**4. Discussion**

Microbial indicator levels assessed in the hot carcass wash section of the study were substantially lower before the hot carcass wash intervention, demonstrating the efficacy of proper sanitary dressing procedures in the facility (Table 1). Since the initial concentration of microorganisms was so low, no significant reductions in indicator bacteria concentrations were observed in the early stages of sampling and no effects were observed after subjection to the different treatment combinations. Because of this, the concentration of *Enterobacteriaceae, E. coli*, and coliforms were below detection limits (<0.25 CFU/cm2) after the hot water wash intervention in most samples collected at day 0. Under the conditions evaluated in this study, the hot water carcass intervention was not found to significantly reduce APC and PSY counts compared to no-wash treatments. This finding shows that despite significantly reducing a small number of bacteria on the surface of the carcass, washing the carcass may also redistribute the bacteria throughout the whole carcass surface and that can contribute to further differences during prolonged storage. This may pose a counterproductive result as bacteria will have a greater surface area of contact with the carcass and these may allow for more microbial attachment, growth, and development [21]. Furthermore, the washing of the carcass surfaces may increase available water for microbial growth which in the long term may allow a higher proliferation of bacteria in the striploins, a tendency observed in the long-term storage under refrigerating conditions [22]. Higher reductions may be achieved with alternative physical interventions such as steam vacuuming and trimming which do not use any chemicals for the reduction of bacteria [23,24].

When observing the hot carcass washing and type of carcass chilling, an immediate effect was not observed in any of the five bacteria quantified at day 0. Particularly EB, CO, and EC were all below detection limits within the in-plant sampling at day 0. By the time striploins had undergone shipment and distribution to export markets under refrigeration (day 45), a significant difference was observed between treatments in PSY, APC, and EB, where dry chilling treatments had lower bacterial counts overall. This trend was kept throughout the 135 days of refrigerated storage evaluated in this study.

APC evaluations show an overall count of mesophilic bacteria demonstrating a general picture of the total bacteria counts within the striploins. However, psychrotroph counts represent a more accurate bacterial load of meat, as meat is mostly stored under refrigerated conditions for a prolonged time. Previous research has demonstrated around a 0.5–1.0 log CFU increase in concentration on PSY counts compared to APC [18,25]. Most psychrotrophic enumeration methods require incubation at 7 ◦C for 10 days, or at 10 ◦C for 7 days, among others [19]. Furthermore, methods with incubation at 20 ◦C for 72 h have been used to enumerate carcass and meat subprimal psychrotrophic counts [18,26]. Due to variability in protocols for quantification of psychrotrophic bacteria counts, a trial comparing psychrotrophic counts using incubation at 7 ◦C for 10 days and 20 ◦C for 72 h was performed. Results led to a correlation of 0.96 and 0.98 at 45 and 70 days of aging, respectively (data not shown), thus validating the use of the protocol of incubation for psychrotrophic bacterial counts at 20 ◦C for 72 h. PSY counts were significantly lower on the dry chilling treatments, particularly in the no-washed dry-chilled treatment combination. Furthermore, package bloating and off-odors were less frequently found in striploins subjected to the dry-chilled treatment.

During long-term refrigerated storage of meat, Pseudomonas, Enterobacteria, and lactic acid bacteria become the main microorganisms that cause spoilage [27]. In the past, EB has also been used as an indicator of the risk of *Salmonella* spp. contamination. A higher concentration of EB may increase the risk of *Salmonella* spp. presence [28]. However, EB presence does not confirm *Salmonella* spp. presence. Similarly, pathogens of public health interest are within the EB family classification, such as *Shigella*, *E. coli*, and *Klebsiella*. In this context, all treatments were effective at mitigating EB presence at day 0 of storage; however, as the long-term storage continued, evident differences between treatments were observed, where dry chilling treatments more effectively mitigated EB proliferation in the striploins. This mitigation of EB growth throughout time may sugges<sup>t</sup> that dry chilling not only prolongs shelf life but also further ensures food safety through lower rates of bacterial growth with the potential of increasing bacterial injury [8,29,30].

No difference between treatments was observed in CO and EC due to the low concentration encountered within sampling dates throughout the trial. EB and EC counts

serve as a Gram-negative indicator of fecal contamination. Generic *E. coli* serves as an indicator of process control; the FSIS has published minimal sampling requirements for beef processors indicating that final carcasses must have negative results of *E. coli* to be considered acceptable; moreover, if more than four samples are between 1–100 CFU/cm<sup>2</sup> in a window of 13 consecutive samples or a sample is over 100 CFU/cm<sup>2</sup> a corrective action is warranted [31]. In this context, the beef processing plant is well within the acceptable limits of *E. coli* enumeration, having over 95% of the carcasses below the detection limit and all below 100 CFU/cm<sup>2</sup> after harvest and throughout the long-term storage of striploins under refrigerated conditions. This is an outstanding indicator of proper hygiene procedures and sanitary dressing procedures within the plant. Previous research effectively validated process controls within beef slaughter operations using EC as an indicator of process control alongside APC and EB counts [26,32].

Overall, dry chilling procedures prolonged the shelf life of striploins more effectively. Dry aging has been observed to reduce *E. coli* O157:H7 and generic *E. coli* concentrations in carcasses [11,12]. Moreover, air chilling and blast chilling of carcasses have both shown similar results in the reduction of indicator microorganisms in beef and pork carcasses [15,16]. Contrastingly, spray-chilled carcasses have been shown to have no immediate effect on the microbial load of carcasses [17,30], as observed in this study. However, in the long term, spray-washed treatments consistently had higher microbial loads throughout all the treatments and no-washed dry-chilled treatments had consistently significantly lower microbial concentrations, suggesting different slopes for growth curves of microorganisms under different treatments. Importantly, dry chilling procedures are known to reduce cold carcass weight due to the loss of moisture from the carcass surface during chilling, resulting in economic loss for the processing plant [33,34]. The optimization of interventions, chilling techniques, and barrier technologies depending on the end consumer and shelf life requirements of the meat can result in minimization of economic loss, a better microbial quality, and a safer meat product.
