Sarid M. Shefet
Master's Thesis submitted to the Faculty of the North Carolina State University in partial fulfillment of the requirements for the degree of
MS
in
Food Science
Approved
Dr. Brian W. Sheldon, Chair of Advisory Committee
Dr. Todd R. Klaenhammer, Member of Advisory Committee
Dr. William H. Swallow, Minor Representative
July 5, 1994
Raleigh, North Carolina
Abstract
More than 10% of the U.S. population experience at least one incident of foodborne disease annually (Todd, 1989). From 1983 to 1987, Salmonella species were the most often identified bacteria responsible for foodborne disease (Bean et al., 1990) causing an estimated 2 million cases of foodborne salmonellosis annually in the United States at an estimated annual cost of one billion dollars (Cohen, 1978; Roberts, 1988). The Centers for Disease Control estimate that Salmonella infections contribute to at least 1,000 deaths per year in the United States.
Poultry products are considered to be the single most important food source of Salmonella. For example, approximately one third of the foodborne salmonellosis outbreaks for which a mode of transmission was identified have been linked to poultry consumption. Salmonella contamination rates for live chickens can vary from about 13% to 80% of the flock and are invariably higher after processing (Mead, 1976; Roberts, 1988; Budnik, 1990). In 1992, the U.S. was ranked first in the world in poultry consumption with 94.8 pounds per capita, followed by Israel with 83.7 pounds, and Hong Kong with 79.3 pounds (Brown, 1993). In 1993 over 27.6 billion pounds of ready-to-cook poultry products were produced in the U.S. Per capita consumption of poultry products has increased substantially over the last two decades relative to other meat products; therefore, exposure of the consumer to poultry product-associated microorganisms including pathogens has correspondingly increased and no doubt contributes to these foodborne disease statistics.
Besides bacterial pathogens, poultry products are also contaminated with a variety of spoilage microorganisms which can contribute to the development of strong off odors and/or slime formation and shortened product shelf life. These organisms, however, are not generally associated with human illness. A reduction in the population of these microorganisms or suppression of their growth often results in increased product shelf life and greater consumer acceptability. Some reports have estimated that the presence of pathogenic and spoilage microorganisms on poultry may cost the American public over two billion dollars annually in foodborne disease-related expenditures and spoiled products (Roberts, 1988; Todd, 1989).
The bacteriocin nisin was approved by the United States Food and Drug Administration in 1988 as a GRAS (general recognized as safe) substance for use in pasteurized cheese spreads to control outgrowth and toxin production by Clostridium botulinum. Blackburn et al. (1990) and Stevens et al. (1991) reported that nisin's spectrum of inhibitory activity could be extended to include gram-negative bacteria such as Salmonella when combined with chelating agents such as disodium ethylenediamine tetraacetate (EDTA) and citrate. Perturbation of the outer membrane of gram-negative bacteria via chelation of divalent cations located in the lipopolysaccharide layer is believed to sensitize the cells by providing access to the cytoplasmic membrane where nisin-mediated inactivation occurs.
The initial focus of this study was to optimize the inhibitory activity of nisin against a Salmonella typhimurium NAR test strain inoculated on broiler drumstick skin by combining nisin with various chelating agents and a surfactant. Treatment parameters included a fixed nisin concentration (100 µg/ml), acidic solution pH, and varying EDTA, citric acid, and Tween 20 concentrations. Using a simplex search algorithm optimization program, four treatments were identified that significantly reduced the population of S. typhimurium NAR cells on broiler drumstick skin. Salmonella typhimurium NAR skin population reductions ranged from log10 5.18 to 5.56 following immersion of the inoculated skin in the treatment solutions for 30 minutes (37°C). Inhibitory activity was also evident in the treatment solutions where S. typhimurium NAR cells washed from the inoculated skin were significantly inhibited by the treatments. In related studies, the inhibitory activity of these four optimized nisin-containing treatments were compared to either distilled, deionized water or 20 ppm chlorine against S. typhimurium NAR cells inoculated on the skin of broiler drumsticks. Following a 30 minute exposure to the treatment solutions at 25°C, surviving S. typhimurium NAR organisms were recovered from the skin and treatment solutions and enumerated. Log reductions in the skin populations were calculated relative to the control skin samples that were immersed in water under the same conditions. Salmonella typhimurium NAR skin populations were reduced an average of 6.67 logs following exposure to the nisin-containing treatments in comparison to 2.24 logs for the chlorine-treated broiler skin. Studies were also completed that evaluated the efficacy of the optimized nisin-containing treatments to inhibit Salmonella-infected broiler drumsticks. Similar reductions in the S. typhimurium NAR skin population, as o bserved with broiler drumstick skin, were detected following treatment with the four nisin-containing treatments.
Experiments were also conducted to determine the efficacy of the nisin-based treatments against S. typhimurium NAR-infected drumstick skin under varying exposure times and concentrations of nisin. Exposure time significantly influenced the lethality of the treatments and depending on the treatment, nisin concentrations could be reduced from 100 µg/ml to 50 or 25 µg/ml without loss of significant biocidal activity. In other studies, the refrigerated shelf life of broiler drumsticks was extended by 1.5 to 3 days following immersion for 30 minutes in one of the optimized nisin-containing treatments in comparison to drumsticks immersed in distilled, deionized water.
These findings indicate that treatments containing nisin and varying concentrations of chelating agents and/or surfactant at an acidic pH are capable of significantly inhibiting the population of Salmonella microorganisms and spoilage bacteria that contaminate the surfaces of poultry carcasses. Applying the nisin-based treatment as a post chill application; either by dipping, spraying, or by incorporation into or on the surface of primary packaging materials and edible films, might be effectively used by poultry processors to reduce Salmonella populations on raw poultry products. With the poultry industry experiencing increased consumer pressure to produce Salmonella-free poultry products, the identification and implementation of effective preservation methods could result in several long term benefits including greater public confidence in poultry products, an increased market potential, and increased profits for the poultry industry.