High Oxygen as an additional factor in Food Preservation Promotor: Prof. Dr. ir. F.M. Rombouts Hoogleraar in de Levensmiddelenhygiëne en microbiologie, Wageningen Universiteit Copromotors: Dr. L.G.M. Gorris SEAC, Unilever, Colworth House, Verenigd Koninkrijk Dr. E.J. Smid Groupleader Natural Ingredients, NIZO Food Research, Ede Samenstelling promotiecommissie: Prof. Dr. ir. J. Debevere (Universiteit Gent, België) Prof. Dr. G.J.E. Nychas (Agricultural University of Athens, Griekenland) Prof. Dr. J.T.M. Wouters (Wageningen Universiteit) Dr. J. Hugenholtz (NIZO Food Research, Ede) Athina Amanatidou High Oxygen as an additional factor in Food Preservation Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus, van Wageningen Universiteit, Prof. dr. ir. L. Speelman, in het openbaar te verdedigen op dinsdag 23 oktober des namiddags te half twee in de Aula Amanatidou A.-High Oxygen as an additional factor in Food Preservation-2001 Thesis Wageningen University-With summary in Dutch- pp. 114 ISBN: 90-5808-474-4 To my parents, my brother and to Erik Abstract In this thesis, the efficacy of high oxygen as an additional hurdle for food preservation is studied. At high oxygen conditions and at low temperature, significant impairment of growth and viability of bacterial cells is found to occur as the result of free radical attack. The imposed oxidative stress leads - to an increase of intracellularly generated reactive oxygen species (mainly O2 , H2O2 and HO·), which disturbs the cellular homeostasis due to catabolic imbalance and results in growth inhibition. The so- called “free radical burst” probably is responsible for the induction of a host defence mechanism against the destructive impact of high oxygen. Different Lactobacillus sake strains possess endogenous levels of antioxidative properties (metal chelating capacity, hydroxyl radical scavenging and reducing properties) which play an important role in protection against oxidative stress. Superoxide dismutase is demonstrated to be, among others, a main antioxidative enzyme in oxygen insensitive strains and mutants of Lactobacillus sake. Although most aerobic organisms possess biochemical mechanisms that contribute to their resistance to oxygen stress conditions, high oxygen is an efficient preservation strategy if used in combination with other antimicrobial factors (hurdles). The impact of the combined application of high oxygen with refrigeration, carbon dioxide and/or high pressure on microbial stability and physicochemical properties of minimally processed fresh produce (carrots and salmon) is investigated. Contents Outline of this thesis ................................................................................................v Chapter 1 Introduction...............................................................................................................1 1.1 Background ...............................................................................................................................1 1.2 Hurdle technology for preservation of minimally processed foods......................................1 Definition and history................................................................................................................................1 Basic aspects of hurdle technology............................................................................................................2 Risks related to hurdle technology and minimal processing .....................................................................3 1.3 Oxidative stress as an additional hurdle.................................................................................4 Oxidative stress and food preservation .....................................................................................................4 Oxidative stress and food quality ..............................................................................................................5 1.4 High oxygen modified atmospheres and food preservation..................................................6 Modified atmosphere packaging ...............................................................................................................6 Applications of high O2 MA for food preservation ....................................................................................6 1.5 Antimicrobial activity of high oxygen.....................................................................................7 O2 chemistry - Free radical generation and O2 toxicity ............................................................................7 Biochemical effects of high O2 on micro-organisms..................................................................................8 Cellular defences against damage in bacteria ........................................................................................11 1.6 Combination of high oxygen MA with other hurdles..........................................................13 Combined applications of O2 MA with CO2 ............................................................................................14 Combined applications of O2 MA with high pressure..............................................................................14 Chapter 2 Effect of elevated oxygen and carbon dioxide on the surface growth of vegetable-associated micro-organisms................................................................17 2.1 Introduction ............................................................................................................................17 2.2 Material and methods ............................................................................................................18 Micro-organisms .....................................................................................................................................18 Growth conditions ...................................................................................................................................18 Storage and preparation..........................................................................................................................18 Experimental set-up.................................................................................................................................19 Enumeration of viable counts..................................................................................................................19 Quantification of microbial growth parameters......................................................................................19 2.3 Results......................................................................................................................................19 Effect of high O2 and CO2 on the growth characteristics of selected micro-organisms ..........................19 Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium and Escherichia coli ...........19 Lactobacillus plantarum, Leuconostoc mesenteroides and Lactococcus lactis.......................................20 Pseudomonas fluorescens, Enterobacter agglomerans and Aureobacterium strain 27 ..........................23 Candida sake and Candida guilliermondii..............................................................................................23 2.4 Discussion ................................................................................................................................24 Chapter 3 Antioxidative properties of Lactobacillus sake upon exposure to elevated levels of oxygen.................................................................................................................27 3.1 Introduction ............................................................................................................................27 3.2 Material and methods ............................................................................................................28 i High Oxygen as an additional factor in Food Preservation Strains and growth conditions.................................................................................................................28 Preparation of cell-free extracts..............................................................................................................28 Determination of anti-oxidative properties of cells and cell-free extracts ..............................................29 Extracellular H2O2 toxicity......................................................................................................................29 Extracellular generation of H2O2 ............................................................................................................29 Intracellular generation of H2O2 .............................................................................................................30 Effect of extracellular H2O2 on L. sake NCFB 2813 and L. sake DSM 6333...........................................30 Other methods .........................................................................................................................................30 3.3 Results......................................................................................................................................30 Growth of L. sake strains under different gas conditions........................................................................30 Anti-oxidative properties of cells and cell-free extracts ..........................................................................31 Toxicity