**1. Introduction**

The world beef market is heavily influenced by consumer demands and choices; therefore, the beef industry must adapt to the consumers' needs and concerns and provide meat products that fulfill such needs. Certain consumer demands have created niche opportunities for a variety of meat product offerings. An important market niche for beef products is the "fresh meat" "never frozen" alternatives. This has led meat processors to seek schemes for increasing the shelf life of fresh meats by combinations of proper cold management, barrier technologies, and application of antimicrobial interventions (chemical or physical) [1,2]. The growing demand for fresh products has put pressure on the cold supply chain and quality control at all steps in the processing plant [3]. Such a trend has evolved rapidly, and now regulatory agencies have developed a series of

**Citation:** Casas, D.E.; Manishimwe, R.; Forgey, S.J.; Hanlon, K.E.; Miller, M.F.; Brashears, M.M.; Sanchez-Plata, M.X. Biomapping of Microbial Indicators on Beef Subprimals Subjected to Spray or Dry Chilling over Prolonged Refrigerated Storage. *Foods* **2021**, *10*, 1403. https:// doi.org/10.3390/foods10061403

Academic Editor: Mohammed Gagaoua

Received: 5 May 2021 Accepted: 11 June 2021 Published: 17 June 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/).

labeling requirements for never frozen meat and poultry products. The U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) has defined that any poultry product below −3.3 ◦C (26 ◦F) or red meat that has ever been frozen cannot be labeled as fresh, not frozen [4]. To address these market trends, beef processors need to explore novel processing schemes, product protection options, and process modifications that have been properly validated in commercial settings to extend product shelf life, especially when long transport regimes are necessary under refrigerated conditions due to significant distances between production and market locations.

Australia is one of the leading beef exporters in the world. As an important market player, the Australian beef industry has been continuously assessing new market opportunities and has been exploring fresh, never frozen beef alternatives for competitive markets worldwide. In 2018, Australia's bovine meat exports accounted for 43.2% of animal product exports with a market value of approximately \$6.47 billion for the country's economy. Australia's biggest beef export market is Japan with a market share of 36.8% in 2018. With the recent interest of the United States to significantly increase its beef exports to Japan [5,6], Australia has sought opportunities to expand its presence in the European Union. This high-income market shows significant consumer interest in the fresh, never frozen beef products [7]. Unfortunately, the distance between the meat source and the EU market has created a challenge, due to the long-haul transportation needs and rigorous chilled conditions necessary for product arrival and suitability for fresh distribution. Consequently, extending the shelf life of chilled meat products has become of the utmost interest.

Meat shelf life extension has been achieved through the use of several antimicrobial interventions, chilling methods, and barrier technologies in the past [1,2,8,9]. At the same time, these interventions and barrier technologies mitigate the growth of indicator and pathogenic bacteria that are responsible for product deterioration. The hot water wash of carcasses has been observed to reduce 2.7–3.0 log CFU/cm<sup>2</sup> of *L. monocytogenes*, *Salmonella*, and APC counts [10]. Dry aging has been observed to reduce over 2 log CFU/cm<sup>2</sup> of generic *E. coli* and *E. coli* O157:H7 in beef carcasses and subprimals [11,12]. Air chilling has been shown to reduce total viable counts by 03–0.7 log CFU/cm<sup>2</sup> [13], and up to 2 log CFU/cm<sup>2</sup> of *E. coli* [14]. Moreover, indicator and pathogenic microorganisms have been reduced after the air conventional chilling and blast chilling of carcasses [15,16]. Comparatively, spray chilling has been observed to have no immediate effect in microbial populations [17]. Thus, in this study, we evaluate the use of hot carcass washing and different carcass chilling systems to assess Australian chilled beef's extended shelf life in export settings that require product viability for more than 130 days of refrigerated storage and distribution.

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

### *2.1. Sample Collection*

Samples were taken at Teys Australia beef processing plant located in Beenleigh, QLD, Australia. A total of 200 carcasses were evaluated. Swab samples were taken using EZ-Reach™ Sponge Samplers hydrated with 25 mL buffered peptone water (BPW, World BioProducts, Mundelein, Illinois) by swabbing an over 100 cm<sup>2</sup> area on the striploin region of each carcass. Samples were taken before the hot carcass wash, after the hot carcass wash for washed samples, and 24 h after being subjected to one of the chilling methods described below (spray vs. dry chilling). Edible ink was used to mark the area where the sample was taken to avoid re-sampling of the same surface. The hot water carcass wash consisted of spraying 85 ± 2 ◦C water onto the surface of the carcass through eight nozzle sprayers, four per side of the carcass. Water temperature was recorded on the pipes feeding the water to the sprayers right before sample collection. The chilling methods evaluated consisted of 18–24 h storage in a refrigerated chamber subjected to continuous spraying of water at 0–2 ◦C in the room at 15 min intervals, following the processing plant's protocols. Dry chilling consisted of 18–24 h storage in a refrigerated room at 0 ◦C with constant airflow while the sprayers were completely turned off. After 24 h chilling, either under water

spray conditions or dry refrigerated storage, striploins were taken and cut into 4 sections. Individual sections were vacuum packaged and assigned a date for further sampling at 0, 45, 70, and 135 days of refrigerated storage. Samples collected for day 0 were analyzed in an in-plant laboratory setup at the processing facility. Striploins were shipped via sea to the ICFIE Food Microbiology Laboratory at Texas Tech University (TTU) in Lubbock, Texas, USA for the long-term shelf life section of the study corresponding to storage at days 45, 70, and 135 under refrigerated conditions. Striploins were kept at 0–4 ◦C from carcass fabrication to meat reception at TTU. On day 40, striploins were received at TTU and the refrigerated temperature was raised to 7 ◦C, simulating abusive counter temperatures common in retail stores. On each sampling day, striploin packages were opened with sterile scalpels and an area of 100 cm<sup>2</sup> of the product was swabbed for sample collection.

### *2.2. Sample Processing*

Swab samples collected were stomached for 30 s at 230 rpm. Serial dilutions for each swab sample were made with 9 mL BPW tubes. A volume of 1 mL was plated onto Petrifilm™ plates (3M, Saint Paul, Minnesota) in duplicate corresponding to *Enterobacteriaceae* (EB), *Escherichia coli* (EC), coliforms (CO), and aerobic plate counts (APC). In addition, aerobic plate count Petrifilm was also used to estimate psychrotroph counts (PSY) by incubating separate plates at 20 ◦C for 72 h [18,19]. *Enterobacteriaceae* Petrifilms were incubated for 24 h at 37 ◦C before counting. Coliforms were counted after 24 h incubation at 37 ◦C. *Escherichia coli* counts were recorded after 48 h incubation at 37 ◦C following the manufacturer's recommendations. APC plates were incubated for 48 h at 37 ◦C.

### *2.3. Experimental Design and Statistical Analysis*

The hot water wash section of the study had a completely randomized design with a factorial arrangemen<sup>t</sup> of 2 factors, feed regime and carcass wash, at 2 levels each: grass vs. grain and washed vs. not washed, respectively. Three sampling points were evaluated, before wash, after the washing stage, and after a 24 h chilling period. For each repetition, 10 samples were taken per treatment (Table S1) at each sampling point. A total of 5 repetitions were conducted.

The section of the study regarding the extended shelf life of the striploins was arranged in a completely randomized design with a factorial arrangemen<sup>t</sup> of three factors, feed regime, hot water wash application, and chilling method, at two levels each: grass vs. grain, washed vs. not-washed, and dry vs. spray-chilled, respectively. For each repetition, 5 samples per treatment were taken at each sampling date (Table S2). A total of 5 repetitions were conducted resulting in 200 samples per sampling date. An ANOVA by sampling date was used to analyze the data when parametric assumptions were satisfied. The Kruskal–Wallis (nonparametric ANOVA) test was used to analyze the data when parametric assumptions were not met. When the ANOVA or Kruskal–Wallis was significant, pairwise comparisons were done using a pairwise T-test on significant ANOVAs or a Wilcoxon rank-sum test on significant Kruskal–Wallis tests [20]. Statistical significance was evaluated at a 0.05 probability level.
