# Comparative Study of Fresh and Frozen Broiler Neck Skin Sampled for Process Hygiene Purposes
## 1 Introduction  
Broiler carcasses at poultry abattoirs and processing plants can be contaminated with different bacteria, e.g., Gram-negative Enterobacteriaceaeaeae like _Escherichia coli_ and _Salmonella_ spp. as well as other potential human pathogens such as _Campylobacter_ spp. [1, 2]. Other sources of bacterial contamination of broiler meat can be slaughter and processing plant equipment and environments as well as slaughter personnel, from which a severe risk of cross-contamination emanates [3, 4, 5]. To monitor the hygienic status of broiler meat, it is necessary to reliably evaluate the microbial status of broiler carcasses in a standardized way. Neck skin taken at slaughter can be seen as a pooled sample of the carcass' surface, as broilers are processed head down, with all fluids running over the neck skin [6]. Moreover, following the European Regulation (EC) No. 2073/2005 [7], the use of neck skin samples is required for process controls in broiler abattoirs. Neck skin tissue can be seen as the preferred sample type because neck skin can be taken easily without removing carcasses from the slaughter line [8]. Additionally, this carcass part has low nutritional and economic value, and taking this part of a broiler carcass as a sample for analysis is less destructive than other sample types/methods [9]. Furthermore, skin tissue removal was previously shown to be the most precise and accurate sampling method representing the skin contamination of poultry carcasses [10]. Bacterial multiplication requires certain physical conditions depending on the species. One of these conditions is the right temperature. Most zoonotic pathogens are adapted to the human body temperature and multiply and grow within a temperature range of 20 to 40 \({}^{\circ}\)C [11]. Because low temperature measurably controls microbial activity, especially mitosis, freezing has become a very important procedure for food preservation [12; 13]. The two conditions that do not allow microbial multiplication are appropriately low water activity (a\({}_{\text{W}}\)) and low temperature [14]. In meat, microbial multiplication stops at temperatures lower than \(-10\) to \(-12\)\({}^{\circ}\)C [15; 16; 17], while the total inhibition of cellular metabolism in animal tissues occurs at \(-18\)\({}^{\circ}\)C [16]. Following [PERSON] and [PERSON] (2007) [12], up to 60% of a microbial population is killed during freezing, but the proportion of the remaining bacteria gradually increases during the freezing process [18]. A reduction of 60% of the aerobic mesophilic cell count occurred after long-term storage for one year at \(-30\)\({}^{\circ}\)C [19]. The surviving microbial species found in thawed frozen meat were almost identical to those found before freezing, but they can occur in lower numbers. Moreover, after thawing, the surviving bacteria can multiply to such an extent that their count can reach the initial value [20]. [PERSON] and [PERSON] (1969) [19] found no influence on the quantitatively measured total viable count (TVC) after one year's storage at \(-30\)\({}^{\circ}\)C, even though the proportions of Gram-positive and Gram-negative bacteria changed compared to their initial proportions. [PERSON] and [PERSON] (1977) [14] concluded that most bacteria are only damaged during frozen storage, but not killed. Influencing factors are the speed of freezing, the storage temperature, and duration and, in addition to the type of bacteria, the protective substances contained in the surrounding matrix [15; 21]. If freezing occurs quickly, it is more likely that only small crystals form in the matrix surrounding the bacteria. When compared to large crystals, which occur during slow freezing, small crystals lead less frequently to the death of the bacteria present [14]. Higher temperatures, around \(-2\)\({}^{\circ}\)C, were more likely to kill bacteria [12; 22] during storage. In general, Gram-positive bacteria are more resistant to freezing death than Gram-negative bacteria, e.g., Enterobacteriaceaeaeae [12; 15].