The Pennsylvania State University The Graduate School College of Agricultural Sciences SURVIVAL OF SPOILAGE AND PATHOGENIC MICROORGANISMS ASSOCIATED WITH THE PRODUCTION OF PICKLED SAUSAGE USING A COLD FILL PROCESS A Thesis in Animal Science by Nelson J. Gaydos © 2015 Nelson J. Gaydos Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2015 i The thesis of Nelson J. Gaydos was reviewed and approved* by the following: Jonathan A. Campbell Assistant Professor of Animal Science Extension Meat Specialist Thesis Advisor Catherine N. Cutter Professor of Food Science Food Safety Extension Specialist – Muscle Foods Hassan Gourama Associate Professor of Food Science Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Animal Science *Signatures are on file in the Graduate School ii ABSTRACT Pickling has been used for centuries to preserve and extend the shelf life of different foods, ranging from fruits and vegetables, to eggs and meat. The pickling process inhibits microorganism growth by reducing product pH with organic acids, as well as the addition of salt to the product being pickled. Food items are suspended in a brine solution containing an organic acid, such as citric, lactic or acetic (vinegar) acid and combined with salt, spices and other preservatives, such as potassium sorbate. The majority of pickled foods undergo what is known as a hot fill process, where the brine solution is heated before being introduced to the food item. When the hot brine is applied to pre-cooked, ready-to-eat (RTE) meat sausages, the brine solution can change from clear to cloudy, becoming turbid and unappealing to consumers. To avoid these quality defects, pickled sausage manufacturers may use a cold fill pickling process. Cold fill pickling utilizes a room-temperature brine instead of a heated pickling solution and does not cause the aforementioned quality defects. Since very little is known about the safety of the cold fill pickling processes, a series of separate experiments was performed using a brine solution (5% acetic acid and 5% salt at ~22- 23°C) to pickle pre-cooked, RTE beef and pork smoked sausages inoculated with various spoilage and pathogenic microorganisms. In two separate experiments, spoilage microorganisms consisting of lactic acid bacteria (LAB) (Lactobacillus curvatus and L. sakei); yeasts (Debaryomyces hansenii and Candida zeylanoides); molds (Penicillium nalgiovense and Cladosporium cladosporioides) and pathogenic bacteria (Salmonella Typhimurium, S. Senftenberg, S. Montevideo, Listeria monocytogenes, and Staphylococcus aureus) were inoculated onto RTE sausages prior to pickling. Microbial populations were evaluated initially (day 0) and up to 28 days after pickling and storage at room temperature. Other parameters, such as pH of the sausage, pH of the brine, water activity of the sausages, and percent salt of both the sausage and the brine, were measured over time. Results demonstrated that all microorganisms experienced a significant (p≤0.05) reduction within the first 24 hours of pickling. LAB populations were reduced ca. 6.58 log10 CFU/g in 7 days, while yeasts were reduced ca. 3.89 log10 CFU/g in 48 hours and molds were reduced ca. 4.09 log10 CFU/g in 24 hours. Pathogenic bacteria were reduced ca. 6.80 log10 CFU/g in 72 hours when plated on non-selective media and >6.33 log10 CFU/g when plated on selective media. Likewise, pH of sausages decreased significantly (p≤0.05) throughout the length of the iii study. No significant differences were observed for water activity (p=0.2062; p=0.1291), or % salt of the sausages (p=0.0640; p=0.1445) or brine (p=0.1046; p=0.3180) for all experiments. It was concluded from the observed data that the cold fill pickling process is capable of inhibiting and destroying contamination from both spoilage and pathogenic microorganisms within 24 hours after initial pickling. Parameters, such as the diameter of the sausages, casing type, and brine concentration were chosen in order to implement a worst case scenario. These results suggest that processors may experience an even more effective inhibition of spoilage and pathogenic microorganisms by using a higher concentrations of salt or acetic acid, pickle smaller diameter sausages or use natural or skinless casings, which will result in a faster penetration of the brine into the sausages and decrease in product pH. Results of these studies may provide scientific validation to manufacturers that cold fill pickled sausages in order to meet requirements for their HACCP systems and for regulatory compliance with the USDA-FSIS. iv TABLE OF CONTENTS List of Tables .....................................................................................................................................viii List of Figures ....................................................................................................................................x Chapter 1: Literature Review ............................................................................................................1 Introduction ............................................................................................................................2 Hurdle Technology ................................................................................................................2 Introduction ................................................................................................................2 Microbial Inhibition ...................................................................................................2 Effect of Temperature on Preservatives .....................................................................4 Pickled Foods .............................................................................................................5 Acetic Acid ................................................................................................................5 Introduction ....................................................................................................5 Action of Acetic Acid ....................................................................................6 Microbial Defenses Against Acid ..................................................................7 Anion Accumulation ......................................................................................8 Salt and Osmotic Stress .............................................................................................9 Introduction ....................................................................................................9 Function in Meat and in Combination with Acid ..........................................9 Growth Stimulation ........................................................................................10 Developed Resistance to Preservatives ..................................................................................10 Introduction ................................................................................................................10 Reaction to Stresses ...................................................................................................11 Government Agencies and Food Safety ................................................................................13 Background Information ............................................................................................13 Regulation of Meat and Acidified Foods ...................................................................14 Studies of Pickled Foods ........................................................................................................15 Spoilage Microorganisms ......................................................................................................17 Lactic Acid Bacteria ..............................................................................................................18 Background Information ............................................................................................18 Effects of Atmosphere, Acid and Salt........................................................................18 Lactobacillus curvatus and Lactobacillus sakei ........................................................19 Inhibition of Other Microorganisms ..........................................................................20 Yeast ......................................................................................................................................21 Background Information ............................................................................................21 Effects of Acid and Salt .............................................................................................22 Adaptation and Growth ..............................................................................................22 Debaryomyces hansenii and Candida zeylanoides ....................................................23 Studies Involving Meat Spoilage ...............................................................................24 Mold ......................................................................................................................................25 Background Information ............................................................................................25 Penicillium nalgiovense .............................................................................................26 Mycotoxins ....................................................................................................27