Downloaded from orbit.dtu.dk on: Oct 06, 2021 Microbial changes and safety of lightly preserved seafood Mejlholm, Ole Publication date: 2007 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Mejlholm, O. (2007). Microbial changes and safety of lightly preserved seafood. DIFRES, DTU. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Microbial Changes and Safety of Lightly Preserved Seafood By Ole Mejlholm Danish Institute for Fisheries Research, Department of Seafood Research, Technical University of Denmark Preface The present Ph.D.-project has been carried out at the Danish Institute for Fisheries Research (DIFRES) in Kgs. Lyngby, Denmark under the supervision of senior scientist Paw Dalgaard. The presented work was financed by the Danish Directorate for Food, Fisheries and Agri Business. I wish to thank Paw Dalgaard for his support and many valuable discussions, and for introducing me to predictive microbiology. I would like to thank our industrial partner Royal Greenland Seafood for being supportive throughout the project. Special thanks should go to Niels Bøknæs for his great enthusiasm and for many interesting discussions on “seafood” as well as other essential things in life. Thanks to Prof. Johanna Björkroth and her group for making my stay at the University of Helsinki, Finland both educational and enjoyable. I wish to thank Nadereh Samieian and Tina Dahl Devitt for their skilful and valuable technical assistance, and our librarian Søren Tørper Christensen for finding literature. Last but not least, I would like to thank my beloved wife Lise for her patience and endless support, and Nikolai, Valdemar and Augusta – you are truly precious and unpredictable. Ole Mejlholm Kgs. Lyngby, July 2007 II Microbial changes and safety of lightly preserved seafood Summary This thesis deals with microbial changes and safety aspects of lightly preserved seafood including cold-smoked and gravad fish, and shrimp products. Lightly preserved seafood is of significant economical importance to the Danish seafood industry. To maintain this position management of spoilage microorganisms and Listeria monocytogenes is important as they affect the shelf life and the safety of these products. The objective of the present Ph.D.-project was to identify means to reduce and prevent growth of spoilage microorganisms and L. monocytogenes, respectively, in lightly preserved seafood. Furthermore, it was the aim to develop mathematical models to predict the effect of product characteristics and storage conditions on growth of spoilage microorganisms and L. monocytogenes in lightly preserved seafood. The antimicrobial effect of modified atmosphere packaging (MAP) and organic acids against L. monocytogenes was examined in challenge tests with cold-smoked and gravad salmon and Greenland halibut. Growth of L. monocytogenes was prevented in MAP cold-smoked fish by using sensorially acceptable concentrations of diacetate and lactate. Thus, it is concluded that diacetate and lactate can be used as additional growth hurdles in lightly preserved seafood in order to prevent growth of L. monocytogenes. A mathematical model was developed to predict the effect of diacetate, lactate, CO2, smoke components (phenol), nitrite, pH, NaCl, temperature, and interactions between all these parameters on growth and the growth boundary of L. monocytogenes in lightly preserved seafood. Validation studies revealed that both growth and the growth boundary of L. monocytogenes in lightly preserved seafood were accurately predicted by the developed model. The growth and growth boundary model correctly predicted 73 of 76 growth and no growth responses of L. monocytogenes in lightly preserved seafood. The developed model can be used by the seafood industry in order to identify combinations of product characteristics and storage conditions that prevent growth of L. monocytogenes, and thereby facilitate production of lightly preserved seafood in compliance with the EU regulation on ready-to-eat foods (EC 2073/2005). A review of the literature showed that lactic acid bacteria (LAB) are most frequently found as the dominating spoilage microflora of lightly preserved seafood and that they are able to cause spoilage of e.g. cold-smoked salmon. Thus, a predictive model III Microbial changes and safety of lightly preserved seafood for growth of LAB in lightly preserved seafood was developed including the effect of diacetate, lactate, CO2, smoke components (phenol), pH, NaCl, temperature, and interactions between all parameters. Growth of LAB in lightly preserved seafood was correctly predicted by the developed model. The LAB-model can be used by the seafood industry to predict the time for LAB to reach a specified concentration. The availability of validated models for growth of L. monocytogenes and LAB made it possible to predict the effect of microbial interaction on growth (i.e. maximum population density, MPD) of the pathogen in lightly preserved seafood. The performance of the previously developed model for L. monocytogenes was improved when the effect of microbial interaction was taken into account. The observed and predicted MPD of L. monocytogenes in naturally contaminated vacuum-packed cold-smoked salmon were 0.7 and 0.6 log (CFU/g) when a relative lag time of 4.5 was used. The combined LAB-Lm model can be used to assure and document that the critical limit of 102 CFU/g of L. monocytogenes (EC 2073/2005) is not exceeded within the declared shelf life of e.g. cold- smoked salmon. Furthermore, it can be used to predict the concentration of L. monocytogenes in lightly preserved seafood e.g. at the time of consumption, and thereby be useful for exposure assessment studies. The shelf life of cooked and peeled shrimp in MAP at chill temperatures is limited by the potential growth of L. monocytogenes as established in challenge tests. This pathogen reached critical concentrations prior to sensory spoilage and predicted time to toxin production by Clostridium botulinum. Carnobacterium maltaromaticum and Brochothrix thermosphacta were identified as the dominating spoilage microflora of cooked and peeled shrimp in MAP. Inoculation experiments with cooked and peeled MAP shrimp showed that a mixture Cb. maltaromaticum and B. thermosphacta produced a distinct off- flavour resembling the spoilage of naturally contaminated products. The importance of brine composition on growth of L. monocytogenes in chilled brined shrimp was studied in challenge tests. Growth of L. monocytogenes was prevented in brined shrimp when benzoic, citric and sorbic acids were used as preservatives in concentrations resembling those of commercial products. However, it was shown that brined shrimp are sensitive to even small changes in the preserving profile, especially the concentration of benzoic acid. Thus, it would be relevant to expand the existing model for L. monocytogenes with the effect of benzoic, citric and sorbic acids, to be able to predict IV Microbial changes and safety of lightly preserved seafood how growth of the pathogen is affected by changes in brine composition. Storage trials were carried out to examine the shelf life and to identify the spoilage microflora of brined shrimp. The composition of the spoilage microflora of brined shrimp was affected by process hygiene, preserving parameters and storage conditions. Eighty-two isolates from the spoilage microflora of brined shrimp was identified and they included 53 LAB, 6 coagulase negative Staphylococcus spp., 18 Pseudomonas fluorescens and 5 yeast isolates. Lactobacillus sakei dominated the spoilage microflora of brined and drained shrimp in MAP whereas a more diverse microflora was found on shrimp in brine. V Microbial changes and safety of lightly preserved seafood Sammendrag (summary in Danish) Denne afhandling omhandler mikrobielle ændringer og sikkerheds aspekter for letkonserverede fiskeprodukter herunder koldrøget og gravad fisk samt rejeprodukter. Letkonserverede fiskeprodukter er af stor økonomisk betydning for fiskeindustrien. For at opretholde denne position er det vigtigt at kunne styre vækst af fordærvelsesbakterier og Listeria monocytogenes da disse påvirker holdbarheden og sikkerheden af produkterne. Formålet med det foreliggende Ph.D.-projekt var, at identificere metoder til at reducere og forhindre vækst af henholdsvis fordærvelsesbakterier og L. monocytogenes i letkonserverede fiskeprodukter. Derudover var det formålet at udvikle matematiske modeller til at forudsige effekten af produktkarakteristika og lagringsbetingelser på vækst af fordærvelsesbakterier og L. monocytogenes i letkonserverede fiskeprodukter. Den antimikrobielle effekt af modificeret atmosfære pakning (MAP) og organiske syrer overfor L. monocytogenes blev undersøgt i podningsforsøg med koldrøget