**4. Discussion**

Each treatment has different active ingredients; therefore, the overall spectrum, the mode of action, and the efficacy against microorganisms are highly dependent on the chemical and physical properties of the antimicrobial [6]. Treatment with Washing Solution 750 ppm is a biosurfactant within the glycolipid category known as rhamnolipids, which are produced mainly by *Pseudomonas aeruginosa* [16,17]. Rhamnolipids is an amphipathic surface-active molecule composed of ß-hydroxy fatty acid connected to a rhamnose sugar molecule used for a broad range of applications, such as antimicrobial agents [18,19]. Its antimicrobial activity is related primary by damaging the cytoplasmic membrane, causing an increase in its permeability due to the release of lipopolysaccharides from the outer membrane [20–22].

In a study where rhamnolipids were tested at different concentrations for Grampositive and Gram-negative bacteria, the antimicrobial effect was completely indistinguishable for Gram-negative bacteria at all concentrations tested, while Gram-positive bacteria were inhibited at most concentrations, explaining the lower antimicrobial efficiency when compared to other antimicrobials that have a larger overall spectrum of action [22,23].

In a pork chop shelflife study using organic (citric or ascorbic) acid applications and vacuum packaging system, psychrotrophic enumeration was performed in order to see the effect of these interventions in storage time up to 14 days. Results showed that, despite of the intervention and packaging system, psychrotrophic bacteria were still capable of growing over the storage period [24]; however, our study presented a clear decrease in log counts for APC-M and APC-P after 14 days of storage time when compared to the Water application. Evidence in this study suggests that antimicrobial interventions are effective

during the 14-day period, but once the antimicrobial effects are depleted, the remaining bacteria have less competition to multiply, increasing the rate of growth up to the point that counts at 42 days of storage did not differ from water application.

Treatment Bovibrom (225 and 500 ppm) is a commercial name for the active ingredient 1,3-dibromo-5,5-dimenthylhydantoin (DBDMH) [25]. DBDMH is a bromanine polymer, which hydrolyzes to hypobromous acid (HOBr) in presence of water [26]. This hypobromous acid has the same biocide property as hypochlorous acid (HOCl), and they both combine with organic compounds to form bromanines or chloramine, respectively; however, bromanines are more potent than chloramines and, therefore, show more effectiveness in the presence of organic matter [6]. Halogen's mechanism of action is not well defined, but theories such as interference in cell metabolism by oxidation of SH groups (sulfhydryl group) essential for bacterial enzymes due to pH or oxidation of purine and pyrimidine bases causing mutation are the most well-known and accepted [27]. Further, treatment with Fit Fresh 3 ppm is a commercial name for the active ingredient that in, presence of water, is known as chlorine dioxide. Chlorine dioxide differs from normal chlorine compounds, as it does not form HOCl, but it presents similarities in the antimicrobial activity due to the oxidation-reduction potential [6]. The mechanisms of action are not well defined, but it is theorized that protein synthesis and disruption of the outer membrane to be highly responsible for their antimicrobial activity [6].

Counts performed at the end of each dark storage time suggested that Fit Fresh, Bovibrom 500 ppm and Washing Solution 750 ppm performed better until 14 days of storage despite the wide spectrum of chlorine dioxide and hypobromous acid when compared with rhamnolipids. In addition, there is a clear increase in counts for all microorganisms as dark storage time increased, which is not surprising, because increased storage time in a vacuum bag will result in increased bacterial proliferation [28]. The use of antimicrobials is mainly to inhibit the growth of microorganisms by extending the lag phase of their lifecycle, and according to literature found, a meat product around 6 log CFU/cm<sup>2</sup> is at a level in which it could be considered spoiled, even though microbial loads is not the only attribute to be considered for shelf life [29]. During the dark storage period, treatments with Bovibrom 500 ppm, Fit Fresh 3 ppm, and Washing Solution 750 ppm presented values below this limit, and it is not until 42 days of storage that the pork section approached this limit, suggesting that the increase of shelf life, considering microbiological characteristics using these antimicrobials, is accomplished. When compared to other studies where, after 28 days of aging this limit was reached, our study delayed reaching the 6-log population limit until 42 days of storage [30].
