on the phenolic content of strawberries. UV and US led to significant increases (p<0.05, n=108) in phenolic content after 30- min treatment.
Page 189 Results
Figure 3.3.2.6: AA of Strawberries before and after conventional, alternative and combined disinfection technologies.
In all treated samples the AA content was decreased. However, a slight difference was observed between UV and US treated samples, where the decrease was higher for US , US+NaOCl 50 ppm and US+NaOCl 200ppm treated samples (-0.18, -0.16, -0.17 respectively) after the longest treatment times (60-min, 33-min, 33-min), compared to UV and UV+NaOCL treated samples (-0.12, -0.14, -0.1) at the same treatment times respectively. UV and US decreased the AA content significantly (p<0.05, n=108) after 3-min treatment, whereas all combinations of alternative disinfection technologies were statistically significant (p<0.05, n=36).
Page 190 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Cherry Tomatoes
Figure 3.3.2.7: TAC of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies.
It was observed that the TAC remained constant or slightly decreased when conventional treatments were used. However, when alternative disinfection treatments were used, UV resulted in greater values compared to US. In both technologies the increase was significant (p<0.05, n=108) from the first minute of treatment. After the longest exposure time (60-min) of UV, 155 μmol Fe2+ /g increase of antioxidants was observed, whereas 115 μmol Fe2+ /g, after 60-min treatment with US. Moreover, the combined effect of UV+US did not exhibit an additive effect, compared to single treatments, however the increase was significant (p<0.05, n=36) when tested with pairwise t-test. Furthermore, when combined alternative and conventional technologies were used, the effect of sodium hypochlorite did not affect the final value of TAC on cherry tomatoes (p>0.05, n=156).
Page 191 Results
Figure 3.3.2.8: TPC of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies.
The values for TPC of cherry tomatoes slightly increased (p>0.05, n=44) after conventional technologies, whereas a higher increase (p<0.05, n=108) is observed after Ultrasound and UV technology, with increase of TPC by 3,07 mg gallic acid/g and 2,31 mg gallic acid/g respectively. The increase was significant from the first minute of US treatment (p=0.003) and first minute of UV treatment (p=0.034). When combined technologies were used, a statistical increase (p<0.05, n=180) was also observed, with the higher increase (2,48 mg gallic acid/g) observed when US+NaOCl 200ppm was implemented.
Page 192 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.3.2.9: AA of Cherry Tomatoes before and after conventional, alternative and combined disinfection technologies.
The concentration of AA was generally remained constant or was slightly decreased. However, no statistically significant differences (p>0.05, n=310) observed before and after the use of different disinfection technologies, as far as their ascorbic acid content is concerned.
Finally a more generalized statistical analysis was carried out. For instance, Tukey HSD test was used in conjunction with an ANOVA to find disinfection technologies that are significantly different from each other for each physicochemical parameter.
TAC did not differ significantly (p>0.05, n=620) when all disinfection methods were used for strawberry and cherry tomatoes. However, differences between conventional and other technologies (p<0.05) were recorded for lettuce.
TPC was different (p<0.05, n=310) when lettuce was immersed in NaOCl solutions compared to other methods. However, the differences were not significant (p>0.05) for the rest of disinfection technologies. As far as strawberry is concerned, TPC observed
Page 193 Results when US method was used, was different compared to conventional methods (p=0.0001), as well as compared to UV method followed by immersion in NaOCl 50 ppm solution (p=0.003). For cherry tomatoes, difference in phenolic content was significant between US and NaOCl 50 ppm (p=0.002) and NaOCl 200 ppm (p=0.24), and between US and combined UV followed by NaOCl 50 ppm (p=0.003) and UV followed by NaOCl 200 ppm (p=0.001).
Differences in AA content among different technologies were observed in cherry tomatoes. For instance, AA content between US and NaOCl 50 ppm and 200 ppm was significant (p=0.0001 and p=0.019 respectively). Whereas in strawberries significant differences were detected only between US and US followed by NaOCl 200 ppm (p=0.043). No significant differences (p>0.05, n=310) were found in AA content of lettuce when treated with different disinfection technologies.
The Pearson correlation was finally used to examine any correlation between physicochemical values irrespectively of the disinfection method used. The relationship between TAC and TPC values was found to be positive and near to linear for strawberry (r=0.738, n=620) and cherry tomatoes (r=0.792, n=620) but a weak correlation (r=0.505, n=620) between the two aforementioned values was found for lettuce.
Page 194 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 3.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain
Three experts evaluated the effect of nine critical points to the final produce (C10=lettuce).
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 (OUTPUT)
C1 - M - - - VS M M W -VS
C2 VS - M M - VS VS VS M -VS
C3 - M - - M - - - - -W
C4 - M ------
C5 - - M ------W
C6 - M - - - - W - - W
C7 - M - - - W - W - W
C8 - M - - - - W - - W
C9 ------W
C10(OUTPUT) ------
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 (OUTPUT)
C1 - VS - -W - VS VS -M W -VS
C2 VS - - W - S S M M S
C3 -M W - - -S - - - -M -M
C4 ------W -
C5 -W W -S ------S -M
C6 - - - - W - M M -S -M
C7 - - - - - M - W S -M
C8 - - - - - M W - -M -W
C9 ------M -M - -W
Page 195 Results
C10(OUTPUT) ------
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 (OUTPUT)
C1 - S - - - VS VS -M W -VS
C2 VS - W W - VS VS S M S
C3 -W M - - -M - - - -W -M
C4 - W ------W -
C5 -W W W ------W
C6 - W - - - - W W -M W
C7 - W - - - W - W W W
C8 - W - - - W W - -W W
C9 ------W -M - -W
C10(OUTPUT) ------
Table 3.4.1: Evaluation of three experts, where W: Weak, M: Medium, S: Strong, VS: Very Strong
The concepts that were selected to be tested during the lettuce production procedure were extracted from questionnaires that were filled from experts. The methodology described extracts the knowledge from experts and exploits their experience of the process. Each expert based on his/her experience knows the main factors that contribute to the decision. Experts described the existing relationship firstly as “negative” or “positive” and secondly, as a degree of influence using a linguistic variable, such as “low”, “medium”, “high” etc. More specifically, the causal interrelationships among concepts are declared using the variable influence which is interpreted as a linguistic variable taking values in the universe of discourse U = [-1, 1]. Its term set T (influence) is suggested to be comprised of nine variables. Using nine linguistic variables, an expert can describe the influence of one concept on another in detail and can discern it between different degrees. The nine variables used here are: T (influence) = {negatively very strong, negatively strong, negatively medium, negatively weak, zero, positively weak, positively medium,
Page 196 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki positively strong, positively very strong}. With this method the purpose was to diagnose and predict the effect of different factors during the lettuce production chain in their contribution to a final safe fresh lettuce.
Figure 3.4.1: The FCM Model
Figure 3.4.2: Fuzzy Cognitive Map.
Page 197 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki FCMs are a combination of methods of fuzzy logic and neural networks. It is a flexible computational method, which is able to consider situations in which human reasoning process includes fuzzy and uncertain descriptions. FCMs are fuzzy-graph structures for representing causal reasoning. Their fuzziness allows hazy degrees of causality between causal objects (concepts). The effect and the interrelationships between the nodes should be calculated, in order to create a FCM. Each node is a concept, a main feature of the system. Each interrelationship between the nodes represents a cause-effect relationship that exists between concepts and determines the manner that one concept influences on the value of the interconnected concepts.
Wij are the weights among concepts and they take their values in the universe of discourse U = [-1, 1]. Each expert described the interconnections using linguistic variables, which with a defuzzification method are transformed to a numerical weight
Wij, belonging to the interval [-1, 1].
• Wij>0 positive causality, which means that when the value of concept Ci
is increased the value of the concept Cj is also increased.
• Wij<0 negative causality, which means that when the value of concept Ci
is increased the value of the concept Cj is decreased.
• Wij=0 no relationship between the concepts.
Generally, the value of each concept at every simulation step is calculated, computing the influence of the interconnected concepts to the specific concept, by applying the following calculation rule:
N (k+ 1) () kk() Ai = f() kA21i + k∑ Aj W ji ji≠ j=1
(k+1) (k) where Ai is the value of the concept Ci at the iteration step k+1, Ai is the value of the concept Cj at the iteration step k, Wjj is the weight of interconnection from concept
Ci to concept Cj and f is the sigmoid function. “k1” expresses the influence of the interconnected concepts in the configuration of the new value of the concept Ai and k2 represents the proportion of the contribution of the previous value of the concept in the computation of the new value.
The sigmoid function f belongs to the family of squeezing functions, and the following function is usually used to describe it:
Page 198 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 1 = f −λx 1+ e This is the unipolar sigmoid function, in which λ>0 determines the steepness of the continuous function f(x).
As mentioned above, each expert estimated each weight Wij between nodes i and j, according to his/her experience. In order to be sure about experts’ reliability, an algorithm was used to calculate both the weights of each interconnection and the credibility of experts. Each expert constructed his/her own weight matrix. Each weight Wij was collected and then they were compared according to the algorithm that followed.
The above variables were converted into numerical values with a defuzzification method. The Center of Area (COA) defuzzification method is one of the most commonly used defuzzification techniques. In this method, the fuzzy logic controller first calculates the area under the scaled membership functions and within the range of the output variable. The fuzzy logic controller then uses the following equation to calculate the geometric center of this area.
where S is the support set of the membership function of the output μ(y).
After COA defuzzification method the final weight matrix was presented.
Figure 3.4.3: Subsequent values of concepts till convergence of 1st case.
Page 199 Results
In the 1st Case, the experts decided as initial values of the inputs the following: C1, C2, C3, C5, C6, C7: very strong, C4, C8, C9: strong
The initial values for the concepts after COA defuzzyfication method were:
A(0)= [1 1 1 0.75 1 1 1 0.75 0.75]
The iterative procedure is being terminated when the values of concepts Ci have no difference between the latest two iterations. Considering λ=1 for the unipolar sigmoid function and after N=9 iteration steps the system reaches an equilibrium point, where the values do not change any more from their previous ones (figure 3.4.3). The calculated value of the decision concept was C10=0.951, which corresponds to the 95.1% of the output. Consequently the lettuce could be safe for consumption with 95.1% certainty.
Figure 3.4.4: Subsequent values of concepts till convergence of 2nd case.
In the 2nd Case, the representation of the concepts till their convergence were illustrated (figure 3.4.4). The experts decided as initial values of the inputs the following: C1, C2, C3, C5, C6, C7: strong, C4, C8: medium, C9:weak
The value of the decision concept was C10=0,818 which corresponds to the 81.8% of the output. It needed 10 iteration steps in order to reach to an equilibrium point. It can be concluded that the lettuce was also safe with 81.8% certainty for consumption in this case.
Page 200 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
Figure 3.4.5: Subsequent values of concepts till convergence of 3rd case.
In the 3rd Case, the experts decided as initial values of the inputs the following: C1, C3, C4: medium, C5, C6, C7: strong, C2, C8: weak, C9: zero
The output is C10=0.43, which corresponds to the 43% of the output (figure 3.4.5). This means that the lettuce was not guaranteed (with 43% certainty) for human consumption.
Page 201 Results 3.5 Assessment of disinfection technologies based on infectivity doses
Taking all the results into consideration, tables have been constructed which include the initial microorganism load, the best disinfection value produced with each disinfection technology and the infectious dose that is recorded in literature for each microorganism (tables 3.5.1, 3.5.2, 3.5.3).
Data on pathogen-specific relative infectivity were collected from the U.S. Food and Drug Administration’s (FDA), Public Health Agency of Canada (PHAC) and the European pathogen fact sheets. Given that relative infectivity cannot be expressed as a single numerical input but is, instead, a range of concentrations that depend on factors such as individual susceptibility and pathogen species, strain, or subtype, a worst-case scenario approach was taken whereby the relative infectivity categorization reflected the lower end (highest risk) of this range.
UV+NaOCl US+NaOCl NaOCl INFECT UV (30+3min, US (30+3 min, (200 ppm, UV+US IOUS LETTUCE Control (60min) 200ppm) (60min) 200ppm) 3min) (30min) DOSE 6 5 5 4 5 7 E. coli 10⁸ 10 (**) 10 (*) 10 (*) 10 (*) 10 (*) 10 (***) 10⁶ 5 5 5 5 6 7 5 6 S. aureus 10 10 (**) 10 (**) 10 (**) 10 (**) 10 (**) 10 (***) 10 -10 104(***) 104(***) 100(*) 103(**) 104(***) 105(***) 102-103 S. Enteritidis 10⁸⁶ 5 4 4 3 5 6 2- 3 L. innocua 10⁷ 10 (***) 10 (***) 10 (***) 10 (**) 10 (***) 10 (***) 10 10 2 3 6 5 2 4 1 2 HAdV35 10 10 (**) 10 (***) 10 (***) 10 (***) 10 (**) 10 (***) 10 -10
Table 3.5.1: Values⁸ (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in lettuce. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***).
The combined technology of US followed by NaOCl was found to be the best disinfection technology for reducing bacteria in lettuce. Moreover, UV and NaOCl exhibited promising results in disinfecting lettuce from HAdV35. The combined technology of UV+US recorded to have low disinfection efficiency for all microorganisms, compared to the other technologies.
Page 202 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
UV+NaOC l (30+3 US+NaOCl NaOCl INFEC STRAW- UV min, US (30+3 min, (200 ppm, UV+US TIOUS BERRY Control (60min) 200ppm) (60min) 200ppm) 3min) (30min) DOSE 6 5 4 4 6 7 E. coli 10⁷ 10 (**) 10 (*) 10 (*) 10 (*) 10 (**) 10 (***) 10 5 4 5 4 5 6 5 6 S. aureus 10⁷ 10 (**) 10 (*) 10 (**) 10 (*) 10 (**) 10 (**) 10 -10⁶
104(***) 103(**) 100 (*) 102(**) 104(***) 105(***) 102-103 S. Enteritidis 10 104(***) 104(***) 100 (*) 104(***) 105(***) 104(***) 102-103 L. innocua 10⁶⁷ 2 5 5 6 3 3 1 2 HAdV35 10⁷ 10 (**) 10 (***) 10 (***) 10 (***) 10 (***) 10 (***) 10 -10
Table 3.5.2: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in strawberries. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***).
US seemed to be the most promising disinfection technology for reducing bacteria population in strawberries, followed by US+NaOCl technology. However, UV was recorded as the best option for reducing HAdV35 in strawberries.
UV+NaO US+NaO Cl (30+3 Cl (30+3 NaOCl INFEC CHERRY UV min, US min, (200 ppm, UV+US TIOUS TOMATOES Control (60min) 200ppm) (60min) 200ppm) 3min) (30min) DOSE 4 3 3 2 3 4 E. coli 10⁷ 10 (*) 10 (*) 10 (*) 10 (*) 10 (*) 10 (*) 10 4 3 3 2 4 4 5 6 S. aureus 10 10 (*) 10 (*) 10 (*) 10 (*) 10 (*) 10 (*) 10 -10⁶ 104(***) 104(***) 103(**) 103(**) 104(***) 103(**) 102-103 S. Enteritidis 10⁶⁷ 4 3 3 2 4 4 2- 3 L. innocua 10 10 (***) 10 (**) 10 (**) 10 (*) 10 (***) 10 (***) 10 10 103(***) 104(***) 104(***) 104(***) 102(**) 104(***) 101-102 HAdV35 10⁶⁷
Table 3.5.3: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different disinfection methods at the longest exposure times and infectious doses for each microorganism inoculated in cherry tomatoes. Stars show the severity of infection: low infectivity (*), medium infectivity (**), high infectivity (***).
The best disinfection results were recorded for cherry tomatoes, where the majority of the disinfection methods exhibited promising results in reducing populations. However, NaOCl seemed to be the only method that could reduce HAdV35. On the contrary, E. coli and S. aureus were adequately reduced with all the disinfection methods. In general terms US+NaOCl seemed to be a valuable disinfection method as it reduced the four bacteria.
Page 203 Discussion Chapter 4. DISCUSSION
Four approaches of experiments were conducted in order to evaluate the efficiency of different disinfection technologies on different fresh produces. The final scope was to ensure food safety and public health. For this reason, hurdle approaches (such as UV, US, NaOCl, and combined disinfection treatments) were used in order to determine their individual as well as potential synergistic/additive mode of action against foodborne bacteria and viruses.
In recent years, consumer demand for safe, and natural products without chemical residues has been of great importance. It is well-known that processing of vegetables and fruits promotes a faster physiological deterioration, changes and microbial degradation of the product even when only slight processing operations can be used, which may result in degradation of their color, texture and flavour (Martin-Belloso, 2007). While conventional food-processing methods extend the shelf-life of fruit and vegetables, the minimal processing to which fresh RTE fruit and vegetables are subjected renders products highly perishable, requiring chilled storage to ensure their reasonable shelf-life (Martin-Diana et al., 2008). The vast majority of fresh minimally processed produce manufacturers use chlorine in washing and decontamination procedures (Seymour, 1999). There is controversy about the formation of carcinogenic chlorinated compounds in water (chloramines and trihalomethanes), calling into question the use of chlorine (Wei et al., 1995). As a consequence, alternative, non-thermal treatments gain more and more ground as promising technologies for food disinfection (Martin-Diana et al., 2008).
The first experimental approach of the present thesis evaluated the effectiveness of three non-thermal light technologies (NUV-Vis, continuous UV, and HILP) on their ability to inactivate two pathogens on a liquid matrix. The scope was to select the most effective light technology for use in fresh produce companies. The second experimental approach involved the use of non-thermal technologies (UV and US) as well as conventional sodium hypochlorite (NaOCl) solutions, in order to evaluate the disinfection efficiency of three RTE produces (romaine lettuce, strawberry and cherry tomatoes). The series of these disinfection technologies included also combinations of the above technologies. More precisely, UV+US, UV+NaOCl and US+NaOCl combined technologies were used. RTE produces were inoculated with different concentrations of bacteria and virus commonly found in many foodborne diseases and their disinfection efficiency with
Page 204 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki selected disinfection technologies was tested. Furthermore, with the aim of investigating how the above pathogens survive during refrigerated storage, the three fresh produces inoculated with the cocktail of the above four microorganisms were treated with selected disinfection technologies and were kept in refrigerated conditions for 15 days. The microbial load of romaine lettuce, strawberry and cherry tomatoes was recorded after 3, 7 and 15 days of storage at 6˚C.
In the third experimental approach, the quality and the physicochemical characteristics of the above fresh RTE produces were tested before and after the use of disinfection technologies, in order to extract conclusions about their nutritional properties in combination with the disinfection technology used.
The fourth approach was a computerized model, which was proposed, in order to obtain a risk assessment software tool to ensure food safety and public health.
Finally, conclusions based on infectivity doses for each pathogen and the results obtained from the present study, were exported, with the final scope to assure public health.
Page 205 Discussion 4.1 In Vitro Experiments with 3 Light Technologies
The initial aim of this study was to test the relative susceptibility of two bacteria (one gram negative and one gram positive) using three different light techniques. Then, a determination of the effectiveness of three light equipments on inactivation efficiency of selected types of bacteria when different dosages were implemented, followed.
The current study demonstrated that both E. coli and L. innocua are susceptible to all three light technologies investigated. Previous studies have investigated the lethal effects of high-intensity ultraviolet 405 nm light on Escherichia, Salmonella, Shigella, Listeria, and Mycobacteria as well as on Saccharomyces cerevisiae, Candida albicans, and spores of Aspergillus niger (Murdoch et al., 2012, Murdoch et al., 2013). As the mechanism of inactivation by visible light is believed to be through the production of ROS, the susceptibility of both E. coli and L. innocua to ROS may play an important role in the inactivation of these organisms by NUV-vis light of 405 nm. The mode of action is based on the stimulation of endogenous microbial porphyrin molecules to 1 produce oxidizing reactive oxygen species (ROS), predominantly singlet oxygen ( O2) that damages cells leading to microbial death (Maclean et al., 2008a). Specifically, 405 nm light has been shown to be capable of inactivating a range of predominantly nosocomial pathogens and also Gram-negative food-related pathogens (Enwemeka et al., 2008). When NUV-vis light was implemented, L. innocua proved to be the most readily inactivated organism compared to E. coli (p < 0.05). Murdoch et al. (2012) found that L. monocytogenes was most readily inactivated in suspension, whereas S. enterica was most resistant. They concluded that 395±5 nm light inactivates diverse types of bacteria in liquids and on surfaces, in addition to the safety advantages of this visible (non-UV wavelength) light. Furthermore, it has been reported (Murdoch et al., 2013) that fungal organisms may be somewhat more resistant to 405 nm light than bacteria.
The results obtained in this study are consistent with other studies which have reported that Gram-positive species, in general, were more susceptible to 405 nm light inactivation than Gram-negative species (Maclean et al., 2009). The prokaryotic bacteria also exhibit considerable variability in susceptibility achieving 5-log10 order reductions, with doses as low as 18 J/cm2 with Campylobacter jejuni was tested (Murdoch et al., 2010). When doses around 50–300 J/cm2 were implemented, Gram-positive species were generally more susceptible than Gram-negatives (Maclean et al., 2009).
Page 206 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki Microbial inactivation by 405nm light exposure has been found to be dose-dependent (Murdoch et al., 2012), and in applications where more rapid inactivation is desirable, the use of a much higher power light source, would significantly reduce the exposure times required for effective treatment. It must be emphasized that at the highest dosage 2 (36 J/cm ), 1.37 log10 CFU/mL reduction was achieved for E. coli and a greater log reduction (2.74 log10 CFU/mL) was achieved for L. innocua. Our results are in accordance with another study (Murdoch et al., 2012), where L. monocytogenes was 2 completely inactivated at an average dosage of 128 J/cm , whereas a 2.18 log10 reduction was achieved for E. coli at 192 J/cm2 dosage.
In the present study it was shown that, in order to achieve 2.66 log10 CFU/mL reductions 2 for E. coli and 3.04 log10 CFU/mL for L. innocua, respectively, a dosage of 2.832 J/cm with continuous UV equipment was needed. However, the samples were not treated further due to the temperature arise (>50°C). Our results are not in agreement with other studies (Schenk et al., 2011) where better reductions (7.2 log10 CFU/mL reduction and 2 4.6 log10 CFU/mL reduction for E. coli and L. innocua, respectively, at 1.2 kJ/cm ) were achieved, perhaps due to different E. coli and L. innocua strains that were used. UV light creates mutated bases that compromise cell functionality, but bacteria have developed DNA repair mechanisms to restore DNA structure and functionality (Friedberg et al., 2006). This phenomenon is reflected in the shape of the inactivation curves of our experiment (Gayán et al., 2013). The killing effects of HILP are caused by the rich and broad-spectrum UV content, the short duration, and the high peak power of the pulsed light produced by the multiplication of the flash power manifold (Cheigh et al., 2013, Gómez-López et al., 2007). Other researchers found that a significant reduction of 3.6 log10 CFU/mL for E. coli K12 and 2.7 log10 CFU/mL for L. innocua (p = 0.001) was achieved with HILP technology (3.3 J/cm2) (Muňoz et al., 2012). Our results are similar to another study (Muňoz et al., 2012) where 2.57 log10 CFU/mL reduction for E. coli 2 and 2.14 log10 CFU/mL reduction for L. innocua were achieved when 2.832 J/cm dosage was implemented. Other researchers (Chun et al., 2010, Krishnamurthy et al., 2007) have also studied the application of HILP in a continuous system. Moreover, Huang and Chen (2014) studied pulsed light in fresh produce. It is known that the surface structure of fresh produce is usually complex and bacterial cells may lodge in surface irregularities or crevices, such as calyx. As a consequence, the efficacy of HILP can be reduced by preventing the highly directional, coherent PL beam from reaching its target (Lagunas-Solar et al., 2006). Therefore, Huang and Chen (2014) impose the
Page 207 Discussion importance of selecting the representative inoculation site in a microbial challenge approach study.
Moreover, although HILP treatment is considered “non-thermal”, this is valid for treatments of short durations. For longer treatment periods, the temperature of sample increased to a level high enough to cause thermal inactivation of microorganisms. Hence, it should be taken into account that somehow the temperature must be kept low. In the study of Bialka and Demirci (2007), the HIPL-treated blueberries had a cooked appearance and lost structural integrity when samples were treated at a high fluence level. Moreover, in the study of Bialka and Demirci (2008), the maximum temperature of 80° C was reported after treatment with HILP at fluence level of 72 J/cm2. Darker cut- apple surfaces treated with HILP were observed in the study of Gomez et al. (2012). This can be attributed to temperature increase during treatment. In another study of Ramos-Villarroel et al. (2011), the use of 12 J/cm2 pulsed light resulted in a browning and softening of fresh-cut avocado. Whereas, Fine and Gervais (2004) reported a modification of color in pulsed treated pepper and flour, due to oxidation that might have been caused by pulsed light.
Page 208 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 4.2 Food Disinfection
The Mediterranean diet is based largely on plant-based foods. The diet is built on the cooking and food habits of people living in Mediterranean areas such as Greece, Spain, and Italy. A special emphasis on fruits and vegetables is given when the Mediterranean diet is followed, which should be included in the daily eating plan. The trick of the Mediterranean diet is to eat vegetables that are steamed, grilled or raw. People, that like the Mediterranean diet, have been found to have a lower risk of heart disease. Fruits and vegetables can become contaminated with pathogenic microorganisms while growing in fields, orchards, vineyards, or greenhouses, or during harvesting, post-harvest handling, processing, distribution, and preparation in food service or home settings. Manure used as a fertilizer or soil amendment, as well as in irrigation water, represent potential sources of pathogens that can contaminate fruits and vegetables. E. coli O157:H7 and Salmonella are carried by animals and shed in their feces (Beuchat et al., 2001). Despite considerable progress made in improving the safety of fresh fruits and vegetables, frequent foodborne outbreaks continue to occur. Among the major etiological agents responsible for outbreaks in RTE fruits and vegetables are E. coli, Staphylococcus spp., Salmonella spp, and Listeria spp. Once attached, bacteria can survive on RTE produces during postharvest storage (Sapers and Jones, 2006), and are capable of growing to populations exceeding 107 CFU/g, provided that appropriate conditions exist (Wei et al., 1995, Zhuang et al., 1995). Consumers are becoming more demanding and are looking for safe food products with high quality retention. Thus, due to absence of processing for the majority of these foods, disinfection remains one of the critical aspects in food industries. For this reason, a number of washing and sanitizing agents have been approved for fruits and vegetables disinfection. Food industries have currently started using emerging, non-thermal technologies in food processing such as Ultraviolet radiation, Ultrasound, pulsed light, cold plasma, ultrasounds and novel packaging practices (Ramos et al., 2013). When microorganisms are stressed, through the implementation of the above treatments, an adaptive response may follow which can increase the organisms’ tolerance to the same or to a different type of stress. Many bacteria react to stress by inducing the synthesis of various proteins (Jones and Inouye, 1994). Buchanan and Edelson (1999a), reported a cross protective effect of acid shock and acid adaptation of enterohaemorrhagic E. coli (EHEC) against heat or other stresses but also observed that the determination of survival of EHEC in acidic foods should consider the strain and its ability to induce stress responses. The resistance or adaptation
Page 209 Discussion of microorganisms to acid conditions can have implications for food safety (Buchanan and Edelson, 1999a).
The objective of inoculating RTE produces with cocktail microorganisms was to simulate real conditions that can occur during the food production chain of the lettuce, the strawberry, and cherry tomatoes since all bacteria are considered important for the food industry. E. coli O157:H7, Salmonella spp., and L. monocytogenes are the main pathogens implicated in several foodborne outbreaks related to fresh produce (Griffin and Tauxe, 1991, Sagong et al., 2011). Moreover, S. aureus is important as it concerns the contamination of food by food handling (Oliveira et al., 2011). There are few studies that have examined the effect of non-thermal technologies in the disinfection of one or two pathogens (Alexandre et al., 2011, Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Bialka et al., 2008, Oliveira et al., 2011, São José and Dantas Vanetti, 2012, Syamaladevi et al., 2012, Yaun et al., 2004).
4.2.1 Bacteria Disinfection
All the RTE foods were washed and then left to dry. Spot inoculation was the method used to inoculate the bacteria and viruses on romaine lettuce and strawberries, as it is more consistent and produces more reproducible results for the inoculation of a known number of pathogen cells on fresh produce surfaces than the dipping inoculation method (Beuchat et al., 2001). However, cherry tomatoes were dipped inoculated. Topography of fruits and vegetables is a critical parameter for the adhesion of bacterial cells (Wang et al., 2009). Studies have shown that E. coli O157:H7 was better attached to coarse, porous or injured surfaces of green peppers, than to those without injuries (Wang et al., 2009). Moreover, it has been shown that smooth surfaces (apple) are easy for bacterial removal, but when the roughness is higher with some deep valleys (oranges and avocados), bacteria is not totally exposed to the mechanical forces of washing. In products with high roughness with valleys and big cavities (cantaloupe) bacteria are well protected from mechanical forces and disinfection agents (Wang et al., 2009). Motility of microorganisms facilitates pathogen entry into wounds, stomata and other existing fruit surface openings (Deering et al., 2012). Internalization through the naturally existing opening is considered as one of the major route of pathogens to entry the plant tissue (Deering et al., 2012). Incidences of internalization depend on concentration of bacteria, their location on the plant, age, integrity and stages of plant development, as
Page 210 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki well as indigenous agonistic/antagonistic bacteria present on plant have been reported (Erickson, 2012).
Initial average populations for all microorganisms were about 7 log10 CFU/g of fresh produces (7.87, 7.96, 6.53, 7.52 log10 CFU/g for E. coli, S. aureus, S. Enteritidis, L. innocua respectively, for lettuce 7.85, 7.14, 6.14, 7.08 log10 CFU/g for strawberry and
7.38, 6.58, 7.42, 7.11 log10 CFU/g for cherry tomatoes). The numbers of E. coli, S. aureus, S. Enteritidis and L. innocua recovered from lettuce, strawberry and tomatoes samples were similar irrespectively the bacteria were inoculated separately or as a cocktail, which demonstrated that all bacteria retained similar attachment. The bacterial attachment is in accordance with other studies (Yang et al., 2003). Other studies have also selected cocktail inoculums due to their simultaneous prevalence of all these strains in vegetable and fruits (Bialka et al., 2008, Mahmoud, 2010, Ölmez and Temur, 2010, Sagong et al., 2011, Yaun et al., 2004).
Variations between initial populations of bacteria to different produces were apparent, which is in accordance with other researchers (Ziuzina et al., 2014), where with SEM images they confirmed that larger populations of L. monocytogenes adherent cells in addition to clusters of cells, were present. Despite the irregular nature of strawberry surface, which would probably facilitate bacterial attachment, Ziuzina et al. (2014) found that E. coli populations visualized by SEM on strawberry surface were still less dense by comparison with L. monocytogenes. However, in the present study the attachment of S. Enteritidis population was less compared to all other bacteria, which could be explained by the fact that S. Enteritidis interacts with naturally existing indigenous epiphytic bacteria. Depending on the types of epiphyte present, the survival of pathogens can be either enhanced or inhibited (Erickson, 2012). For example, Cooley et al. (2006) demonstrated that Enterobacter asburiae isolated from lettuce inhibited colonization of E. coli, whereas another epiphyte Wausteria paucula had the opposite effect, enhancing E. coli survival.
A common practice for food disinfection in food industry is the rinsing of fresh produce with tap water. However, removal of bacteria from fresh produce with rinsing with tap water is not effective since cells are very well attached and only some disinfectant agents can reach the cells and inactivate them. For instance, E. coli cells are attached to lettuce leaves with EspA filaments, which are the same filaments used to attach to human and bovine cells, following a similar molecular mechanism as the one used to colonize to the
Page 211 Discussion mammalian intestine. Generally, E. coli is characterized by having a strong and intimate attachment to the host cell membrane. It can destroy the microvilli of the bacteria at the bonding site.
According to studies, chlorine remains the most popular disinfection method for fresh produce contamination (Gil et al., 2009, Issa-Zacharia et al., 2010, Rico et al., 2007, Sapers, 2001). Chlorination is applicable to fruits and vegetables, using a postharvest process with flumes, water dump tanks, and spray washers. This postharvest treatment has been applied to various fruits and vegetables such as tomatoes, citrus, apples, pears and peppers (Yoon, 2014).
Among its advantages, it can be concluded that it is an easy method in an industrial setting, the contact time with the food is short and it is cost effective (Goodburn et al., 2013). In our study, chlorine was delivered as sodium hypochlorite solution. It is currently the most common sanitizer used in the fresh-cut produce industry. According to WHO, in order to have an effective disinfection, chlorine needs to be used in concentrations of 50 to 200ppm, at pH<8 and to be in contact with the produce for not less than one minute (WHO, 2008). The experiments conducted in this study used sodium hypochlorite solutions at pH: 6.5 at a low (50ppm) and a high (200 ppm) concentration.
When sodium hypochlorite was used for lettuce disinfection, it was obvious that when 1 and 3-min treatment was used, NaOCl 200ppm exhibited better results (p<0.05), as far as E. coli, S. aureus, S. Enteritidis and L. innocua is concerned. However, the disinfection efficiency between two concentrations of NaOCl was statistically significant (p<0.05) only for S. Enteritidis when 5-min treatment time was used. The main challenge in leafy vegetables, such as romaine lettuce, is that bacteria migrate easily to some points of difficult access for the sanitizers and thus protects the microorganisms (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Published data indicated that population reductions on produce surfaces with chlorine are within the range of 1–2 log10 units (Sapers et al., 2001). In the study of Bermúdez-Aguirre and Barbosa-
Cánovas, 2013, the highest inactivation for romaine lettuce was 3.5 log10 reductions and was achieved after 15-min in contact with the NaOCl solution of 100 ppm. They concluded that 2-3 log10 reduction of E. coli was achieved for lettuce, whereas 5-6 log10 E. coli reduction for tomatoes. In another study, where iceberg lettuce was used, the vegetable was washed for 1-min with 100ppm chlorine solution and inactivation of 1.4
Page 212 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki log10 for coliforms occured (Allende et al., 2008). Chlorinated water is by far the most common disinfectant method for washing produce. However, generally, it is believed to have minimal effectiveness in sanitizing the vegetables, with less than 2-3 log10 reduction (Chang and Schneider, 2012, Park et al., 2008). In another study with lettuce leaves, it was reported that 1.79, 2.48 and 0.33 log10 reductions were achieved for Salmonella, E. coli 0157:H7 and aerobic mesophilic population respectively, when lettuce leaves were immersed in 200 ppm chlorine for 10-min (WHO, 2008). Other researchers studied the difference between 100 ppm chlorine solutions of temperatures 47°C and 4°C, and concluded that higher temperature resulted in greater microbial reduction (Delaquis et al., 1999). Zhang and Faber (1996), using 200 ppm chlorine for
10-min at 4°C and 22°C found log10 reductions of L. monocytogenes of 1.3 and 1.7 on lettuce and 0.9 and 1.2 on cabbage, respectively. Lang et al (2004) demonstrated a 1.42 log10 reduction of E. coli 0157:H7 when lettuce was immersed in 200 ppm chlorine solution for 5-min. Bae et al. (2006) reported that 200 mg/L of chlorine, reduced populations of E. coli, S. aureus, L. monocytogenes and S. Typhimurium to 2.08–2.66 log10. Iturriaga et al (2010) has also found a 4-5 log10 reduction for S. Montevideo achieved by chlorine (200-100 ppm) on tomatoes. Ge et al. (2013) recorded the disinfection of S. Typhumurium in lettuce which resulted in 0.92 log10 reduction. Lopez- Galvez (2009) clearly indicated the difference between rinsing with water and chlorine disinfection, showing that 2 log10 reduction was achieved for lettuce when chlorine was used, compared to rinsing with water. Ölmez et al (2009) found a greater log10 reduction for E. coli (3.7 log10) inoculated in lettuce when 100 ppm chlorine was used and the temperature was 10 °C. Similar results have been exhibited by many researchers (Baur et al., 2004, Mahmoud et al., 2010, Pereira et al., 2013, Weissinger et al., 2000). Mahmoud et al. (2010) has studied the effect of chlorine dioxide gas to inactivate E. coli (2.5 log10), L. monocytogenes (3 log10), S. enterica (2.7 log10) on strawberries. Pereira et al. (2013) support that chlorine is a promising disinfection method for effectively reducing pathogens in fresh produces. Baur et al. (2004) investigated the effectiveness of cold and warm (50 °C) chlorinated water, as well as warm water without chlorine, for prewashing trimmed, cored iceberg lettuce. The use of warm water resulted in a significant log reduction of initial population of total aerobic bacteria, pseudomonads and enterobacteriaceae. However, this was not an issue in this study as only non-thermal technologies were selected, due to the fact that temperature more than 50 °C, can cause more stress to the organisms as it is above their normal growth temperature.
Page 213 Discussion
Chlorine has been widely used as a sanitizer in commercial produce wash (Ahvenainen, 1996, Beuchat, 1998), and hypochlorous acid is one of the most efficacious form of chlorine. However, studies have shown that chlorine used at concentrations between 50- 200 mg/L permitted by the FDA lacked efficacy in inactivation of human pathogens and spoilage microorganisms (Beuchat, 1998, Zhang and Farber, 1996). The technical challenge in a commercial fresh-cut wash operation has been how to maintain a relatively stable level of hypochlorous acid as this weak acid reacts quickly with organic materials present in wash solution (Gil et al., 2009). However, the reaction of chlorine with organic residues can result in the formation of potentially mutagenic or carcinogenic reaction products. This is a cause for concern since some restrictions in the use of chlorine might eventually be implemented by regulatory agencies. Increasing public health concerns regarding the possible formation of chlorinated organic compounds has resulted in a demand for alternatives to chlorine (Singh et al., 2002). Undesirable effects such as an unpleasant odor, softening of lettuce tissue and a browning reaction may occur when foods are treated with high chlorine concentrations (Kim et al., 2008). Chlorination of fruits and vegetables leads to side reactions between - active chlorine compounds (Cl2, HOCl, ClO ) and natural organic material, resulting in the production of chlorinated disinfection by-products (DBPs) such as trihalomethane, which is a carcinogenic substance (Chang et al., 2000). These reports clearly show the need for alternative disinfection methods in order to exceed the apparent population reduction “ceiling” of 1–2 log units. Thus, more effective, environmental friendly methods must be used from industries (Sapers et al., 2001).
Many studies have been published, explaining the increase of use of alternative disinfection techniques, trying to totally replace the conventional ones in the food industry (Alexandre et al., 2012, Allende and Artés, 2003, Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Sagong et al., 2011, Syamaladevi et al.,2012).
Short-wave UV radiation (200–280 nm) –UV light– is considered the most germicidal region of the UV spectrum for a great variety of microorganisms, having greater effect at wavelengths between 250 nm and 260 nm (Kowalski, 2009). At these wavelengths, UV photons are produced, which are mostly absorbed by thymine and cytosine nitrogenous bases of the deoxyribonucleic acid (DNA). The result is the formation of cross-linking photoproducts that interrupt the transcription and replication of DNA, thus leading to cell death (López-Malo and Palou, 2005). To cope with DNA damage, bacteria have developed repair mechanisms, including photorepair or photoreactivation and light- Page 214 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki independent or dark repair systems. Photorepair is carried out by photolyase enzymes which reverse DNA damages using the energy of visible light (Sinha and Hader, 2002). UV light sensitivity varies significantly among different types of microorganisms. Most authors agree that Gram-negative bacteria are more sensitive than are gram-positives, followed by yeasts, bacterial spores, molds, viruses and protozoa (López-Malo and Palou, 2005). These variations in UV resistance have been attributed to several intrinsic microbial factors, including the cell wall thickness, cell size, pigment production, composition, size and conformation of the genetic material, and cell DNA repair ability (López-Malo and Palou, 2005, Tran and Farid, 2004). In addition, the physiological state of cells, such as their growth phase, also determine their microbial sensitivity to UV radiation (Bucheli-Witschel et al., 2010, Wassmann et al., 2011). Ultraviolet (UV) light is one of the most promising technologies, due to its ability to inactivate a wide range of spoilage and pathogenic microorganisms, its minimal loss of the nutritional and sensorial quality of foods and its low energy consumption compared with other non-thermal technologies (Guerrero-Beltrán and Barbosa-Cánovas, 2004). Short wave ultraviolet (UVC, 254 nm) irradiation, is a process that has gained increasing attention after the USFDA approved its use in 1999 as an alternative to thermal pasteurization of fresh juice products (Flores-Cervantes et al., 2013, US FDA, 2000,)
Both Salmonella and E. coli in lettuce showed similar log10 reductions when treated for the same period with UV, which is in accordance with the study of Yaun et al. (2004). Also, when UV dosages (7.56 kJ/m2) have been used in other fruit surfaces (peaches and pears), better reductions of E. coli (up to 2.91 and 3.70 log10 CFU/g for peaches and pears respectively) have been achieved (Syamaladevi et al., 2012). In addition, reductions of E. coli and Salmonella in strawberries were achieved up to 2.5 log10 reduction, with dosages up to 64.4 J/cm2 of pulsed UV light (Bialka et al., 2008). The low log reduction recorded in our experiment was due to the UV lamp choice. The choice of a low dosage UV lamp technology was made in order to evaluate UV technology for disinfection taking into consideration that the food should not be affected in its color and quality in general. In agreement to other findings (Allende and Artés, 2003, Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Yaun et al., 2004), our work showed that higher UV doses resulted in a greater decrease of bacterial growth in ‘romaine’ lettuce, strawberry pieces and cherry tomatoes. The present results are in accordance with Bermúdez-Aguirre and Barbosa-Cánovas (2013) who showed that inactivation at short working distance and higher fluence (1.6 mW/cm2) was higher from
Page 215 Discussion the first-min of treatment. They found that after 60-min of treatment the inactivation achieved for E. coli was about 2.8 log10 when samples were closer to the radiation source. The doses needed to reduce E. coli O157:H7, L. monocytogenes, S. enterica and
S. flexneri, by 5 log10 reduction, on the surface of roma tomatoes were less than those needed on other produces (Mahmoud, 2010). Sanitizers can achieve better contact with smoother surfaces than with rough surfaces (Koseki et al., 2004). The germicidal effect of UV light in fresh-cut fruit and vegetables with rough surfaces (strawberry and lettuce) is usually within 1 and 2 log cycles, whereas for cherry tomatoes the reduction is greater. Moreover, it has been stated that treatment of tomatoes with short wavelength ultraviolet light has been shown to have a number of benefits. These include delayed senescence, as manifested by the maintenance of both firm texture and green pigmentation, and induction of resistance against phytopathogens such as Rhizopus stolonifer and Botrytis cinerea (Barka et al., 2000, Obande, 2011, Stevens et al., 2004).
The efficacy of surface disinfection by UV on food surfaces is influenced by several factors including: UV dose, UV dose rate, exposure time, surface characteristics, initial bacterial inoculum level and bacterial type (Otto et al., 2011). Despite the known limited ability of UV light to penetrate rough food surfaces, this study demonstrated that UV light has the potential to reduce bacterial contamination on food surfaces such as lettuce and strawberry surface and therefore has the potential to be used as post lethality treatment to control pathogens in ready to eat foods. However, it has been shown that UV was less effective at reducing populations of all bacterial types in strawberries when compared to lettuce. To predict UV disinfection rates on food surfaces, more kinetic inactivation data need to be obtained for pathogen and spoilage microorganisms, taking into account interactions between microorganisms and surface materials, such as shielding effects from incident UV and their dependency on surface structure or topography. Considering the bacteria inoculated into both lettuce and strawberry, more bacteria could have colonized deeper inside the strawberry, which could have reduced the chance of bacterial exposure to the UV light. Therefore the internalized bacteria in strawberry were possibly less affected by the UV-C light. It is known that UV-C light can not penetrate deeply into the fresh produce (Morgan, 1989). Hadjok et al. (2008) 2 showed that UV (37.8 mJ/cm ) combined with 1.5% H2O2 continuous spraying at 50 °C achieved a 2.84 log10 reduction of the internalized S. Montevideo in iceberg lettuce. Ge et al. (2013) showed that UV irradiation with higher fluencies (150, 450, 900 mJ/cm2) can significantly reduce the internalized S. Typhimurium in iceberg lettuce. Mahmoud
Page 216 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki (2010) studied the effect of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri on whole roma tomatoes by X-ray doses. Bermúdez-Aguirre and Barbosa- Cánovas (2013) studied the effectiveness of UV only on E. coli, artificially inoculated on grape tomatoes. Other researchers have evaluated the survival of S. Enteritidis inoculated on tomatoes using disinfection methods such as ozone, gamma-irradiation, chlorine dioxide, pulsed UV (Aguiló-Aguayo et al., 2013, Daş et al., 2006, Todoriki et al., 2009, Trinetta et al., 2010).
However, the mechanism of UV against internalized bacteria in the plant has not been clearly illustrated and needs further exploration. Moreover, it has been shown that more irregular and complicated surfaces are less decontaminated (Luksiene et al., 2012). Since UV light has limited penetration and depth, plant morphological characteristics such as roughness and presence of wounds on fruit surfaces impact microbial inactivation. Understanding these influences is needed if this technology is to be commercialized (Schenk et al., 2008). However, limited information is available on the influence of fruit surface properties on the efficacy of UV for surface decontamination.
Furthermore, the interactions encountered between indigenous microorganisms derived from the natural flora of foods and foodborne pathogens found in either the planktonic and biofilm states have been studied for Salmonella spp., L. monocytogenes and E. coli O157:H7 (Al-Zeyara et al., 2011, Møller et al., 2013 ). Indigenous microorganisms on fresh produce alone can form biofilms (Liu et al., 2013) and shows antagonistic effect with S. enterica on baby carrot (Liao, 2007). This could be an explanation of low disinfection efficiency achieved when UV was used. Biofilm is common phenomenon for fresh produce and difficult to eradicate using disinfectants (Jahid and Ha, 2012). The efficacy of a disinfectant toward fresh produce microorganisms depends on the ability of them to attach to plant tissues and form biofilms on the surface, stomata, trichomes, and cutting edges of the plant material (Jahid and Ha, 2012, Olaimat and Holley, 2012). The attachment and formation of biofilms on lettuce by Salmonella spp. have been reported previously (Kroupitski et al., 2009, Patel and Sharma, 2010). S. Typhimurium (ST) and E. coli O157:H7 are internalized through natural openings such as stomata, lenticels, and lateral roots (Deering et al., 2012, Jahid, 2014).
From a practical point of view, the UV chamber plays an important role in food disinfection. UV treatment is a simple and inexpensive method of processing which leaves no residues behind and may prove worthy for use in post-harvest situations to
Page 217 Discussion improve safety and to maintain quality of fresh ready to eat produces. It has been stated that the specific location of pathogens on produce surface influences the effectiveness of UV lamps, thus different levels of reduction can be achieved (Mudkoparnahyaya et al., 2014). One of the major concerns for the produce industry is limited shelf life. Several studies have indicated that significant improvement of shelf life for fruits and vegetables can be achieved by UV treatment due to inactivation of spoilage organisms and delayed ripening process (Arvanitoyannis et al., 2009).
US is based on cavitation, which enhances the mechanical removal of attached or entrapped bacteria on the surfaces of fresh produce by displacing or loosening particles through a shearing or scrubbing action (Seymoor et al., 2002). The results demonstrated that the reduction was greater as the treatment time was increased. São José and Vanetti (2012) studied the effect of ultrasound (45 kHz, 25 °C) on cherry tomatoes. The reduction of the total viable count, yeast and mold count, and inoculated S. enterica typhimurium that adhered to the surface of the tomatoes was evaluated. Treatments for
30-min with ultrasound alone, led to reductions of 1.73 log10 for S. enterica (São José and Vanetti, 2012). In the study of Bilek and Turantas (2013) ultrasound treatment (37 kHz, 25 °C) removed an average of 1.60 log10 CFU/g of the Salmonella population in cherry tomatoes after 30-min and the increase in contact time from 30 to 60-min of treatment reduced contamination even further. Similar findings were observed in the present study. In this study when US was used, better disinfection efficacy was presented in strawberries than lettuce for the majority of microorganisms. This may be attributed to different food surface properties such as hydrophobicity, electric charge, and roughness that can influence the adhesion and distribution of bacterial cells on food surface (Araujo et al., 2010). The survival of microorganisms depends upon several other factors such as type of strain, initial inoculums level, surface characteristics, and growth conditions (Guerrero-Beltrán and Barbosa-Cánovas, 2004). Microbial reduction by ultrasound is very important from the stand point of green decontamination and the hurdle concept of inhibition and elimination methods for food preservation technologies in fruits and vegetables (Bilek and Turantas, 2013). The lack of effectiveness that might be caused by the ultrasonication system is correlated with the operational procedures used in those disinfection treatments. Some key factors play an important role when applying ultrasound to a produce. For instance, dissolved gas in a washing solution is known to decrease the cavitation activity in a cleaning operation (Awad et al., 2012), and, therefore, degassing is essential for any ultrasonic cleaning applications. Moreover,
Page 218 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki the acoustic field distribution in an ultrasonic treatment chamber or tank is not uniform, mainly due to a standing wave formation. The nonuniform ultrasound field distribution and hence the nonuniform cavitation will result in variations in microbial inactivation activities at different locations in a washing tank. Consequently, during a wash treatment, those produce lettuce leaves that have received a good dose of ultrasound treatment and thus have a low microbial count would be easily cross-contaminated by neighboring leaves that have received less ultrasound treatment due to blockage of produce leaves to ultrasound propagation in the wash liquid and hence have a high microbial population due to an un-even acoustic field distribution. A good understanding of the underlining principles of power ultrasound, as well as a good design in wash system and operation procedure is absolutely a prerequisite for fully utilizing the power of ultrasound in produce decontamination applications. In addition, the efficacy of ultrasound is also affected by ultrasound frequency, power level, the size and shape of the ultrasonic bath, the depth, volume, temperature and nature of the liquid, and treatment time (Zhou et al., 2009). Zhou (2010) has shown that the 75 kHz ultrasound treatment was significantly less effective compared to two other frequencies (p < 0.05). The acoustic power density was 79.41 W/L for 25 kHz, 68.95 W/L for 45 kHz and 33.64 W/L for 75 kHz under the maximum power level. Mason and Lorimer (2002) mentioned, an increase in the gas content of a liquid can lead to a lowering of both the cavitational threshold and the intensity of the shock wave from the collapse of the bubble because of the increased number of nuclei (or weak spots) present in the liquid and the greater “cushioning” effect in the microbubble. Therefore, a degassing step is necessary in any practical ultrasonic cleaning application in order to remove the gases and enhance the effectiveness of the process.
Finally, combined disinfection technologies were used. As reported by Seymour et al. (2002), the combination of ultrasound with water or chlorinated water enhanced the removal of S. Typhimurium attached to iceberg lettuce by 1 log10 CFU/g compared to water or chlorinated water alone. However, data (Nastou et al., 2012) demonstrated that in an ultrasound-assisted wash, the decay of chlorine is further accelerated. Therefore, precautions must be taken to regularly monitor chlorine concentration during ultrasound and chlorine combined treatment. For this reason, in the present study the sodium hypochlorite immersions followed the UV or US treatments. These observed differences are likely to be due to differences in the microenvironment (topography, presence of stomata, chemical composition) in which the bacteria find themselves on the different
Page 219 Discussion surfaces, which could either affect the washing processes directly (such as the presence of crevices and fissures, a surface of low wettability) or indirectly by influencing the physiological state of the bacteria (Nastou et al., 2012). Ajlouni et al. (2006) demonstrated that washing Cos lettuce in combined treatments with ultrasound (40kHz) with various sanitizers at different concentrations reduced the microbiological populations by 1 to 2.5 log10 CFU/g immediately after washing, but there was little effect of ultrasonication alone on Cos lettuce regarding total or psychrophilic counts (p > 0.05), even during storage at 10°C (Zhou, 2010). However, they observed that after long-time ultrasonication (20 min) significant (p < 0.05) damage to the quality of Cos lettuce tissues was caused. Huang et al. (2006) reported that the treatment of 170-kHz ultrasonication resulted in 2.97 log10 reduction in Salmonella and 2.26 log10 reduction in
E. coli O157:H7 on inoculated lettuce, while using combined ClO2 and ultrasonication to treat inoculated apples with Salmonella and E. coli resulted in bacterial reductions of
3.12 to 4.25 and 2.24 to 3.87 log10, respectively (Zhou, 2010). Other researchers, have demonstrated that ultrasound in combination with 1% calcium hydroxide enhanced the decontamination efficacy on alfalfa seeds inoculated with S. enterica and E. coli O157:H7 (Scouten and Beuchat, 2002). Contradictory results regarding ultrasound disinfection treatments have been proposed by many researchers. Dehghani et al. (2005) investigated the impact of sonication as a disinfection method for determining the effectiveness of ultrasound on the E. coli inactivation. Ugarte-Romero et al. (2006) achieved a 5 log10 reduction of E. coli with power ultrasound in apple cider. D’Amico et al. (2006) studied the inactivation of microorganisms in milk and apple cider and demonstrated that ultrasound technology was a promising processing alternative for the reduction of microorganisms in liquid foods. However, Pagan et al. (1999) found that ultrasonic treatment (20 kHz) at ambient temperature was not very effective against L. monocytogenes. It has been reported that Gram-negative were more sensitive than Gram- positive bacteria (Patil, 2010). Lee et al. (2009) concluded that the combination of lethal factors (heat and/or sonication, with and without pressurisation) could significantly shorten the treatment time needed to achieve a 5-log10 reduction in the survival count of E. coli K12. Wang et al. (2010) investigated that, Alicyclobacilli had a higher resistance to ultrasonic treatments in apple juice than in buffer solutions, indicating that resistance to ultrasound depends on their environment. Adekunte et al. (2010) indicated sonication alone at moderate temperatures can achieve the desired 5 log10 reductions in yeast cells (Patil, 2010). Page (2003) studied the effect of combined technologies on inactivation of B. subtilis spores. No cumulative inactivation was presented, when treatment of UV was
Page 220 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki followed by free chlorine, implying that the effects of the two processes were just additive with no occurrence of synergy. In another study, the combined treatments of ultrasound and organic acids were employed to inhibit pathogens on organic fresh lettuce. More precisely, the combined treatment of ultrasound and organic acid for 5-min achieved an additional 0.8 to 1.0 log10 reduction of the three pathogens compared to organic acid treatment alone and the combined treatment of ultrasound and 2.0% organic acid (2.67 log10 CFU/g) was the most effective in reducing the number of pathogens (Sagong et al., 2011).
In the study of Yoon et al. (2014) reported that the combined chlorine–ionizing radiation disinfection treatments result in synergistic benefits in regard to reducing the numbers of natural microflora. Similarly, in this study, the combined methods result in a greater reduction than when the single methods are used alone.
4.2.2 Adenovirus Disinfection
Even though no foodborne outbreak with HAdV has been documented so far, potential viral transmission by foods is possible as they have been already detected in raw vegetables (Cheong et al., 2009), and are believed to be possible indicators for HAV and noroviruses. Different strategies have been developed to eliminate HAdV, mostly based on inactivation of virus by UV technologies (Baxter et al., 2007, Thurston-Enriquez et al., 2003b), ozone (Thurston-Enriquez et al., 2005), or chemical disinfectants such as free chlorine (Baxter et al., 2007, Cromeans et al., 2010, Thurston-Enriquez et al., 2003a), monochloramine (Baxter et al., 2007, Cromeans et al., 2010, Sirikanchana et al., 2008) or by combination of these technologies (Shin and Lee, 2010).
In this study, lettuce and strawberry achieved better reductions compared to cherry tomatoes, when immersed in sodium hypochlorite solutions. For instance after the longest exposure time, lettuce and strawberry achieved 4.95 and 5.02 log10 GC/g reduction, whereas cherry tomatoes achieved 3.76 log10 GC/g reduction. Baert et al
(2009) concluded that 200 ppm chlorine was able to render an additional 1 log10 reduction of MNV-1 present on lettuce, compared to washing with tap water. On the contrary, Gulati et al (2001) found that the same treatment of strawberries and lettuce did not result in an additional reduction of FCV compared to tap water. Baert et al. (2009) reported a significant lower decline by chlorination in the case MNV-1 lysate
Page 221 Discussion when it was directly spotted onto lettuce than when the inoculum was ten-fold diluted in tap water. Less free chlorine was available in the first case showing that chlorine reacted with the organic matter originating from the inoculum suspension. These observations are in accordance with the present study and can explain the low disinfection of cherry tomatoes by NaOCl solutions. Moreover, the use of only 1-2 pieces to determine the efficacy of sanitizers is indicative, but the efficiency of these sanitizers could change dramatically when introduced in an industrial process. Therefore, the produce/treatment ratio is important in order to evaluate conditions that are observed in fresh produce industries. In another study of Casteel et al. (2008), 1.7 log10 reductions of both MS2 and HAV were observed on strawberries, tomatoes and lettuce treated with 20 ppm chlorine. Inactivation rates of MS2 on lettuce differed significantly between studies (Casteel et al., 2008, Dawson et al., 2005) and the present study. Factors such as produce:treatment solution ratio, the presence of organic matter, inoculation method and produce type are reported to influence the efficacy of chlorination towards bacterial pathogens as well as viruses (Beuchat et al., 2004, Francis and O'Beirne, 2002, Lang et al., 2004). Butot et al. (2007) found a significantly higher inactivation of FCV after treatment of berries blueberries, strawberries, raspberries, basil and parsley with 200 ppm chlorine, suggesting the influence of produce type (Baert et al., 2009). Other factors play an important role on the effectiveness of disinfection strategies on viruses. For example, pH and water activity of foods can significantly influence the inactivation of microorganisms by High Hydrostatic Pressure (HHP) (Patterson, 2005). Low pH, which is characteristic for several fruits (e.g. berries) and vegetables (e.g. tomato), and pressure can act synergistically leading to enhanced microbial inactivation (Patterson, 2005). However, protection effect of lower pH in murine norovirus (MNV-1) was also previously observed (Lou et al., 2011). Furthermore, some food components such as proteins, lipids, carbohydrates or cations can confer a protective effect (Patterson, 2005). Sucrose, which is an important nutrient of berries has been linked to virus-borne outbreaks (Maunula et al., 2009), was recognized to protect feline calicivirus (FCV) when treated with HHP (Kovac et al., 2012).
High chlorine levels would be required to achieve a 2 to 3 log10 reduction of viruses on fresh produce. The application of higher concentrations (more than 200 ppm) is limited due to sensorial aspects. According to other studies (Duizer et al., 2004, Gulati et al., 2001), it seems useless to increase the efficacy of chlorination since they showed that a contact time beyond 10-min made little difference in antiviral activity towards FCV. In
Page 222 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki the present study, the treatment time of 3-min was sufficient for inactivating HAdV as far as cherry tomatoes are concerned. An additional 0.79 log10 reduction was achieved for another 2-min immersion in sodium hypochlorite solution. For strawberry and lettuce the treatment time plays an important role in inactivation efficiency. The difference between 3 and 5-min was not found to be statistically significant (p>0.05). However, when 10-min treatment time was implemented, the inactivation rate was significantly enhanced (p<0.05). For instance, in strawberries a double inactivation log10 GC/g of
HAdV was achieved. Whereas, in lettuce an additional 1.23 log10 reduction GC/g of HAdV was reported when the treatment time was increased from 3 to 10-min.
Viruses were likely sheltered from UV light by the strawberry matrix. UV inactivation of micro-organisms is probably due to the absorption of UV by nucleic acids causing dimerization of thymine in DNA or uracil in RNA (Nuanualsuwan and Cliver, 2003a, Sommer et al., 2001). At higher doses (≥1000 mW●s/cm2) UV light can also affect the capsid proteins. The combined effect of size/type of the virion and nucleic acids are thought to be factors determining the resistance/sensitivity of viruses towards UV (Sommer et al., 2001). A dose of 1 J/cm2 (corresponding with 1 W●s/cm2) reduced HAV and poliovirus by 5.7 and 6.7 log10 in PBS (Roberts and Hope, 2003). Fino and Kniel, (2008) concluded that the UV light treatment on lettuce at a dose of 40 mW s/cm2 achieved 4.3, 4.0 and 3.5 log10 reduction of respectively HAV, aichivirus and FCV. They also studied strawberries which were found to have a lower disinfection efficiency compared to lettuce. This is in accordance with the present study where the inactivation of strawberries compared to lettuce was greater in all treatments, with the only exception of UV at 60-min treatment time.
Bidawid et al. (2000a) found that 3 kGy of γ-irradiation was needed in order to achieve 1 log10 reduction of HAV on lettuce or strawberries. However, UV was less effective at reducing viral populations in lettuce. It was observed, that when the time was doubled (from 30 to 60-min), the mean reduction of HAdV was also doubled for strawberry
(from -1.26 to -3.98 log10 Genome Copies/g) and cherry tomatoes (from -0.92 to -2.22 log10 GC/g). Moreover, it has been shown that more irregular and complicated surfaces, such as lettuce are less decontaminated (Luksiene et al., 2012). Since UV light has limited penetration and depth, plant morphological characteristics such as roughness and presence of wounds on fruit surfaces impact microbial inactivation (Schenk et al. 2008).
Meng and Gerba (1996) found 3 log10 inactivation of adenovirus type 40 at a UV dose 2 2 of 90 mJ/cm and 4 log10 reduction at 120 mJ/cm . Whereas, Thurston-Enriquez et al. Page 223 Discussion
2 2 (2003a) found that Ad40 requires over 150 mJ/cm for 3 log10 and over 200 mJ/cm for 2 4 log10 inactivation. Ad1, Ad2, and Ad6 require 120 mJ/cm for 3 log10 inactivation (Nwachuku et al., 2005). Variation between studies can occur as a result of viral preparation methods and complexity of the adenovirus capsid.
Treatment with US was less effective (p<0.05) compared to UV. After the longest exposure time, lettuce exhibited the greatest reduction (1.79 log10 GC/g) compared to other fresh produces. However, the treatment time also played an important role, as far as virus reduction is concerned. Reduction of viruses by US is mainly due to the physical phenomenon called cavitation (Alegria et al., 2009, Piyasena et al., 2003, Seymour et al., 2002). Chrysikopoulos et el (2013) showed that the bacteriophages X174 and MS2, which were used as model viruses, were inactivated adequately when relatively high US frequencies (i.e., 582, 862, and 1142 kHz) were used.
The synergistic or additive effect of disinfectants has been investigated in some studies (Cho et al., 2011, Chrysikopoulos et al., 2013, Lotierzo et al., 2005) by carefully selecting the primary and secondary disinfectants and avoiding long contact times and high concentrations. However, the mechanism of action by which the combination of two disinfectants affects the disinfection action is still not clear. For instance, the sequential application of ozone (or ozone/H2O2) followed by free chlorine (Cho et al., 2006) was shown to achieve a higher level of inactivation of Bacillus subtilis spores than the sum of the inactivation level achieved with individual ozone (or ozone/ H2O2) and free chlorine application. This enhanced inactivation is referred to as a synergism, which is beneficial since it leads to a reduction in the amount of disinfectant and reaction time as well as a potential decrease in the formation of disinfection by-products (Rennecker et al., 2000).
However, there are few reports in the literature regarding the synergism involved in sequential disinfection processes employing UV or US followed by free chlorine (Cho et al., 2011, Chrysikopoulos et al., 2013). Chrysikopoulos et al. (2013) found that, for the case of MS2 US+UV was more effective than US alone. For the case of X174, US and UV did not provide any synergistic effects, on the contrary, the inactivation of X174 was hindered. Therefore, the combined use of US and UV should be employed only on specific cases.
Page 224 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki In this study, a synergistic effect was observed when UV and US were followed by immersion in sodium hypochlorite solutions, however, no additive effect was observed. The synergy was enhanced more when UV was followed by sodium hypochlorite, rather than when US followed by sodium hypochlorite (p<0.05). Moreover, the sequential treatment of alternative methods exhibited more promising results compared to the combination of an alternative and a conventional treatment, in strawberries and cherry tomatoes. In all cases the sequential application of two alternative technologies depended on the time used for each method.
It could be concluded, that there is a need for alternative sanitizers to be used for fresh RTE fruits and vegetables not only for the organic food sector but also for the conventional food processors (Ölmez et al., 2009). Thus, non-thermal technologies, alone or in combination, could offer attractive benefits to food disinfection.
Several researchers reported that microorganisms can attach in inaccessible sites like the stomata of leaves of leafy vegetables (Ells and Hansen, 2006, Itoh et al., 1998). Koseki et al. (2001) stated that microorganisms on the surface of lettuce leaves were easily disinfected, but bacteria inside the biofilms or cellular tissue, such as stomata could not be disinfected (Ölmez and Temur, 2010). This could probably explain the lower reductions of virus observed in lettuce, when combined technologies were used, compared to other fresh produces.
In order to assure the results obtained by PCR, tissue culture assays for lettuce followed. Plaque-based assays were selected in order to determine virus concentration in terms of infectious dose. Viral plaque assays determine the number of plaque forming units (pfu) in a virus sample, which is one measure of virus quantity. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with DMEM medium. A viral plaque was formed when a virus infects a cell within the fixed cell monolayer. Then, the virus infected cell lysed and spread the infection to adjacent cells where the infection-to-lysis cycle was repeated. The infected cell area created plaque (an area of infection surrounded by uninfected cells) was seen with an optical microscope as well as visually (figure 3.2.2.5). 4.2.3 High and Low Initial Load Disinfection Treatments
In order to assure that the disinfection efficiency was independent of the high bacteria inoculum that was selected throughout the experiments, selected experiments with
Page 225 Discussion conventional, alternative and combined technologies were conducted with different initial bacteria inoculum. It was observed that in all RTE produces the bacteria populations were under the limit of detection (<0.22 log10 CFU/g), when three effective treatments (NaOCl 3-min, US+NaOCl 33-min, US 60-min) and low initial bacteria inocula were selected. In cherry tomatoes, the disinfection efficiency was even better, and bacteria were shown to be under the limit of detection (<0.22 log10 CFU/g), with almost all disinfection treatments, since a low initial microbial load was inoculated. In another study where cold plasma was used as a disinfection technology, different initial concentrations complicated the comparison of the bacterial sensitivity to the ACP treatments (Zuzina et al., 2014). In the study conducted by Fernandez et al. (2012) the increase of concentration of S. Typhimurium from 5 to 8 log10 CFU/filter reduced the inactivation efficiency of ACP.
4.2.4 Storage Conditions
To examine the shelf life of the treated foodstuffs and test for possible microbial reactivation after disinfection treatments, the foods after treatments were stored in fridge (6°C) for 15 days.
During storage, the microflora on shredded iceberg lettuce leaves gradually increased for untreated and treated samples. However, treated samples maintained microbial populations significantly at a lower level compared to the untreated control. Mahmoud et al. (2010) have studied the treatment with 2.0 kGy X-ray, and concluded that the population of mesophilic, psychrotrophic, and yeast and mold was maintained under the detectable limit for 12, 20, and 9 days storage, respectively. These results are in agreement with another study Zhang et al. (2006) who reported that aerobic mesophilic bacteria on fresh-cut lettuce irradiated with 1.0 kGy gamma rays were reduced by 2.4 log10 CFU. The limited shelf life of fresh processed leafy lettuce is one of the greatest problems faced by commercial marketers (Mahmoud et al., 2010). Oliveira et al., (2010) studied the effect of modified atmosphere packaging on survival of E. coli, Salmonella and Listeria on lettuce. In another study of De Oliveira et al. (2012), significant differences on the behavior of L. monocytogenes on shredded organic lettuce were observed throughout storage period (p<0.05) depending on the initial load of background microbiota. Populations of L. monocytogenes on the ‘high’ counts increased slightly from 3.8 to 5.0 log10 CFU /g after 8 days. Numbers on the ‘medium’ mesophilic counts Page 226 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki were similar with levels ranging from 3.8 to 5.3 log10 CFU/g. On the contrary, when the initial mesophilic count was ‘low’, populations of L. monocytogenes increased rapidly with significant differences between other treatments during storage period, reaching final population of 6.1 log10 CFU/g. Our results are in accordance with Koseki and Isobe, 2005 where an increase was observed in all microorganisms after storage at refrigerated conditions. The increases were obvious after the 3rd day of storage (Koseki and Isobe, 2005). Finally, Mahmoud et al., (2010) studied the effect of mesophilic bacterial counts on untreated and irradiated treated lettuces after a total time of 30 days storage period.
As far as strawberries is concerned, the effect of disinfection treatments total mesophiles and yeasts and moulds, has also been studied by Alexandre et al. (2012). More precisely, the impact of ozone, ultrasound, UV radiation, NaOCl, H2O2 was studied. Strawberries washed with hydrogen peroxide solutions (before storage) had the highest total mesophiles reduction: 2.26 ± 0.38 and 1.59 ± 0.41 log10 unit reductions, for H2O2 at 5% and 1%, respectively. During storage at refrigerated temperature, these two washing treatments resulted in strawberries with lower microbial loads, when compared to the results obtained with the remaining treatments. At the end of refrigerated storage, samples pre-treated with ozone presented lower microbial loads than samples ultrasonicated or treated with UV radiation. Throughout refrigerated storage, strawberries pre-washed with all sanitizer solutions and with ozone presented lower microbial loads (p<0.05) than untreated, ultrasonicated, UV irradiated or water-washed samples. In the present study an increase in E. coli, S. aureus, S. Enteritidis and L. innocua was obvious after the 7th day for all treatments. Miguel-Pintado et al. (2013), studied the effect of HPP on storage of tomatoes and found that at day 30, fruits submitted to HPP showed lower microbial counts while non-treated presented microbial spoilage (Miguel-Pintado et al., 2013) Mukhopadhya et al. (2014) studied the effect of UV on mold and yeast population of tomatoes, during storage for 21 days at 5°C. A slight increase was observed from day 14th to day 21th. However, when the effect of UV studied for total aerobic count on tomatoes, where the initial loads were higher, an increase was observed for days 7, 14 and then the microbes decreased until day 21th. Similar results were observed in the present study. Moreover, the effect of storage on tomatoes was also studied by Aguiló-Aguayo et al. (2013), were pulsed light was used for disinfection of tomatoes, was similar to our results. A steadily increase in total aerobic mesophilic bacteria was observed from day 0 to day 15, with the control sample
Page 227 Discussion to have the highest values and treated samples to have lower. Finally, the study of Daş et al. (2006), suggested that the storage plays an important role on the survival of S. Enteritidis on tomatoes. All the treatments that were implemented in RTE produce in this study, exhibited similar reaction, for all the microorganisms. The only difference observed in S. Enteritidis inoculated on lettuce and strawberries and treated with combined disinfection technologies. For example, Salmonella population in lettuce showed a reduction from day 7 to day 15, when combined treatments were used (UV+NaOCl, US+NaOCl), whereas all the other microorganisms were increased in all the treatments. Moreover, Salmonella population in strawberries when treated with UV+NaOCl and stored, remained constant from day 7 to day 15, compared to the increase that was observed for all microorganisms treated with all the other disinfection technologies.
Page 228 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki 4.3 Food Quality parameters
4.3.1 Color
Color is one of the most important attributes affecting consumer perception of quality. Moreover, it plays a key role in food preference and acceptability and may even influence taste thresholds and sweetness perception (Clydesdale, 1993). When comparing the color of the outer leaves of lettuces, mini romaine had more greenish (higher hue) but less intense (lower chroma) color than romaine for instance. Tiwari et al. (2009) reported a slight increase (1–2%) in the anthocyanin content of sonicated strawberry juice at lower amplitude levels and treatment times. The color parameter related to browning (Castaner et al., 1999) and to the breakdown of chlorophyll (Bolin and Huxsoll, 1991) is a* value. A significant increase in a*, indicates a shift from greenness to redness. Samples treated with chlorine showed higher levels of potential browning (Martin-Diana et al., 2008). In the present study, the highest net change of color (ΔΕ) for lettuce was observed when the samples were treated with US at longest time intervals, which indicated that a significant non-enzymatic browning reaction was present (Cao et al., 2010). Similar results were observed in another study for UV treated lettuce (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Strawberry color is a mix of red and yellow. Thus, Hunter a* and b* values or some combination of a* and b* may be considered as the physical parameters describing visual color degradation (Rodrigo et al., 2007). Both a* and b* values showed significant differences from 45-min of treatments (p<0.05) with both non-thermal disinfection methods (UV, US). It was obvious that C* value, which shows the degree of saturation, purity and intensity of color changed significantly (p<0.05) for the strawberry samples treated with UV for 45 – min and 60-min as well as for strawberry samples treated with ultrasound at the same times, compared to the control sample. The color of the surrounding liquid was also changed after 60-min of Ultrasound treatment and a slightly pink color was obvious (data not shown). Moreover, the texture and the general appearance were modified after 45-min of Ultrasound treatment, whereas there was no obvious modification of the appearance after 45-min of UV treatment for lettuces and strawberries. The decrease of C* value may be attributed to enzymatic oxidation of anthocyanins leading to losses of color brilliancy (Holzwarth et al., 2012). In another study, thermosonicated strawberry samples retained their color (Alexandre et al., 2011). Aday et al. (2013) reported that changes in L* values in US treated strawberries were not significantly important.
Page 229 Discussion
However, 90 W treatment had an adverse effect on the anthocyanins' stability. Moreover they reported that the bright color of strawberry was preserved when 30 W and 60 W treatments were implemented for 5 and 10-min. In our study, the treated samples with 30 W/L ultrasonication, retained their bright color (L*, a*, b*, C*) for treatments of 10, 20 and 30-min , which is in accordance with the study of Aday et al. (2013). Aguiló- Aguayo et al. (2013) studied the effect of Pulsed Light on physical characteristics (color, firmness and fruit weight) as well as on nutritional composition of tomatoes. More precisely, they concluded that PL treatments did not affect the color of tomatoes, by observing the luminosity and hue values before and after treatments. Luksiene et al. (2012) also reported that no difference in color of tomatoes was observed when PL treatments were used for tomatoes. Bialka and Demirci (2008) also did not report any significant difference in the skin color of strawberries when were treated with PL. However, non-thermal PL technology resulted in a loss of firmness and loss of weight for tomato samples, according to the study of Aguiló-Aguayo et al. (2013). In this study no significant differences in net color of cherry tomatoes were observed, when treated with UV and US which is in accordance with the aforementioned studies. It was noted that the color readings in general have relatively large standard errors, which can be attributed, in part, to the heterogenous composition of different tissues in lettuce samples (Baur et al., 2004).
When sodium hypochlorite immersions were used, the color was degraded as the treatment time was increased. The highest net color differences were observed in lettuce, after the longest exposure time in NaOCl 200ppm, and the lowest differences were observed in cherry tomatoes, compared to lettuce and strawberries. The chlorinated samples showed higher levels of the red parameter (an indicator of browning), which is similar to the findings of Martin-Diana et al., (2007). Low levels of luminosity indicate high levels of browning since darkness is related to browning (Chen et al., 2010, Martin-
Diana et al., 2005,). However, ClO2 has been proven to be effective in inhibiting enzymatic browning of fruit and vegetables (Chen et al., 2010).
As far as combined disinfection technologies are concerned, the non-thermal combined treatments (UV+US) exhibited slight differences in color of all RTE produces. UV+NaOCl was a more severe treatment regarding the quality retention of lettuces, whereas US+NaOCl was more severe for cherry tomatoes. For strawberries, all combined treatments resulted in same differences in color. Total Color difference was
Page 230 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki also observed for lettuce after combined treatment with chlorine and US in the study of Salgado et al., (2014).
4.3.2 Physicochemical Parameters
It is well known that fruits and vegetables are essential components of the human diet. There is considerable evidence of the health and nutritional benefits associated with their consumption (Abadias et al., 2008). Due to their high nutritional value (presence of high levels of micronutrients and fibers), their consumption is recommended by many organizations (WHO, FAO, USDA and EFSA) to reduce the risk of cardiovascular diseases and cancer (Allende et al., 2006a, Ragaert et al., 2004). Among the primary metabolites found in vegetables, soluble sugars and organic acids are important components, and both greatly contribute to their flavor characteristics and nutritional value. Moreover, carbohydrates promote ascorbic acid stability, and thus enhance the vitamin content (Lopez et al., 2014). Moreover, consumers demand healthy, fresh-like and easy to prepare products, as a consequence of their lifestyle changes. Thus, minimally processed, RTE fresh-like fruits and vegetables have been developed (Allende et al., 2006a, Allende et al., 2006b, Froder et al., 2007, Tournas, 2005). RTE fruits and vegetables constitute a suitable meal as they provide a great variety of nutrients, minerals, and vitamins and they do not need preparation in order to be consumed (Froder et al., 2007). Furthermore, the freshness, economic handling and attractive presentation of these types of foods are factors that enhance their marketing (Little and Gillespie, 2008).
Lettuce in one of the most consumed vegetables in many countries. Romaine lettuce is an important dietary leafy vegetable, which contains appreciable amounts of water- soluble antioxidant compounds such as vitamin C, phenolic compounds and lipid- soluble antioxidants, such as lutein and tocopherols (Rice-Evans et al., 1995, Rice-Evans et al., 1996, Szeto et al., 2002). Moreover, it is believed that lettuce consumption is correlated with an improved lipoprotein profile and antioxidant status, leading to a prevention of lipid peroxidation in tissues thus having a cardiovascular protective effect (Nicolle et al., 2004). Many antioxidants and phenolic components have been detected in lettuce (Heimler et al., 2007). However, the concentrations of flavonoids and phenolic acids in lettuce are sensitive to environmental conditions (Liu et al., 2007). Differences observed in the nutritional composition of the studied romaine lettuces between the experiments of this study, could be explained in part by differences in the head structure
Page 231 Discussion and size. Opener lettuce heads such as those of romaine and mini-romaine lettuces have a higher photosynthetic area, which would contribute to increasing chlorophylls and sugars as well as other related metabolites (Lopez et al., 2014, Mou, 2009).
Antioxidants deactivate radicals by two major mechanisms, single electron transfer (SET) and hydrogen atom transfer (HAT) (Prior et al., 2005). FRAP is a SET-based method, ABTS assay utilizes both HAT and SET mechanisms. The response of the different antioxidants to these assays depends on their ability to quench free radicals by hydrogen donation and/or their ability to transfer one electron to reduce any compound. According to Gil et al. (2000), the major organic acids present in lettuce, malic and citric acids, do not show antioxidant capacity when they are evaluated with the FRAP assay. This may explain the different results observed when different methods are used. Lopez et al. (2014) have found that mini- romaine type lettuces, show higher antioxidant capacity with the ABTS method due to their high organic acids content, whereas romaine type showed higher antioxidant capacity with the FRAP method, probably due to their higher phenolic compound content.
The initial values of TAC, TPC and AA in this study are similar or slightly lower than values from other studies (Tiveron et al., 2012, Zhan et al., 2013). This can be attributed to many factors such as cultivating conditions of the produce, the extraction conditions as well as differences in varieties of the lettuce produce.
Moreover, TAC, TPC and AA greatly can vary among different leaf positions. For instance, outer leaves exhibit significantly higher TAC, than middle and inner leaves in both red and green color lettuce where the differences were more distinct particularly in red color cultivars (Ozgen and Sekerci, 2011). It has been demonstrated that outer leaves have the highest phytonutrient content and antioxidant properties, compared to inner leaves (Ozgen and Sekerci, 2011). Gobbo-Neto and Lopes (2007), reported that several factors such as seasonality, temperature, water availability, soil nutrients, pollution, and pathogen attack can affect the content of secondary metabolites in vegetables, such as phenolic compounds. In a study of Tiveron et al. (2012), they evaluated the phenolic content and antioxidant activity of a great variety of vegetables commonly consumed in Brazil and they found that the highest ability to reduce Fe3+ to Fe2+ was found in lettuce (447.1 μmol Fe2+/g), watercress (277.4 μmol Fe2+/g), and spinach (273.3 μmol Fe2+/g). The above results are similar to the findings of this study, where 319 μmol Fe2+/g was the average initial concentration of TAC in lettuce. The phenolic content found in lettuce
Page 232 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki in other studies was 16.9 mg GA/g DW lettuce, which was different from findings of Chu et al (2002) and Llorach et al (2008) which found lower and higher TPC in lettuces respectively.
The issue nowadays is to increase the shelf-life of food products by preventing loss of sensory and nutritional quality at the same time. Thus, many industries tend to add antioxidants (Ponce et al., 2004). So, disinfection technologies should not alter the nutritional properties of lettuce. In the present study, TAC was enhanced when non- thermal disinfection technologies were used. The increase was more intense when UV was used. It is worth mentioning that after 30 and 60-min treatment with UV the concentration of TAC was increased by 500 and 829 μmol Fe2+/g respectively, whereas 545 and 674 μmol Fe2+/g was the TAC increase when US treatment for 30 and 60–min respectively was achieved. Karaca and Velioglou (2014) also studied the effect of disinfection treatments on different minimally processed fruit and vegetables. The enhancing effect of UV on the total phenolic compounds of fruits and vegetables has been well studied over the last few years. Scientific evidence shows that the DNA- damaging effect of UV light induces the accumulation of UV-absorbing flavonoids and other phenolics compounds, predominantly in the epidermal tissues of fruit (Bravo et al., 2012).
Kenny and O’Beirne (2009) reported decreased levels of ascorbic acid in lettuce treated with chlorine, compared to tap-rinsed lettuce, which is in accordance with the results of the present study. Ascorbic acid degradation reactions are often responsible for important quality changes that occur during the storage of foods, limiting shelf-life. When products are treated with strong oxidizers, loss of antioxidative compounds like ascorbic acid can be induced. The results of this study indicated ascorbic acid degradation in lettuce after disinfection treatments, which are in accordance with studies on rice leaves (Imai and Kobori, 2008) and strawberries (Allende et al., 2007). Phenolics are believed to extend shelf life and increase the stress tolerance of plants, leading to lower postharvest losses (Hodges and Forney, 2003). Such a property is based on their ability to scavenge reactive oxygen species that are known to be involved in leaf senescence and in the plant antioxidant defense system. Phenolic acids may occur in multiple conjugated forms with sugars, acids and other phenolic compounds (Robbins, 2003). In the present study TPC was increased when all emerging non-thermal technologies were used. This increase in the concentration of phenolic compounds can be attributed to the wounding stress from the treatment (Martin-Diana et al., 2008). Page 233 Discussion
Finally, lettuce is a relatively poor source of vitamin C compared with other vegetables (Bahorun et al., 2004). In another study, vitamin C (AA plus DHAA) showed an average concentration of 78 mg/g FW (Nicolle et al., 2004). Generally, differences in vitamin C concentration have been found among lettuce cultivars (Lopez et al., 2014). AA content of romaine lettuce in this study was found to be 0.09 mg/g FW. Generally it remained constant throughout non-thermal disinfection technologies, whereas it was decreased when immersion in NaOCl solutions followed.
Anthocyanins can be found in many fruits and vegetables, and they are largely responsible for the red color of ripe strawberries (Allende et al., 2007, Ayala-Zavala et al., 2004). Moreover, ascorbic acid (AA) has long been considered an important nutritional component of strawberries. In the present study, the AA content of strawberries was 53 mg/100 g FW. In Selva strawberries the initial AA content was found to be 86.4 for organic and 71.2 mg/100 g FW for conventional strawberries, respectively (Crecente Campo et al., 2012). It is known that strawberries have an antioxidant capacity up to 10-fold greater than that of other fruits (Szeto et al., 2002). Although the literature suggests that the exposure of plant foods to stressful situations could modulate the synthesis of such defense substances as polyphenols and anthocyanins, an increased concentration of these substances was presented in our study, which is in accordance with other studies (Crecente Campo et al., 2012). Different studies have shown that the TPC varies depending on the variety of the strawberry studied. Meyers et al. (2003) indicated that Earliglow, Evangeline, and Annapolis varieties had the highest free phenolic contents, averaging 273 mg gallic acid/100 g FW, whereas the lowest content was measured in Mesabi, Jewel, and Allstar, averaging 202 mg gallic acid/100 g FW. Wang and Lin (2000) reported TPC values of the juice from strawberry fruits (cv Allstar) at different stages of maturity, which was found to be 256 and 103 mg of gallic acid equiv. (GAE) per 100 g fw for green and ripe fruit, respectively. The TPC also varies with the attachment of the stem (Crecente Campo et al., 2012).
Erkan et al. (2008) studied the effect of UV on antioxidant capacity of strawberries fruits. UV enhanced the total phenolic content of strawberry, which is in accordance with the present study. Moreover, the phenolic content of strawberries increased during the 15 day storage period. However, this increase was relatively lower in control fruit when compared to illuminated fruit (Erkan et al., 2008). Moreover, UV treatment has been shown to cause a significant increase in the antioxidant capacity of peppers Page 234 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki (Vicente et al., 2005) and blueberry fruits (Perkins-Veazie et al., 2008). Fernandez et al. (2012) observed a difference among phenolic compounds and anthocyanins in strawberry samples, and depended on different agricultural practices. Also, the radical scavenging ability and the reducing capacity assayed by the DPPH and FRAP methods, respectively, were higher in strawberries of organic farming (Gündüz and Özdemir, 2014). In another study of Li et al. (2014) the effect of UV on antioxidant capacity and anthocyanins in strawberries was studied. The effect of alternative disinfection technology such as high hydrostatic pressure on antioxidant activity, total phenolic compounds, vitamin C and color of strawberry have been recorded in many studies (Nunez Mancilla, 2013, Patras et al., 2009).
The lower or higher values of AA of cherry tomatoes compared to other studies, can be attributed to the fact that often it has been oxidized to dehydroascorbic acid before measurement (Szeto et al., 2002). Moreover, some of the differences may be owing to differences in seasonality or the variety tested, or can also be related to geographical factors (Szeto et al., 2002). The initial values of TAC, TPC and AA (5.97 μmol Fe2+/g, 1.19 mg/g and 0.25 mg/g FW respectively) for cherry tomatoes of this study are in accordance with Hanson et al., (2004) where 0.87-1.53 mg GA/g and 0.18-0.34 mg AA/g were observed for TPC and AA respectively.
The UV radiation had an enhancing effect on total phenolic compounds as well as on antioxidant capacity of tomatoes which is in accordance with the study of Bravo et al. (2012) and Alothman et al. (2009). However, other technologies such as gaseous ozone treatments, have been reported to reduce TAC and TPC (Alothman et al., 2009). On the other hand, antioxidants status and phenolic contents were not affected in tomatoes exposed to ozone concentrations up to 1.0 μmol● mol−1 (Karaca and Velioglu, 2014).
A loss of phenolic-related antioxidant power in vegetables is likely to occur with crushing, chopping or pureeing. Interestingly, disruption of the vegetable matrix has been reported to increase the bioavailability of folate and lutein, but not of b-carotene or AA (van het Hof, 1999). However, cherry tomatoes are consumed whole, as a consequence no loss of nutritional substances is observed. The weight loss and the skin wrinkling of UV -treated tomatoes observed in another study, indicated a reduction of the water content in the fruit, which could be attributed to the increase in the respiratory rate of samples due to stress from the UV treatments (Aguiló-Aguayo et al., 2013). Microstructure changes on the tomato surfaces after UV disinfection are responsible for
Page 235 Discussion partial dehydration of cherry tomatoes in the present study (data not shown). The firmness of tomato was not affected by the UV doses during post treatment storage and also there was no consistent change occurred in tomato color during storage due to UV treatments, which is in accordance with Sagong et al., (2011). An increase in surface temperature of RTE foods was observed when UV light at longest treatment times was used. However, the temperature kept low throughout all treatments (<50° C). According to Luksiene et al. (2012), heat generation is observed during light treatments and has been shown to affect the cell walls of light- treated products during storage, but at high fluences (Gómez et al., 2012). However, when the temperature is kept low, no problems are recorded for RTE foods. The AA content was not significantly affected when all disinfection treatments were used. These results are in accordance to Luksiene et al. (2012) who observed that Pulsed Light (PL) did not affect the ascorbic acid content neither in tomatoes nor in other fruit such as strawberries and fresh-cut mangoes.
In general terms, non-thermal disinfection technologies induce the accumulation of phenolic compounds and flavonoids in fruits and vegetables as a defense mechanism against irradiation. However, the increase in TAC and TPC can also be attributed to the phenylalanine ammonialyase activity, which is one of the key enzymes in the synthesis of phenolic compounds in plant tissues (Alothman et al., 2009). It has been found an increase in the activity of phenylalanine ammonialyase in peaches and cabbage seeds after UV exposure (Brown et al., 2001, Stevens et al., 1998). Moreover, it has been established that various types of environmental stresses promote ethylene production of fruit. Ethylene production increased in tomato leaves and peaches after irradiation with UV-B and UV, respectively (Gonzalez-Aguilar et al., 2004). Ethylene is a well known phytohormone which mediates biotic and abiotic stresses (Kohli et al., 2013), which is important in the strawberry defence system (Derksen et al., 2013). Increase in TPC of treated RTE fruits and vegetables can also be attributed to depolymerization and dissolution of cell wall polysaccharides, which facilitated higher extractability (Bhat et al., 2007). Increased TAC and TPC of US-treated RTE produces can also be attributed to better extraction. Finally, Vitamin C content decreased with the increase in treatment time of non-thermal treatments. Vitamin C is a heat-sensitive bioactive compound, which in the presence of oxygen gets degraded by oxidative processes, which are stimulated in the presence of light, oxygen, and enzymes like ascorbate oxidase and peroxidase (Davey et al., 2000). While all treatments in the present study were carried out directly in the presence of air, oxidation of vitamin C might have occurred
Page 236 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki contributing significantly to the observed slight reduction. Also, during extended exposure to UV, and US mild heat might also have been generated, which has led to decreased vitamin C contents. González-Aguilar et al., (2007) also reported the same negative effect of UV irradiation on vitamin C content in mango “Tommy Atkins” fruits.
The correlation between frap and folin methods has been studied by many researchers (Fu et al., 2011). Pantelidis et al. (2007) found a negative correlation between total antioxidants and ascorbic acid in blackberries and raspberries. The present results exhibited a negative correlation (r=-0.946, p>0.05) between TPC and AA in lettuce as well as a negative correlation between TAC and TPC (r=-0.946, p>0.05), when Pearson correlation analysis was done. However, a positive correlation was found in strawberries fruits between TAC and TPC (r=0.853, p>0.05), and a negative between TAC and AA (r=-0.853, p>0.05) whereas no correlation was found in the same parameters for cherry tomatoes (r=0.355, p>0.05). However between TPC and AA in cherry tomatoes a positive correlation was recorded (r=0.836, p>0.05). According to a study by Kalt et al., (1999), no direct correlation between AA content and TAC can be established examining the antioxidant properties of small fruits. However, a strong correlation (r =0.930–0.960, p<0.05) between total phenolics and antioxidant activity has been also reported in stone fruits (Gil et al., 2002).
Tiveron et al. (2012) also recorded that phenolic compounds present in many vegetables have no direct relationship with the antioxidant activity. From the above finding, it can be concluded that different phenolic compounds and some non–phenolic compounds present different antioxidant activity. As a consequence, it is not always guaranteed a high antioxidant capacity, when certain phenolic compounds exist in RTE foods. Moreover, differences in final results of TAC and TPC, can be attributed to differences among methodologies. Furthermore, the chemical composition of vegetables plays also an important role. For this reason, more than one method is suitable for the analysis of in vitro antioxidant activity of RTE foods (Tiveron et al., 2012).
Results indicate that, in terms of antioxidant capacity, phenolic content and AA, a single serving of some fruits and vegetables is worth several servings of others. Furthermore, results show a rapid loss of antioxidants following fragmentation of some vegetables, and this is prevented by mild acidification. While we do not yet know if increased antioxidant intake is directly beneficial to human health, there is nevertheless a strong
Page 237 Discussion inverse relationship between dietary antioxidants and mortality, as reported by Khaw et al. (2001).
4.4 A user-friendly theoretical mathematical model for the prediction of food safety in a food production chain
In the present study, a computerised food decision support system was described. Soft computing is a set of computing techniques, such as Fuzzy Logic (FL), Artificial Neural Networks (ANNs), and Genetic Algorithms (GAs). These computing techniques, unlike hard computing, which refers to a huge set of conventional techniques such as stochastic and statistical methods, offer somewhat “inexact” solutions of very complex problems through modelling and analysis with a tolerance of imprecision, uncertainty, partial truth, and approximation (Huang et al., 2010).
FCMs were used, which are a combination of methods of fuzzy logic and neural networks. FL is a form of multi-valued logic derived from fuzzy set theory to deal with reasoning that is approximate, rather than precise. In contrast to yes/no or 0/1 binary logic (crisp), FL provides a set of membership values inclusively between 0 and 1 to indicate the degree of truth (fuzzy). ANNs provide a way to emulate biological neurons to solve complex problems in the same manner as the human brain (Huang et al., 2010).
The computation of the weights was undertaken by experts and their cooperation decided the weights. Three experts who assessed and evaluated the relationships between each critical control point by using linguistic variables. Experts participated in the European FP7 project VITAL. During the project (3,5 years period) the experts filled in background information questionnaires from a vertical production enterprise located in Western Peloponnesus, which produces lettuces for the Greek market and also exports to a few EU countries, performed fact finding missions, and participated in monthly sampling campaigns. Thus, the data used to feed the presenting model were “real-world” data.
As mentioned above, each expert estimated each weight Wij between nodes i and j, according to his/her experience. In order to be sure about experts’ reliability, an algorithm was used to calculate both the weights of each interconnection and the credibility of experts. Each expert constructed his/her own weight matrix. Each weight Wij was collected and then they were compared according to the algorithm that
Page 238 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki followed. First of all, if the number of the weights with the same sign is less than pi*N, where N is the number of experts (N=3), then it was not clear if there was positive or negative causality between the nodes and the experts should redefine their weights. Otherwise, the process continues and the proposed weights were used to decide the weight eventually. Every expert who defined a weight that abstains from the average weight that the rest of experts have proposed was penalized, and his reliability was reduced. Moreover, his specific weight was not taken into consideration. The same procedure followed for each one element in the matrix separately. The use of this algorithm gives credence to the method. The experts should be sure for their decisions in order to retain their level of confidence high.
The above variables were converted into numerical values with a defuzzification method. The Center of Area (COA) defuzzification method was one of the most commonly used defuzzification techniques. In this method, the fuzzy logic controller first calculated the area under the scaled membership functions and within the range of the output variable. The fuzzy logic controller then used the following equation to calculate the geometric center of this area.
where S is the support set of the membership function of the output μ(y).
After COA defuzzification method the final weight matrix was ready.
It was considered that k1=k2=1 and λ=1.
In the 1st case, the experts decided as initial values of the inputs the following: C1, C2, C3, C5, C6, C7: very strong, C4, C8, C9: strong
Initial values for the concepts after COA defuzzyfication method were:
A(0)= [1 1 1 0.75 1 1 1 0.75 0.75]
The iterative procedure was terminated when the values of concepts Ci had no difference between the latest two iterations. Considering λ=1 for the unipolar sigmoid function and
Page 239 Discussion after N=9 iteration steps, the system reached an equilibrium point, where the values did not change any more from their previous ones.
The calculated value of the decision concept is C10=0.951, which corresponded to the 95.1% of the output. Consequently the lettuce was safe for consumption.
In the 2nd case, the experts decided as initial values of the inputs the following: C1, C2, C3, C5, C6, C7: strong, C4, C8: medium, C9:weak
The value of the decision concept was C10=0,818 which corresponded to the 81.8% of the output. It needed 10 iteration steps in order to reach to an equilibrium point. The lettuce was also safe in this case.
In the 3rd case, the experts decided as initial values of the inputs the following: C1, C3, C4: medium, C5, C6, C7: strong, C2, C8: weak, C9: zero
The output was C10=0.43, which corresponded to the 43% of the output. This means that lettuce was not appropriate for consumption.
Undoubtedly, the 1st case includes the most promising results. With other words, there is a 95.1% certainty that the lettuce which will be consumed in this case is safe. At the 2nd case, the lettuce is also safe for consumption, but with less confidence. Finally, the results of the 3rd case, indicate that the lettuce is not safe for consumption, thus, it must be withdrown and not enter the market.
Computer-aided engineering (CAE) tools, where physical reality is replaced by its equivalent computer model, and which allows implementation of ‘‘what if’’ scenarios more quickly, can go a long way to increasing the efficiency and competitiveness of food product, process and equipment design. However, CAE tools that are customized to food processing and integrate several disciplines (e.g. engineering, food science, food technology, etc.) need to be appropriately developed. CAE tools can improve safety and quality, reduce costs and decrease ‘‘time to market’’ (Halder et al., 2011).
Perrot et al., used the fuzzy symbolic approach for an application to a support system at a symbolic level to help the operators to evaluate the degree of cheese ripening during manufacturing on the basis of sensory measurements achieved on-line by the operators (Perrot et al., 2004).
Page 240 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki In the present study, nine concepts were selected as the most important ones concerning the lettuce production. Experts can quantify the risks associated with the practices of lettuce production from ‘farm-to-fork’. The present model should be able to accurately describe the process by which contamination occurs and the impact to the endpoint of interest: human health (Verhaelen et al., 2012).
Currently, uncertainty and ignorance about the hygienic effects of the individual operations during production, processing, and handling limit the applicability of a Decision Support System to specify HACCP criteria in a quantitative manner. The usefulness of the DSS is expected to be more significant with continuous improvement from collaboration of experts with different scientific background. Increased efficiency, productivity, and competitiveness are among the benefits of the use of the mathematical model. Moreover, it can offer cost effectiveness and high reliability. This could give to a vegetable business a comparative advantage over other competitors (Groumpos and Stylios, 2000, Nolan, 1997). One of the biggest challenges for DSS is the ex-ante availability of real, relevant and representative data to base the decisions on complex problems that can arise during the food production. Thus DSS can be a valuable and easy tool that could be used on a daily basis not only from the industry itself but also from food authorities that can easily control the quality standards that must be met by foods provided for consumption in order to protect public health and to prevent consumers from fraudulent practises. It is suggested that all companies involved in the lettuce/leafy supply chains consider the recommendations contained within HACCP guidelines to ensure the safe production and handling of lettuce/ leafy greens products from field to fork (FDA, 2006). However, the use of software tools like Food Science Decision Support Systems (DSS) using theories of Fuzzy Cognitive Maps, which have not been yet widely used in Food Science, can be further explored and problems that can arise during the food production chain can be studied in order to indicate the importance of some critical control points during the food production in real time.
Page 241 Discussion 4.5 Assessment of disinfection technologies based on infectivity doses
Fresh vegetables and fruits have come during the past decade to the forefront as important vehicles of foodborne illnesses, accounting for 13% of reported outbreaks between 1990 and 2005 with an identified food source (Tauxe et al., 2010). Salad greens, lettuce, sprouts and melons were the leading vehicles of illness, with norovirus, Salmonella and E. coli O157 being the most frequently identified pathogens. Although increased consumption of fresh produce and better surveillance and detection of foodborne outbreaks are contributing factors to the increased recognition of vegetables and fruits as vehicles of illnesses, the increased occurrence of outbreaks associated with fresh RTE produce is a common problem (Tauxe et al., 2010).
Information campaigns on how to handle and wash vegetables and fruits, are targeted to increase public awareness and food safety knowledge by consumers. The crucial role of “best practices” (whether GAP or GHP) to improve food safety is identified as the most important control measure strategy to prevent contamination (Beuchat and Ryu, 1997, Brackett, 1999, De Roever, 1998). Strategies for intervention to reduce foodborne diseases include surveillance and monitoring, appropriate training for preventative control and the adoption of food safety management systems and risk models. Finally, critical is the role of companies and the disinfection methods that implement after harvest of these produces.
Taking into account the best microbial reductions that occurred with the disinfection technologies used in this study, and infectious doses of the tested microorganisms that have been reported in the literature, conclusions regarding the effectiveness of the aforementioned technologies and their impact on public health are presented.
The infective dose of ETEC for adults has been estimated to be at least 108 cells, but the young, the elderly and the infirm may be susceptible to lower levels. Because of its high infectious dose, analysis for ETEC is usually not performed unless high levels of E. coli have been found in a food (at least 106 EIEC organisms are required to cause illness in healthy adults). The infectious dose for O157:H7 is estimated to be 10 - 100 cells, but no information is available for other EHEC serotypes (FDA, 2011). In this study almost all disinfection technologies were capable of reducing the microbial load in three RTE
Page 242 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki produces with US+NaOCl, UV+NaOCl and US (60-min) being the most effective ones for the three RTE produces.
The infectious dose of S. aureus has been reported to be at least 100,000 organisms in humans (PHAC, 2011). Staphylococcal foodborne intoxication, in which major symptoms are vomiting and diarrhoea, occurs after ingestion of thermostable staphylococcal enterotoxins (SE) produced in food by enterotoxinogenic strains of coagulase-positive staphylococci mainly S. aureus. SE are normally not only slightly, inactivated during food processing, storage, distribution or during the preparation of the food in the kitchen. Therefore, if enterotoxinogenic staphylococci are able to grow in food to high numbers (more than 105 - 106 CFU/g or /mL) before they are killed there is still a risk for intoxication with consumption. In this study, S. aureus was properly reduced to acceptable levels with all disinfection technologies in cherry tomatoes, combined technologies reduced the S. aureus to acceptable levels in strawberries, whereas no disinfection technology assured the elimination of S. aureus to adequate levels in lettuce.
Because of the high fatality rate and the low infective dose of Salmonella Enteritidis and L. monocytogenes, there is a zero tolerance policy of pathogen in ready-to-eat food products. The current international standards for Salmonella are lack of bacteria in 20 or 25 g. Thus detection of the pathogen is sufficient for routine tests by the food industry (positive/negative results), but for research activities it is crucial not only to detect the pathogen, but also to accurately quantify its levels. In large outbreaks infective dose was often low and the ingestion of as little as 10 to 100 cells could result in illness (Blaser and Newman, 1982). For instance, a nationwide outbreak of Salmonellosis in Germany, which led to about 1000 cases, was traced to paprika and paprika-powdered potato chips. The estimated infectious dose of salmonellae can vary, depending on the bacterial strain ingested as well as on the immuno-competence of individuals. Data from outbreaks of foodborne diseases indicate that infections can be caused by ingestion of 10-45 cells (Kisluk et al., 2012, Lehmacher et al., 1995). The infectious dose of Salmonella Enteritidis can be relatively small, 100 to 1,000 organisms and are enough to cause the infection in some people. Food prepared from infected animals, insufficiently cooked and food contaminated prior to consumption are the principal causes of infection (CDC, 2005). S. Enteritidis was reduced effectively in all RTE produces of this study only when US (60-min) was used.
Page 243 Discussion
The last years, several well documented foodborne outbreaks and sporadic cases have been described and L. monocytogenes has been isolated from a wide range of raw and RTE meats, poultry, dairy products, seafoods and fruits and vegetables and from various food processing environments (McLauchlin, 1996). Several more foodborne outbreaks have been reported recently and the population that is most susceptible to listeriosis (the elderly and immunocompromised), is increasing. There is a need for continued vigilance and surveillance. The infectious dose of a foodborne pathogen depends on many variables including the immune status of the host, the virulence and infectivity of the pathogen, the type and amount of contaminated foods consumed, the concentration of the pathogen in the food and the number of repetitive challenges. It was estimated that low L. monocytogenes concentrations (approximately 1 CFU/g) were too frequent to be responsible for listeriosis, whereas, the probability of exposure to a higher dose (> 1.000 CFU) was large enough to account for the observed rate of listeriosis (EU, 1999). Thus, 102-103 was the probable infectious dose correlated with fresh RTE produce. As a consequence, L. innocua which has been selected as a representative bacteria of pathogen L. monocytogenes, can be lowered or eliminated to acceptable levels in lettuce and cherry tomatoes, when US+NaOCl (33-min) is selected as a combined method, whereas US (60-min) is required for strawberry disinfection.
Viruses are often transmitted directly from person to person but epidemiological investigations indicate that viral diseases can be transmitted by foods, particularly those that receive little or no processing, such as shellfish, fresh fruit and vegetables and salad items. The infective doses are not known but available evidence suggests that they are very low. It has been estimated that NLVs have an infective dose of between 10 and 100 virus particles. Outbreaks associated with fresh produce and Hepatitis A virus have been reported from several countries. Soft fruits, salads, strawberries and diced tomatoes have all been implicated. Norwalk-like viruses have been associated with various items of fresh produce including washed salads, frozen raspberries, coleslaw, green salads, potato salad, and fresh cut fruits. Adenoviridae infectious dose is >150 plaque forming units when given intranasally (PHAC, 2001). NaOCL was the only disinfection method that exhibited promising results for reducing HAdV35 prior inoculated to three RTE produces to acceptable levels, thus ensuring public health.
With the fresh produce being increasingly responsible for outbreaks of foodborne illnesses, more effective food safety interventions are needed throughout the production, processing and distribution of fresh vegetables and fruits. Therefore, many companies Page 244 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki must focus on eco-friendly postharvest technologies that can contribute to replacing the use of chemical fumigation techniques for crop quality preservation. Many alternative treatments have been reported: UV treatments (Vicente et al., 2005), pulsed UV light treatments (Xu and Wu, 2014), US treatments (Birmpa et al., 2013, Sagong et al., 2011), ozone (Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Bialka and Demirci, 2007) etc.
The last decade has witnessed several encouraging trends in the thinking about food safety. Firstly, it has become clear that the responsibility for food safety is distributed along the entire food chain of production, and is not only an issue of the final consumer. Secondly, new strategies have been adopted such as HACCP, GHP, GMP, etc. Thirdly, the implementation of new emerging, sustainable and environmentally friendly disinfection technologies are important. There is a growing recognition that the problems identified in the course of outbreak investigations are of concern to the entire food industry and thus need open and public discussion. Increasingly, good epidemiologic data are being used to guide action that protects the public health. Finally, new surveillance tools are increasing the detection of widespread multi-jurisdictional foodborne outbreaks that may be more common than previously recognized (Tauxe, 2002).
Page 245 Conclusions and Future Recommendations Chapter 5. CONCLUSIONS AND FUTURE RECOMMENDATIONS
5.1 Conclusions
The results of the present study showed that non-thermal as well as conventional and combined disinfection treatments were effective in reducing microbial populations in both liquid suspensions and food matrices.
Among light non-thermal disinfection treatments, HILP treatments were more effective for the inactivation of both E. coli and L. innocua. Furthermore, this technology resulted in more rapid and extensive inactivation than either continuous UVC and NUV-vis light treatments. These observations associated with HILP may be attributable to the comparatively higher penetration and emission power compared to continuous UV and NUV-vis. Moreover it has a high peak power produced by the multiplication of the flash power manifold, producing a light intensity at least 100 times greater than that of other two light technologies during the same operating time. However, research must be performed in real food matrixes, as it is known that HILP light generates off flavors. It can be concluded that short treatment times for decontamination efficiency would be an important factor related to productivity in food industry.
When UV and US treatments were used for food disinfection, both were good alternatives to other preservative techniques that are currently being used by the produce industry, due to their low cost, lack of extensive equipment and low energy consumption. The effectiveness of these disinfection methods, were shown to be influenced by the dose, the exposure time and the surface of the food product. US reduced more effectively bacteria, whereas UV was more efficient in reducing HAdV. Some changes in the color of produce can be controlled if the exposure time is kept as low as possible, so as to inactivate effectively the microorganisms, but to still preserve the quality of the product. Therefore UV and US may be of benefit to those with little capital to invest as a means of ensuring product safety and quality. Combined treatments are quite promising, if off-flavors as well as forming carcinogenic compounds from NaOCl can be eliminated or kept at low levels.
Page 246 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki It should be noted that a comparison between these disinfection technologies is not a straightforward procedure, as a successful comparison should be made in terms of quality and nutritional aspects of the produces that are treated. Thus, non-thermal technologies are sustainable and promising treatments, as they have been found to have a positive effect on product quality.
Microbiological and molecular analyses can support quality management of food product chains. However, the results of these analyses are time-consuming (microbiological analyses) and cost-consuming (molecular assays). Moreover, the results depend on the accuracy and calibration of the equipment. The proposed model based on FCM’s provides to quality and product managers of a vegetable company, a total new approach which is clear, simple, user friendly, real-time, easily accessible, fast, reliable, and of low-cost. In addition, the aforementioned software tool can support the Food Authorities to have on their desk a first evaluation of the products that they are going to inspect.
Foodborne diseases are increasing recently, as the production of Fresh RTE food is also increasing. The public perceives food safety as absolute, and the food industry has to deal also with the ‘‘quality” of the produced foodstuffs. Everybody demand a zero-risk food supply and claim that cost should not be a consideration, in order to ensure public health. Thus, disinfection remains one of the most important aspects. Non-thermal disinfection technologies are promising and sustainable as they can offer safe food to the consumers, ensuring at the same time public health.
Page 247 Conclusions and Future Recommendations 5.2 Future Recommendations
The present study investigated E. coli, S. aureus, S. Enteritidis, L. innocua and HAdV survival during different time intervals of various disinfection treatments.
The disinfection methods that were used in this study can reduce the microbial populations on the surface of the produce by two to five log units. Higher reductions are not achieved in practice due to the ability of microorganisms to attach strongly on the surface of the produce, the presence of biofilms and due to embedding of the cells into inaccessible nooks and crannies or areas such as stomata. Bacterial cells embedded within a biofilm can withstand nutrient deprivation and pH changes, and are more resistant to detachment and disinfectants than the individual cells. This in turn limits the efficacy of disinfection treatments and introduces another challenge in assuring the safety of the fresh produce. Therefore, treatments that are able to eliminate or decompose biofilms should be further investigated. In green leaves such as in lettuce, for instance, the leaf surface is covered by a waxy cuticle layer and thus the hydrophobic interactions should be the main forces affecting the bacterial attachment. The use of surfactants could help in the disruption of such interactions. It is clear that a better understanding of the mechanisms involved in bacterial attachment and the biofilm formation on the surface of fresh produce is necessary for improving the technology and developing new intervention strategies. This can be achieved by exploring the physiology and the morphology of bacteria, investigating the cell–cell and the cell– surface interactions, the adhesion kinetics and biofilm formation, and the sensitivity of bacteria to antimicrobial agents. Moreover, the host–pathogen interactions should be studied on a per product basis, since the nature of the interactions is dependent on the characteristic surface properties of a specific food product. Thus, SEM analysis of untreated bacteria and viruses inoculated on produce surface is of importance, in order to explore if a strong bacterial attachment in the form of clusters exists.
The research presented in this thesis provides comprehensive investigation into certain disinfection technologies on certain strains of microorganisms. As a result it may be of interest to consider the inclusion of multiple strains of the same organisms, as well as to expand the studies in testing other important food related microorganisms.
The new technologies proposed must be better and cheaper than the existing ones to find a place in the fresh RTE produce market. Moreover, since the disinfection efficacy is
Page 248 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki greatly affected by the quality parameters of water itself, it is also important to understand the relation between the pH, temperature, and organic matter contents of fresh produce and the efficacy of disinfection. The chemical cleanliness is also essential for the food matrices, extremely in the case of conventional technologies and combined treatments including NaOCl. Therefore, it is also necessary to test the chemical residues left on the produce surfaces after disinfection operations.
The possibility that interaction of US and UV with different types of organic material present in the food product could result in various radical production profiles should be studied as this could also enhance understanding of how the process can be further optimized. Thus, further research is required to ascertain the interaction of food constituents with US and UV and the role of resulting compounds in the inactivation process.
To reach a point where a holistic understanding of virus inactivation is in hand, a number of matters will first need to be addressed. For example, the specific chemical modifications that take place in the genome and capsid during disinfection and the effects of these modifications on virus structure and function should be further examined. Moreover, other virus strains should be used in order to investigate the differences of disinfectant-induced modifications.
Applied US and UV treatments also did not change the quality (color) and the physicochemical properties of RTE produces. Application of US and UV at higher doses that effectively inactivates bacteria in shorter treatment times may change sensory qualities of food. Hence, the possible impact of US and UV treatments on the sensory quality of US- and UV- treated food matrices as well as upon the organoleptic and structural properties of foods warrants further study.
Although, the efficiency obtained in this study is indicative, the efficiency of these treatments should be further evaluated in industrial processes. Therefore, a produce/treatment ratio equivalent to the fresh produce industry must be addressed using individual pieces of RTE produce. Much research is needed to develop environmentally friendly alternative processing and preservation methods for assuring the quality and safety of fresh RTE products without contradicting with environmental protection, consumer acceptability, sustainable use of resources, cost factors for the Produce Company, and food regulatory provisions.
Page 249 Conclusions and Future Recommendations
Finally, the results obtained from the theoretical mathematical model in the present study must be further correlated and validated with risk assessment models in order to meet the gold standard criteria for quality risk assessment.
Page 250 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki
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BOOKS
Barth, Margaret, Hankinson Thomas, R., Zhuang, Hong, and Breidt, Frederick. (2009). Microbiological Spoilage of Fruits and Vegetables. W.H. Sperber, M.P. Doyle (eds.), Compendium of the Microbiological Spoilage of Foods and Beverages, Food Microbiology and Food Safety, DOI 10.1007/978-1-4419-0826-1_6, Springer Science+Business Media.
Cullen, P.J, Tiwari, B, and Valdramidis, V. (2012). Novel Thermal and Non-Thermal technologies for fluid foods. Elsevier Inc.
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Page 301 Appendix
APPENDIX
Page 302 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki PUBLICATIONS
1. Birmpa A., Sfika V. and Vantarakis A. (2013).Ultraviolet light and Ultrasound as non-thermal treatments for the inactivation of microorganisms in fresh ready-to-eat foods. International Journal of Food Microbiology 167: 96-102.
2. Birmpa Angeliki, Vantarakis Apostolos, Paparrodopoulos Spyros, Whyte Paul and Lyng James. (2014). Efficacy of three light technologies for reducing microbial populations in liquids”. BioMed Research International, Volume 2014, Article ID 673939, 9 pages.
Submitted for Publication
1. Angeliki Birmpa, Spyros Paparrodopoulos, Vasiliki Sfika, Apostolos Vantarakis. Efficacy of non thermal technologies in the microbial inactivation of fresh ready-to-eat cherry tomatoes (Submitted).
2. Angeliki Birmpa, Maria Bellou and Apostolos Vantarakis. Effect of non-thermal, conventional and combined disinfection technologies on the stability of human Adenoviruses as fecal contaminants on fresh ready to eat produce surfaces (Submitted to Journal of Food Protection).
3. Angeliki Birmpa, Apostolos Vantarakis, Antigoni Anninou, Maria Bellou, Petros Kokkinos, Peter P. Groumpos. A user-friendly mathematical model for the prediction of food safety (Submitted to Journal of Food Processing and Technology).
4. A.Birmpa, J.Lyng, P.Whyte, S.Paparrodopoulos, E.Sazakli, M.Leotsinidis and A.Vantarakis. The promising use of non-thermal green technologies and their effect on the quality of foods (Submitted).
Page 303 Appendix
CONFERENCE ORAL PRESENTATIONS
International
1. A. Birmpa, P. Kokkinos, V. Sfika, A. Vantarakis: “Ultraviolet radiation and ultrasonication as non-thermal treatments for the inactivation of microorganisms in fresh ready-to-eat foods."23rd International ICFMH Symposium, FoodMicro2012, 3- 7/9/2012. Global Issues in Food Microbiology, Istanbul, Turkey.
2. M. Bellou, A. Birmpa, P. Kokkinos, A. Vantarakis, “Viral Outbreaks Linked To Fresh Produce Consumption the Last Two Decades: A Systematic Review” 24 th ECCMID European Society of Clinical Microbiology and Infectious Diseases, 10-13 May 2014, Barcelona, Spain.
3. Angeliki Birmpa, Michalis Leotsinidis, Eleni Sazakli, Tzina Tsichlia and Apostolos Vantarakis. Effect of disinfection technologies on quality and nutritional properties of lettuce, strawberries and cherry tomatoes. European Symposium on Food Safety, 7-9 May 2014, Budapest, Hungary.
4. Angeliki Birmpa, Michalis Leotsinidis, Eleni Sazakli, Spyros Paparrodopoulos, Paul Whyte, James Lyng, Apostolos Vantarakis. The promising use of non thermal green technologies and their effect on the quality of foods. 3rd International Iseki Food Conference, 21-23 May 2014, Athens, Greece.
5. Angeliki Birmpa, Spyros Paparrodopoulos, Paul Whyte , James Lyng and Apostolos Vantarakis. Efficacy of three light technologies for reducing microbial populations. IAFP 2014, 3-6 August 2014, Indianapolis, Indiana, USA.
6. Angeliki Birmpa, Panagiotis Pitsos, Spyros Paparrodopoulos, Vasiliki Sfika, Apostolos Vantarakis. Efficacy of non thermal technologies combined with chlorine for reducing microbial populations in ready to eat products. IAFP 2014, 3-6 August 2014, Indianapolis, Indiana, USA.
Greek
1. Πίτσος Παναγιώτης, Μαυρίδου Αθηνά, Μπίρμπα Αγγελική, Βανταράκης Απόστολος. Επίδραση της χρήσης χλωρίου στην απολύμανση έτοιμων προς κατανάλωση τροφίμων. ΠΕΤΙΕ, 5-7 Δεκεμβρίου 2013. Αθήνα.
Page 304 Non-thermal technologies for the disinfection of food and risk assessment for Public Health Birmpa Angeliki CONFERENCE POSTERS
International
1. A. Birmpa, D. Papadopoulou, A. Kokkinos, A. Vantarakis. “Evaluation of disinfectant efficacy by ultraviolet light in a laboratory swimming pool model”. 5th International Conference Swimming pool and Spa, 9- 12/4/2013 Rome, Italy.
2. P. Ziros, P. Kokkinos, A. Birmpa, N. Karagiannis, A. Vantarakis. “Real Time PCR Molecular Monitoring of Zygosaccharomyces bailii During Soft Drinks Production to Determine Contamination Sources”. 23rd International ICFMH Symposium, FoodMicro2012, Global Issues in Food Microbiology, 3- 7/9/2012. Istanbul, Turkey.
3. Birmpa Angeliki, Anninou Antigoni, Nikolaou Alexandra, Kokkinos Petros, Groumpos Peter and Vantarakis Apostolos. “A Theoritical Mathematical Model for the analysis of lettuce quality in a Food Production Chain using Fuzzy Cognitive Maps”. International Conference on Predictive Modelling in Food (ICPMF8), 16-20 September 2013 Paris, France.
4. Angeliki Birmpa, Maria Tselepi and Apostolos Vantarakis. Effect of disinfection technologies on Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and Listeria innocua inoculated on lettuce, strawberries and cherry tomatoes during a refrigerated storage period. European Symposium on Food Safety, 7-9 May 2014, Budapest, Hungary.
5. Angeliki Birmpa, Maria Bellou and Apostolos Vantarakis. Effect of non-thermal, conventional and combined disinfection technologies on the stability of human Adenoviruses as fecal contaminants on fresh ready to eat produce surfaces. ISFEV, 2-5 September 2014, Corfu, Greece.
Greek
1. Μπίρμπα Αγγελική, Κόκκινος Πέτρος, Σφήκα Βασιλική, Βανταράκης Απόστολος. «Χρήση εναλλακτικών μη θερμικών τεχνολογιών για την απολύμανση μικροοργανισμών σε έτοιμα προς κατανάλωση τρόφιμα». 8ο Πανελλήνιο Συνέδριο Βιοεπιστημόνων, Βιοεπιστήμες, Μοχλός ανάπτυξης της κοινωνίας, 18-20 Οκτωβρίου 2012, Συνεδριακό και Πολιτιστικό Κέντρο Πανεπιστημίου Πατρών.
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