**1. Introduction**

In 2017, the United States produced almost 52 billion pounds of red meat, of which 25.6 billion pounds of the total were pork [1]. The United States Department of Agriculture (USDA) data show that, in 2018, the per capita consumption of pork was close to 50.8 pounds per year [2]. Pork has always been one of the major meat sources for people, so it is crucial for the industry to ensure a safe pork supply [3].

The Center for Disease Control and Prevention (CDC), in 2018, estimated that one out of six Americans ge<sup>t</sup> sick, and from those who were sick, 128,000 were hospitalized, and 3000 died of foodborne diseases [4]. Moreover, the contribution of meat to foodborne illnesses caused by bacteria is 23.20% (beef: 13.20%, pork: 9.80%, and game: 0.10%) [4]. Although the contribution of pork in foodborne illnesses caused by bacteria is lower when compared with beef, it remains significant.

The Institute of Food Science and Technology defines shelf life as "*the period of time during which the food product will remain safe; be certain to retain its desired sensory, chemical, physical, microbiological, and functional characteristics; where appropriate, comply with any label declaration of nutrition data, when stored under the recommended conditions*" [5]. In order to determine shelf life of products, there are series of different methods and equipment that can be used in relation with sensory characteristics of a product, such as color, odor, structure, and flavor, and how these attributes change with time. These types of equipment have been developed to obtain an objective measurement at the moment of analyzing sensorial characteristics of a product.

Furthermore, consumers expect that foods are free of foodborne pathogens and have a decently long shelf life, where antimicrobials play a substantial role in order to achieve

**Citation:** Vargas, D.A.; Miller, M.F.; Woerner, D.R.; Echeverry, A. Microbial Growth Study on Pork Loins as Influenced by the Application of Different Antimicrobials. *Foods* **2021**, *10*, 968. https://doi.org/10.3390/foods10050968

Academic Editor: Gary Dykes

Received: 24 March 2021 Accepted: 26 April 2021 Published: 28 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

this demand [6]. Food antimicrobials are classified as preservatives, according to the U.S. Food and Drug Administration, which are any chemicals that, when added to food, tend to prevent or retard deterioration [6]. The most common function of an antimicrobial is to prolong shelf life through the process of killing or inhibiting spoilage microorganisms while maintaining and extending all the organoleptic properties [6]. It is important to consider that antimicrobials are never a substitute for good sanitation practices in food processing plants, since low initial counts will always be ideal. Although antimicrobials extend the lag phase, their effects on the surviving population can be overcome through time [6]. The global economy in which we live leads us to store and transport food and assure that the food arrives in the condition that is expected, and this is where antimicrobials undoubtedly play a role.

The purpose of this study was to determine the impact of selected antimicrobial spray products on the microbial growth of indicator bacteria naturally present on pork loins after long term storage under dark and refrigerated conditions.

### **2. Materials and Methods**

### *2.1. Sample Collection*

The study was repeated three times between January to August of 2019. On each repetition, vacuum packaged boneless pork loins (*n* = 36) were purchased from a commercial pork processing plant located in Oklahoma and transported within five hours in a cooler covered with ice at 0–4 ◦C to the Gordon W. Davis Texas Tech University Meat Science Laboratory (Lubbock, Texas, TX, USA). Pork loins were stored under dark conditions (no light) at 0–4 ◦C and processed 24 h later.

### *2.2. Treatment Preparation*

Treatments were prepared two to three hours before application to the boneless pork loins. For each treatment, three liters of solution was prepared and then stored in a handheld sprayer (Chapin 1-Gallon Plastic Tank Sprayer, Chapin, Batavia, NY, USA). Treatments utilized included: cold water, Bovibrom 225 ppm (1,3-Dibromo-5,5-dimenthylhydantoin; prepared in a mixer provided by Passport Food Safety Solutions, West Des Moines, IA, USA), Bovibrom 500 ppm (prepared the same as Bovibrom 225 ppm), Fit Fresh 3 ppm (chlorine dioxide; prepared following label instructions, Selective Micro Technologies, Dublin, OH, USA), and Natural Washing Solution 750 ppm (rhamnolipid, Jeneil Biosurfactant, Saukville, WI, USA).

### *2.3. Treatment Application*

Pork loins were split into five sections of 8.90 cm in length. Each section was randomly assigned to one of the five treatments. For each treatment, 12 pork loin sections were obtained (*n* = 180). Interventions were sprayed onto the pork loin sections for 30 s using a handheld sprayer (Chapin 1-Gallon Plastic Tank Sprayer, Chapin, Batavia, NY, USA; Flow rate: 5.98 ± 0.75 mL/s). Then, sections were flipped and sprayed for another 30 s, ensuring coverage of the entire loin surface. After 10 min, treated sections were vacuum packaged using Cryovac bags (Sealed Air, Charlotte, NC, USA) and randomly assigned to one of the four dark storages periods (1, 14, 28, and 42 days) and refrigerated at temperatures ranging between 0 and 4 ◦C.

### *2.4. Swab Sample Collection*

Buffer peptone water (BPW) pre-hydrated 25 mL swabs (3M, St. Paul, MN, USA), were taken at multiple periods of time during pork processing: before application of intervention, after application of intervention (10 min after finishing interventions), and at the end of each storage time (immediately after opening the bag). For swabs in sections, a 100 cm<sup>2</sup> template was used. The swabs were taken from the fat and the lean portions of the pork loin sections.

### *2.5. Swabs Sample Processing*

After arrival to the laboratory, pre-hydrated swabs were homogenized for two minutes at 230 rpm using an automated stomacher (Steward Laboratory Systems, Davie, FL, USA), serial dilutions with BPW were conducted and plated in Petrifilm (3M, St. Paul, MN, USA) or plates (Thermo Fisher Scientific, Waltham, MA, USA) according to each microorganism.

### *2.6. Total Aerobic Plate Counts*

For Aerobic Plate Counts, the Association of Official Agricultural Chemists 990.12 (AOAC) official method was used. After serial dilutions were performed, Petrifilms were placed on a flat surface, inoculated with 1 mL of sample dilution following product instructions. Petrifilms were left undisturbed for one minute to permit gel to solidify. Petrifilms were incubated for 48 ± 3 h at 35 ± 1 ◦C for mesophilic bacteria conditions and 72 ± 3 h at 20 ± 1 ◦C for psychrotrophic bacteria conditions. Enumeration was conducted using 3M Petrifilm Plate Reader (3M, St. Paul, MN, USA) and checked in a standard colony counter following the rules of the official method [7–11].

### *2.7. Coliforms and Escherichia coli Enumeration*

For Coliforms and *Escherichia coli*, the AOAC 991.14 official method was used. After serial dilutions were performed, Petrifilms were placed on a flat surface. Then one mL of sample was inoculated onto the center of the film base and covered with the top film in duplicates. Petrifilms were left undisturbed for one minute to permit gel to solidify. Petrifilms were incubated for 48 ± 3 h at 35 ± 1 ◦C. Enumeration was conducted at 24 h for coliforms and 48 h for *Escherichia coli* in a standard colony counter following rules of the official method [12,13].

### *2.8. Lactic Acid Bacteria Enumeration*

After serial dilutions, one mL of sample was inoculated on a petri dish and pour plated with 20 mL of Mann–Rogosa–Sharpe Agar (MRS) in duplicates. Plates were placed in BD GasPak EZ Container Systems (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated at 48 ± 3 h at 35 ± 1 ◦C under microaerophilic conditions (6 to 16% O2 and 2 to 10% CO2) using BD GasPak EZ Campy Sachets (Becton Dickinson and Company, Franklin Lakes, NJ, USA), [14,15]. Enumeration was conducted using a Q-Counter (Spiral Biotech Inc, Norwood, MA, USA)

### *2.9. Statistical Analysis*

Pork loin section swabs before and after interventions was a 2 × 2 factorial design (Sampling point × Treatment) with two levels under sampling point (before and after) and 5 levels under treatment (Bovibrom 225 ppm, Bovibrom 500 ppm, Fit Fresh 3 ppm, Washing Solution 750 ppm, Water). Pork loin section swabs at different storage times was a complete randomized design with repeated measures over time. All counts were analyzed using Kruskal–Wallis nonparametric test (R. Version 4.04), followed by pairwise multiple comparison Wilcoxon's test adjusted by Benjamin & Hochber method. Wilcoxon's test was used to identify the significant variation in microbial level on swab samples collected at different sampling points, storage times, and treatments. A *p*-value of 0.05 or less was selected prior to the analysis to determine significant differences in this study.
