Synergistic effect of ozone and microgard 300 for controlling Listeria monocytogenes in ready-to-eat cooked and cured ham. R. JHALA 1, K. Muthukumarappan, J. L. Julson, and R. I. Dave. (1) Agricultural and Biosystems Engineering, AE #2120 South Dakota State University,, Brookings, SD Abstract The effectiveness of ozone and Microgard® (MG300) on the survival of known number of Listeria monocytogenes in ham sample was investigated in a closed system. The survival rate of Listeria monocytogenes was studied as a function of gaseous ozone concentration, Microgard ®, temperature and storage period for a fixed treatment period of 30 min and storage temperature of 4ºC. The results indicate that a synergistic effect of ozone, MG300 and storage period can inactivate up to 99.94% of Listeria monocytogenes on cooked and cured ham. The temperature does not have any significant effect on %kill of Listeria monocytogenes. The combination of ozone and MG300 can be an effective hurdle to control Listeria monocytogenes in cooked and cured ham. Isolation  Listeria monocytogenes culture was obtained from Alfred Chair’s Laboratory (SDSU, Brookings, SD).  The culture was maintained at –80°C in glycerol-nutrient broth. Propagation  The cultures were given three transfers ( hrs) before replications for the purpose of full activation.  Media used for activation was trypticase soy broth with 0.6% yeast extract.  Probable number of Listeria monocytogenes was determined by optical density measurement (at 603 nm) using an UV spectrophotometer Inoculation  About 0.1ml of the active culture of known number of Listeria monocytogenes is spread on cm 2 area of Hyvee brand reduced fat (97% fat free) cooked cured ham slices.  The inoculated samples are stored for 20 minutes under refrigerated condition, to allow the culture to absorb to the surface of the ham. Experimental Design Ozone (ppm) - 0, 0.2, 0.5, 1.0 MG300 (%) - 0, 1.0, 2.0, 3.0 Temperature (°C) - 10, 15, 20 Exposure time - 30 min Storage days - 1, 5, 10 Experimental set-up  The inoculated samples were exposed to gaseous environment of ozone in a closed system housed in an incubation chamber to control the temperature.  The percent solution of MG300 was prepared in pH – 7.0 phosphate buffer.  0.1 ml of MG300 (%) solution was spread after the ozone treatment to study the synergistic effect.  The samples for the storage study were vacuum packed (Food saver) and stored in a refrigerator at 4°C. Microbial Analysis  Enumeration of the surviving Listeria monocytogenes was done according to the method suggested by murano et al, using the pour plate technique.  The efficacy of the above treatment has been reported as percent kill of (%Kill) Listeria monocytogenes. Statistical Analysis  According to the experimental design a total of 432 samples were treated. The whole experiment was replicated thrice (4 ozone level X 4 MG300 level X 3 temperature X 1 exposure time X 3 storage period X 3 replication).  The mean %kill data was analyzed using split- split- split-plot experimental design model with the help of statistical analysis software (SAS Institute, Cary, NC). Introduction In the recent years, Listeria monocytogenes has caused major listeriosis outbreaks in the U.S. associated with ready-to-eat meat products(). Food pathogens like Listeria monocytogenes may survive the conventional food processing methods like pasteurization and cooking. Moreover, there is a demand for safe and judicious usage of sanitizers, bleaching agents, preservatives and chemicals in food processing (1). Thus, the food industry is currently in need of innovative processing technologies in order to meet consumer’s demand for fresher and safe ready-to-eat meat products (2). There are several methods available for inactivation of microorganisms in foods; thermal, high pressure, pulsed electric field, oscillating magnetic field, irradiation and ozonation. In August 2001, Food and Drug Administration (FDA) has approved ozone as a direct food additive for the treatment, storage and processing of food in gaseous and aqueous phases (3). Ozone’s antimicrobial action is through the oxidation of bacterial cell wall components (4). The technologies used singly to ensure that a food is free of pathogens may cause changes in the sensory attributes of the food. Thus, in the food industry, the use of multiple parameter known as “Hurdle Concept” has been suggested (5). Microgard ® 300 is a bacteriocin like, low molecular weight metabolite material of Propionibacterium shermanii in a skim milk base. It has been used as a preservative in cottage cheese and other food products to inhibit psychrotrophic spoilage bacteria, yeast, mold and gram-positive organisms like Listeria monocytogenes (6). In this study we have hypothesized that application of ozone and Microgard ® 300 hurdle concept can better control pathogens like Listeria monocytogenes in cooked and cured ham. Objectives - To evaluate the individual and synergistic effect of ozone and MG300 for controlling Listeria monocytogenes in cooked and cured ham. - To Study the effect of treatment temperature in improving the synergy - To Study the effectiveness of the synergy of ozone and MG300 for controlling Listeria monocytogenes in cooked and cured ham over the storage period. Materials and Methods Ozone Concentrator - GeneratorPlexiglass Sample Treatment Chamber O 3 in O 3 out Ham sample Results References G.Vignolo, S. Fadda, M.N. de Kairuz, A.A.P. de Ruiz Holgado, G. Oliver, Control of Listeria monocytogenes in ground beef by lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705. Intl. J. Food Microbiol. 29: Barnby-smith, FM Bacteriocins:application in food preservation. Trends in Food Sci. Technol. 3: Khadre. M.A, A.E. Yousef, and J.-G. Kim, Microbiological aspects of ozone application in food: A Review.J. Food Sci. 66: Federal Register, Secondary direct food additives permitted in food for human consumption. Federal Register 66(123): Muthukumarappan, K. F. Halaweish, A.S. Naidu, Ozone. In Natural food antimicrobial systems. Eds. A. S. Naidu, pp CRC Press LLC. 6. Daeschel, M.A Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol. 43: Murano, E.A., P.S Murano, R.E. Brennan, K. Shenoy, and R.G. Moreira Application of hydrostatic pressure to eliminate Listeria monocytogenes from fresh pork sausage. J. Food Prot. 62:  As indicated in Table-1, ozone, MG300 & Days have significant effect on mean %kill of Listeria monocytogenes in cured ham. The table also indicates the significant interaction parameters of the experiment.  The group – 1 graphs shows that as ozone concentration increases from 0.0 to 0.5 ppm microbial inactivation significantly increased but above 0.5 ppm the inactivation was not significant.  Increasing the MG300 level up to 2.0% significantly increases the mean %kill of Listeria monocytogenes.  As the storage days increases from 1 to 5 and from 5 to 10 days the mean %kill of Listeria monocytogenes increases exponentially.  Temperature has no significant effect on mean %kill of Listeria monocytogenes.  Group – 2 graphs show the effect of interaction of experimental parameters.  The difference of least square means indicate that ozone & MG300 has a synergistic effect in inactivating Listeria monocytogenes with ozone being a predominant inactivating parameter.  Storage days have a predominant inactivating effect in the interaction of storage days and ozone. While MG300 has a predominant inactivating effect in the interaction of MG300 & storage days. Conclusions DAY 3 DAY 2 DAY 1 OZONE LEVEL – 0 PPM OZONE LEVEL – 0.2 PPM OZONE LEVEL – 0.5 PPM OZONE LEVEL – 1 PPM MG300 0% MG300 1% MG300 2% MG300 3% INTERACTION OF OZONE AND MG300 INTERACTION OF STORAGE DAYS AND OZONE INTERACTION OF STORAGE DAYS AND MG300 GROUP 2 GROUP 1