In uence of Poultry Carcass Skin Sample Site on the Effectiveness of Trisodium Phosphate against Listeria monocytogenes

The aim of this study was to determine the in uence of skin sample site on the efŽ cacy of trisodium phosphate (TSP) solutions in reducing Listeria monocytogenes populations on chicken carcasses during refrigerated storage. Chicken skin samples from the legs, the breasts, and the dorsal area inoculated with L. monocytogenes (108 CFU/ml) were dipped for 15 min in sterile tap water (control) or in 8, 10, or 12% TSP. L. monocytogenes counts and surface pH values were determined after 0, 1, 3, and 5 days of storage at 28C. For all sampling times and TSP concentrations, the reductions in L. monocytogenes numbers in breast skin were signiŽ cantly larger (P , 0.05) than those in leg skin or dorsal skin. No signiŽ cant differences were found in pH values as an effect of skin site. Our results suggest that skin sampling site is an important factor that needs to be considered when decontamination protocols are developed for poultry carcasses with the TSP treatment. Contamination of raw poultry carcasses with pathogenic and spoilage bacteria is well documented (30). In addition to good manufacturing processes, the microbial load of fresh meat can be reduced substantially by the application of decontaminants. In 1992, the U.S. Department of Agriculture approved the use of trisodium phosphate (TSP) as a processing aid to reduce viable Salmonella spp. on raw poultry carcasses (12). TSP is generally recognized as safe and has been reported to reduce populations of Salmonella spp., Escherichia coli, Campylobacter spp., pseudomonads, coliforms, Enterobacteriaceae, Staphylococcus aureus, Listeria monocytogenes, and total bacterial counts (3, 7, 8, 15, 17, 26, 31, 32, 34) on poultry without affecting the taste of the product (6, 13, 14). The decontaminating activity of TSP on poultry has been evaluated for whole carcasses, chicken parts (legs, wings, and drumsticks), in vitro models, and skin (generally breast skin) samples. However, the effect of skin site on the effectiveness of TSP has not been studied up to now. The aim of this study was to determine the in uence of skin site (leg, breast, or dorsal area) on the effectiveness of TSP in reducing L. monocytogenes populations on poultry during refrigerated storage. MATERIALS AND METHODS Bacterial inoculum. L. monocytogenes 64 d (isolated from a chicken carcass in our laboratory) was subcultured at least twice * Author for correspondence. Present address: Instituto de Ciencia y Tecnologṍ a de los Alimentos, C/ La Serna No. 56, 24007-León, Spain. Tel: Int-34-87-243123 or Int-34-87-238162; Fax: Int-34-87-243123; E-mail: dhtrcg@unileon.es. by loop inoculation of 10-ml volumes of Trypticase soy broth (Oxoid Ltd., Hampshire, UK) at pH 6.2 and was then incubated at 308C for 12 h to achieve viable cell populations of 109 CFU/ ml. The L. monocytogenes inoculum was prepared by diluting 10 ml of the suspension with 90 ml of sterile 0.1% (wt/vol) peptone water (Oxoid) to yield about 108 CFU/ml. Sample inoculation and treatment application. Forty chicken carcasses were collected from a poultry processing plant immediately after evisceration. Carcasses were transported to the laboratory in an ice chest and stored for no longer than 2 h before use. For each carcass, three groups (from the leg, breast, and dorsal areas) of four skin samples (a, b, c, and d) were removed aseptically. All of the samples weighed approximately 10 g. The outer surfaces of the skin samples were immersed for 5 min in suspensions of L. monocytogenes 64 d (108 CFU/ml) at 208C. After inoculation, samples were kept for 30 min at room temperature (228C) to allow the attachment of bacteria. The skin samples were immersed in 0 (sample a), 8 (sample b), 10 (sample c), and 12% (sample d) (wt/vol) TSP (Merck, Darmstadt, Germany) solutions for 15 min at 208C. After immersion, samples were kept at room temperature for 15 min. Afterward, the samples were placed into sterile bags and stored at 28C. Half of the skin groups were used for microbiological counts, and the other half were used for pH determination. Analyses were carried out after 0 (30 groups), 1 (30 groups), 3 (30 groups), and 5 (30 groups) days of storage. Day 0 samples were tested immediately after treatment was completed. Microbiological analysis and pH determination. For the counting of L. monocytogenes cells, skin samples were placed in a stomacher bag containing 90 ml of 0.1% (wt/vol) sterile buffered peptone water (Oxoid) and then blended with a Stomacher 400 D ow naded rom hp://m eridianenpress.com /jfp/article-pdf/65/5174/0362-028x-65_5_853.pdf by gest on 12 M ay 2020 J. Food Prot., Vol. 65, No. 5 854 CAPITA ET AL. FIGURE 1. Reductions in L. monocytogenes counts on different chicken skin sites after immersion in 8, 10, and 12% TSP solutions and storage at 28C for 5 days (n 5 5). (A. J. Seward, London, UK) for 2 min. Decimal dilutions from skin homogenates were prepared in 0.1% (wt/vol) peptone water (Oxoid) for spread plating. An aliquot of 0.1 ml was surface plated in duplicate onto PALCAM medium (Oxoid) and incubated at 308C for 72 h. For pH determination, 5 g of skin was transferred to a stomacher bag with 15 ml of sterile deionized water (MILLI Q) and blended for 2 min in a Stomacher 400. Statistical analysis. L. monocytogenes counts were converted to log10 CFU/g values. The reduction in bacterial populations attributable to TSP treatment was calculated by subtracting the log10 CFU/g values of treated samples (samples b, c, and d) from the log10 CFU/g value of the control sample (sample a). Data obtained were compared for statistical differences (P , 0.05) by an analysis of variance and Tukey’s test. The tests were carried out with the Statistica 6.0 software package (Statsoft Ltd., Chicago, Ill.). RESULTS AND DISCUSSION L. monocytogenes counts for control samples were 7.2 6 0.3 log10 CFU/g, 7.1 6 0.3 log10 CFU/g, 7.9 6 0.5 log10 CFU/g, and 8.2 6 0.2 log10 CFU/g for sampling days 0, 1, 3, and 5, respectively. No signiŽ cant differences were found among control samples taken from different locations on the carcass. We previously reported (4, 5) that TSP concentration and sampling time had a signiŽ cant in uence on reductions of L. monocytogenes attached to chicken skin. The results of the present study show that the skin site is also an important factor in uencing the effectiveness of TSP against L. monocytogenes. Breast skin samples showed the largest reductions of all skin sites for all TSP concentrations and sampling times (Fig. 1). In general, no marked differences in bacterial reductions were found between leg skin and dorsal skin. The effects of TSP treatments on bacterial populations on poultry have been studied for carcasses (2, 8, 25, 26, 33, 34), poultry parts (17, 24), and skin samples (7, 15, 19, 31, 32). Studies on skin pieces are frequently carried out with breast skin, and no work to determine the in uence of the poultry skin site on the decontaminating effect of TSP appears to have been reported. The mechanisms of reductions in bacteria on chicken carcasses with the TSP treatment are still not fully understood. It has been suggested that the high pH and ionic strength of TSP solutions and the ability of these solutions to remove a thin layer of lipids (the ‘‘detergent effect’’) from chicken skin are responsible for the bactericidal activity (1, 15, 24, 34). At present, little is known about the mechanism by which bacteria become attached to the surfaces of food products. It is generally accepted that bacterial attachment involves two stages (10). The Ž rst stage consists of a loose reversible sorption that may be related to physicochemical factors. The second stage consists of an irreversible attachment to surfaces by extracellular polymers (glycocalyx) (16). Moreover, bacteria may be trapped mechanically in ridges, crevices, capillary-sized channels (29), and feather follicles (9, 19). Chemical compounds may not be able to gain access to the bacteria that are more Ž rmly attached in these sites, thus providing physical protection and making their removal more difŽ cult (28). According to Korber et al. (20), crevices and roughness still provided the best protection against TSP in terms of the number of surviving bacteria. The loss of epidermis during scalding could also facilitate the Ž rm attachment of bacteria to chicken skin (27). Our Ž ndings suggest that L. monocytogenes cells formed a strong attachment to leg or dorsal skin or became physically entrapped once they were inoculated, and the attached or entrapped cells apparently were not readily removed by rinsing with TSP solution. The apparently more extensive attachment or entrapment of L. monocytogenes on leg and dorsal skin than on breast skin is possibly due to their differences in topographical structure and speciŽ c physicochemical properties. According to Kim et al. (19), there are speciŽ c sites on poultry skin that have a physical structure suitable for irreversible bacteria attachment. A possible explanation for our Ž ndings is that featherless spaces (apteria) are more abundant in the breast area (apterium cervicale ventrale, apteria pectoralia, and apterium sternale) than in the leg area (crural apteria) or in the dorsal area (apterium scapulare and apterium pelvinum laterale) (21). The smaller number of feather follicles in the breast area could explain the lower level of entrapment in this area. Nevertheless, according to Lucas and Stettenheim (22), the stratum corneum on the midventral line is compact as a consequence of its rubbing against the outer surfaces. D ow naded rom hp://m eridianenpress.com /jfp/article-pdf/65/5174/0362-028x-65_5_853.pdf by gest on 12 M ay 2020 J. Food Prot., Vol. 65, No. 5 SKIN SITE INFLUENCES ON TSP EFFECT 855 T A B L E 1. Sk in pH va lu es fo r L . m on oc yt og en es –i no cu la te d le g, br ea st , an d do rs al ar ea s 0, 1, 3 an d 5 da ys af te r T SP tr ea tm en ts (m ea n 6 st an da rd de vi at io n; n 5 5) a