**3. Results**

#### *3.1. Microbiological Analysis (before and after Treatment Application)*

From pork loin sections, counts for coliforms, *Escherichia coli*, mesophilic aerobic bacteria (APC-M), psychrotrophic aerobic bacteria (APC-P), and mesophilic lactic acid bacteria (LAB-M) were performed before and after treatment application.

For all analysis, coliforms and *Escherichia coli* counts were below detection limit, <0.25 colony-forming unit (CFU)/cm2. Due to low initial counts, both coliforms and *Escherichia coli*, no statistical difference was found before and after treatment intervention.

Low initial counts suggested that the plant from which samples were collected has implemented good manufacturing practices and dressing procedures and thus a good control of possible cross-contamination of endogenous sources of pathogens with pork carcasses.

A treatment by sampling point interaction was found for APC-M and APC-P (*p* < 0.01), (Figures 1 and 2). For these microorganisms, initial counts (before intervention) did not differ between treatments, while after intervention, treatment with Washing Solution 750 ppm did not effectively reduced counts for APC-M and APC-P. For mesophilic lactic acid bacteria (LAB-M), no effect was found by treatment or sampling point (*p* = 0.69). After treatment application, counts for Washing Solution 750 ppm for APC-M and APC-P were statistically higher (*p* < 0.01), when compared to the other treatments, suggesting a lower antimicrobial efficiency immediately after intervention.

**Figure 1.** Mesophilic aerobic plate counts (Log CFU/cm2) before and after treatment application on pork loin sections (*n* = 36 per treatment). In each boxplot, the horizontal line crossing the box represents the median, the bottom and top of the box are the lower and upper quartiles, the vertical top line represents the upper interquartile range, and the vertical bottom line represents the lower interquartile range. Boxes with different letters a,b are significantly different according to Kruskal–Wallis analysis followed by pairwise comparison Wilcoxon's test at *p* < 0.05. The points represent the actual data points. Active ingredients: Bovibrom = 1,3-Dibromo-5,5-dimenthylhydantoin; Fit Fresh = chlorine dioxide; Washing Solution = rhamnolipid.

#### *3.2. Microbiological Analysis (End of Dark Storage Time)*

From pork loin sections, enumeration of APC-M, APC-P, coliforms, *Escherichia coli*, and LAB-M were performed at the end of each of four dark storage periods (1 Day, 14 Days, 28 Days, and 42 Days) at refrigerated temperatures 0–4 ◦C. Similarly, coliform and Escherichia coli initial counts were below detection limit (< 0.25 CFU/cm2) for the first day of storage. After 14 days, enumeration was above detection limit, and a statistical difference was found between counts at 14, 28, and 42 days (0.09, 0.52, 1.44 Log CFU/cm2, respectively; largest standard error: 0.09 Log CFU/cm2).

For APC-M, a dark storage time by treatment interaction was found (*p* = 0.05). As storage time was found to be significant, a statistical analysis was performed per storage time in order to find differences between treatments. Bacterial cells by nature multiply over time; that is why the biological importance lies in the change of treatments over time.

Nonparametric approach tests, also called distribution-free tests, was used in order to analyze the results, because they do not assume that the data follow any specific distribution as parametric tests do. The distribution of the data (Figure 3) suggests neither samples follow a normal distribution, or the sample size was big enough. A Kruskal–Wallis test, the test used when assumptions of ANOVA are not met, was performed to find differences between treatments over the four dark storage times.

**Figure 2.** Psychrotrophic aerobic plate counts (Log CFU/cm2) before and after treatment application on pork loin sections (*n* = 36 per treatment). In each boxplot, the horizontal line crossing the box represents the median, the bottom and top of the box are the lower and upper quartiles, the vertical top line represents the upper interquartile range, and the vertical bottom line represents the lower interquartile range. Boxes with different letters a,b are significantly different according to Kruskal–Wallis analysis followed by pairwise comparison Wilcoxon's test at *p* < 0.05. The points represent the actual data points. Active ingredients: Bovibrom = 1,3-Dibromo-5,5-dimenthylhydantoin; Fit Fresh = chlorine dioxide; Washing Solution = rhamnolipid.

> After one day of dark storage, counts were not different between treatments (*p* = 0.08). There were differences for Bovibrom 225 ppm, Bovibrom 500 ppm, Fit Fresh 3 ppm, and Washing Solution 750 ppm counts at 14 days of storage time when compared with Water (*p* < 0.01). Then, after 28 days of storage time, all treatments presented similar values when compared with Water. For all treatments, there was no immediate effect on the microbial load of pork loin sections (Day 1), but these results sugges<sup>t</sup> that there was a residual effect of all the treatments by the fact that, at Day 14, all counts were lower when compared with Water. Moreover, these results show that this residual effect is lost by the time the pork samples reached 28 days of refrigerated storage time.

Further, for APC-P (Figure 4), a dark storage time by treatment interaction was found (*p* = 0.03). At Day 1 of dark storage, counts were not different between treatments. There were differences for Bovibrom 500 ppm, Fit Fresh 3 ppm, and Washing Solution 750 ppm counts at 14 days of storage time when compared with Water, but in later storage times, all treatments presented similar values compared with Water. Similar results were obtained for APC-M and APC-P related with the residual effect, found by using this type of interventions on pork loins. For mesophilic lactic acid bacteria (LAB-M), no significant effect was found for treatment or dark storage (*p* = 0.45).

**Figure 3.** Mesophilic aerobic plate counts (Log CFU/cm2) after 1, 14, 28, and 42 days of dark storage time on pork loin sections (*n* = 45 per dark storage time). In each boxplot, the horizontal line crossing the box represents the median, the bottom and top of the box are the lower and upper quartiles, the vertical top line represents the upper interquartile range, and the vertical bottom line represents the lower interquartile range. For each sampling point day, boxes with different letters a,b are significantly different according to Kruskal–Wallis analysis followed by pairwise comparison Wilcoxon's test at *p* < 0.05. The points represent the actual data points. Active ingredients: Bovibrom = 1,3-Dibromo-5,5-dimenthylhydantoin; Fit Fresh = chlorine dioxide; Washing Solution = rhamnolipid.

**Figure 4.** Psychrotrophic aerobic plate counts (Log CFU/cm2) after 1, 14, 28, and 42 days of dark storage time on pork loin sections (*n* = 45 per dark storage time). In each boxplot, the horizontal line crossing the box represents the median, the bottom and top of the box are the lower and upper quartiles, the vertical top line represents the upper interquartile range, and the vertical bottom line represents the lower interquartile range. For each sampling point day, boxes with different letters a,b are significantly different according to Kruskal–Wallis analysis followed by pairwise comparison Wilcoxon's test at *p* < 0.05. The points represent the actual data points. Active ingredients: Bovibrom = 1,3-Dibromo-5,5-dimenthylhydantoin; Fit Fresh = chlorine dioxide; Washing Solution = rhamnolipid.
