Clostridium perfringens in Cooling Rate Challenge Studies

Proper temperature control is essential in preventing Clostridium perfringens food poisoning. The U.S. Department of Agriculture Food Safety and Inspection Service cooling guidelines offer two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log CFU/g increase of C. perfringens and no growth of Clostridium botulinum. The latter option requires laboratory challenge studies to validate the efŽ cacy of a given cooling process. Accordingly, the objective of this study was to investigate the role of several methodological variables that might be encountered during typical C. perfringens challenge studies. Variables studied included plastic bag type (Whirlpak or Spiral Biotech), sealing method (Multivac or FoodSaver), initial spore inoculum size (1 to approximately 3 log CFU/g), and growth environment (ground beef or Trypticase–peptone–glucose–yeast extract [TPGY] broth). The major factors that affected growth were sample bag type and growth environment. Samples incubated in Whirlpak bags showed signiŽ cantly less growth than those incubated in Spiral Biotech bags, which was likely due to the former bag’s greater oxygen permeability. C. perfringens spores showed shorter germination, outgrowth, and lag times and C. perfringens cells showed faster growth rates in ground beef compared with TPGY broth. No signiŽ cant difference was observed between two different sealing methods. Initial spore inoculum levels in the range studied had no signiŽ cant effect on Ž nal C. perfringens cell concentration. Clostridium perfringens continues to be a major food safety concern to the food industry. There were an estimated 654 outbreaks involving C. perfringens, resulting in 248,520 cases between 1983 and 1992 (13). This organism has been implicated as the cause of foodborne illness in roast beef, turkey, meat-containingMexican foods, and other meat dishes (4). C. perfringens spores may survive cooking and receive sufŽ cient heat activation to germinate and produce cells that may subsequently multiply in cooked foods if the rate and extent of cooling are not sufŽ cient. C. perfringens generation time is as rapid as 7.4 min in autoclaved ground beef with an optimal growth range of 37 to 458C, and growth has been reported at temperatures as low as 68C (20). The Food Safety and Inspection Service (FSIS) guidelines for the cooling of meat products specify that the internal product temperature should not remain between 130 and 808F (54.4 and 26.78C) for more than 1.5 h or between 80 and 408F (26.7 and 4.48C) for more than 5 h during cooling. If meat processors are unable to achieve this timetemperature schedule, they should prove that the alternative cooling regimen used will result in less than a 1-log CFU increase in C. perfringens. When processors deviate from these guidelines, it may be necessary to conduct challenge studies to determine if the performance standard has been met. * Author for correspondence. Tel: 732-932-9611, Ext 214; Fax: 732-9326776; E-mail: [email protected]. Many studies have investigated the enumeration and plating methods of C. perfringens from ground beef and broth systems (1, 2, 11, 16). The effect of methodological differences typically encountered in challenge studies have yet to be described in the published literature, although studies with other pathogenic bacteria have shown that the growth medium and experimental conditions can have a large impact on growth (3, 15). Preliminary experiments conducted at Rutgers, the State University of New Jersey, contradicted those published by the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Eastern Regional Research Center (ERRC). The preliminary Rutgers results showed no growth in cooked ground beef, whereas Juneja et al. (9) reported that cooling ground beef from 54.4 to 4.48C in 15 h (or more) allowed greater than a 1-log CFU increase of growth. This discrepancy prompted a collaborative investigation into methodological differences that might have a large impact on the growth of C. perfringens during cooling. The objective of this study was to investigate several variables normally encountered when conducting C. perfringens challenge studies and to provide recommendations for future studies of this type. MATERIALS AND METHODS Sample preparation and inoculation of meat samples. Three strains of C. perfringens—NCTC 8238 (Hobbs serotype 2), NCTC 8239 (Hobbs serotype 3), and NCTC 10240 (Hobbs seJ. Food Prot., Vol. 67, No. 6 METHODOLOGICAL FACTORS INFLUENCING GROWTH OF C. PERFRINGENS 1129 TABLE 1. Comparisons of the growth of C. perfringens in ground beef during a 21-h cooling cycle using different sampling bags and spore stocks