Introduction
Freezing is widely used in the food industry to preserve quality and extend shelf life. Mechanistically speaking, freezing can damage muscle cell membranes by forming extracellular ice crystals and changing the concentration of solutes found in meat (Leygonie et al., 2012; Lu et al., 2022). Recent research on freezing and subsequent thawing events of red meat has focused on the interactive effects of chilled storage and freeze-thaw events (Aidani et al., 2014; Coombs et al., 2017; Zhang et al., 2018) as well as the critical role that rates of freezing and subsequent thawing events contribute to product quality attributes (Mortensen et al., 2006; Yu et al., 2010; Kim et al., 2018). In addition to this, there has been an emerging interest in research related to repeated freeze-thaw events in recent years (Curry et al., 2023; Rehman et al., 2024a; Rehman et al., 2024b). While much of the research on repeated freeze-thaw events has been conducted in beef, there are 2 studies that have evaluated repeated freeze-thaw events in pork (Xia et al., 2009; Tippala et al., 2021). Results from these studies suggest greater levels of purge loss and cooking loss and improved levels of instrumental tenderness in samples undergoing freeze-thaw events compared with never-frozen counterparts. Yet, studies evaluating repeated freeze-thaw events have reported subtle differences in purge loss, cooking loss, and instrumental tenderness between a single freeze-thaw event and multiple freeze-thaw events.
In addition to product storage conditions, product quality attributes can be greatly influenced by endpoint cooking temperature. It has been suggested in recent years that endpoint cooking temperature plays a very meaningful role in instrumental tenderness and sensory attributes of pork loins. For example, lowering endpoint cooking temperature from 71°C to 63°C significantly improved shear force values and sensory attributes (Bryan et al., 2019; Honegger et al., 2019). To date, there are limited data on the influence of freezing and thawing events in combination with endpoint cooking temperature on pork tenderness and water-holding capacity. Thus, the objective of this study was to determine the interactive effect of freeze-thaw events and endpoint cooking temperature on instrumental tenderness and water-holding capacity of aged pork loins.
Materials and Methods
Approval from the Institutional Animal Care and Use Committee was not required because live animals were not used in this study.
Treatment design
Sixty-four pork loins (longissimus thoracis et lumborum [LTL] muscles) from 8 different genetic sire-lines were obtained from a commercial packing plant that uses electrical stunning and conventional chilling (20–24 h of exposure to ambient temperatures of approximately 4°C). Upon arrival at the Ohio State University Meat Science Teaching Laboratory, the individually vacuum-sealed loins were aged in cardboard boxes in a 2°C refrigerated room until 14 d postmortem. At 14 d postmortem, the vacuum-sealed packaging was removed, the spinalis dorsi and other associated muscles were trimmed to isolate the LTL muscle, and each LTL muscle section was divided into four 8-cm thick sections and individually vacuum packaged (3-mm thick nylon/polyethylene vacuum pouch, Ultra Source LLC, Kansas City, MO, USA). The four 8-cm sections from each LTL muscle were randomly assigned to one of 4 treatments: aged 14 d and never frozen (A14-NF), aged 21 d and never frozen (A21-NF), aged 14 d followed by one freeze-thaw event and 7 additional d of aging (A21-F1), or aged 14 d followed by one freeze-thaw event, 7 additional d of aging followed by a second freeze-thaw event (A21-F2). Postmortem aging for all treatments was conducted in cardboard boxes in a 2°C refrigerated room. During each freeze-thaw event, samples were placed in a single layer on a plastic tray in a −25°C freezer (Crown-Tonka walk-in freezer; Everidge, Plymouth, MN, USA) for at least 12 h before thawing was allowed to occur by placing samples in a single layer on a plastic tray in a 2°C refrigerated room for 24 h.
Quality analysis
Purge loss was measured immediately after each assigned aging/freezing period had elapsed. The loin sections were removed from vacuum-sealed packages, blotted dry using paper towels, and weighed. Purge loss was evaluated using the packaged-muscle cut weight, out-of-package muscle cut weight, and the weight of the dried package, using the following equation: Purge loss = [(packaged-muscle cut weight − dried package weight − out-of-package muscle cut weight)/(packaged-muscle cut weight − dried package weight)] × 100.
After each assigned aging/freezing period elapsed, each muscle section was then fabricated perpendicular to the ventral surface into two 2.54-cm thick pork chops, and randomly assigned to an internal endpoint cooking temperature of either 63°C or 71°C using the sous vide cooking technique in a water bath (precision general purpose water bath; Thermo Fisher Scientific, Waltham, MA, USA) using similar techniques as those described previously by Bryan et al. (2019). The water bath was set and maintained at a temperature of 80°C. Samples allotted to an endpoint temperature of 63°C were cooked for approximately 20 min, while those assigned to an endpoint temperature of 71°C were cooked for approximately 30 min. Internal cooking temperature was continuously monitored during cooking by inserting a digital temperature logger with 2 thermocouples into the center of a non-study reference pork chop sample of comparable weight and size to the study samples. Upon reaching the targeted endpoint cooking temperature, pork chops were immediately arranged in a single layer on a plastic tray and transferred to a 2°C refrigerated room for a period of approximately 12 h. The cooked pork chops were weighed after removal from the vacuum packages, and cooking loss was calculated as the percentage of weight difference between the initial raw weight and the cooked weight. Six 1.27-cm diameter cores parallel to the muscle fiber orientation were obtained from each cooked pork chop using a hand-held coring device. Cores were sheared perpendicular to the fiber direction using a Warner-Bratzler shear attachment on a TA-XTplusC texture analyzer (Texture Technologies Corp., Hamilton, MA, USA). Pre-test and test speeds of the instrument were set to 2 mm/sec, while post-test speed was set to 10 mm/sec. The force was measured for a set distance of 20 mm. The peak force required to shear through each core was recorded, and the value for the 6 cores from each pork chop was averaged and reported as Warner-Bratzler shear force (WBSF).
Statistical analysis
The assumptions of analysis of variance (ANOVA) were assessed by evaluating the residuals for normality and homogeneity of variance using the UNIVARIATE procedure in SAS v9.4 (SAS Institute Inc., Cary, NC, USA), with consideration given to the Shapiro-Wilks test for normality, along with evaluating the Student Panel graphs generated by the GLIMMIX procedure of SAS. Purge loss data were analyzed using a randomized complete block design, with aging/freezing treatments serving as the fixed effect and genetic sire-line serving as a block. Cooking loss and WBSF were analyzed using a split-plot design. The processing treatment served as the whole-plot factor, and endpoint cooking temperature served as the sub-plot factor. Genetic sire-line was considered as a random effect. Data were analyzed using the GLIMMIX procedure in SAS, and multiple comparisons tests were completed using a Tukey-Kramer adjustment.
Results and Discussion
Purge loss was significantly influenced (P < 0.01) by aging/freezing treatment (Figure 1). Never-frozen treatments (A14-NF and A21-NF) had less (P < 0.05) purge loss compared with frozen treatments (A21-F1 and A21-F2). No differences (P > 0.05) were observed within the 2 never-frozen treatments (A14-NF and A21-NF) or within the 2 frozen treatments (A21-F1 and A21-F2). This suggests that freezing and subsequent thawing events lead to significantly greater purge loss compared with samples that were not subjected to freezing and subsequent thawing events, regardless of the number of freezing and subsequent thawing events.
Effects of aging/freezing treatment on purge loss of pork loins; purge loss was evaluated using the packaged-muscle cut weight, out-of-package muscle cut weight, and the weight of dried package, using the following equation: purge loss = [(packaged-muscle cut weight − dried package weight − out-of-package muscle cut weight)/(packaged-muscle cut weight − dried package weight)] × 100. a,bLeast-squares means with different letters are significantly different (P < 0.05); error bars show standard error of the means. 1Loins were randomly assigned to 4 aging/freezing treatments (A14-NF, aged 14 d and never frozen; A21-NF, aged 21 d and never frozen; A21-F1, aged 14 d followed by one freeze-thaw event and 7 additional d of aging; A21-F2: aged 14 d followed by one freeze-thaw event, 7 additional d of aging followed by a second freeze-thaw event).
Cooking loss was significantly influenced (P < 0.01) by the interaction between aging/freezing treatment and endpoint cooking temperature (Figure 2A). Treatment A14-NF exhibited the greatest (P < 0.05) cooking loss compared with other treatments within both respective endpoint cooking temperatures. The interaction may be further described by studying the relationship among the 3 treatments aged for 21 d. Cooking loss was similar (P > 0.05) among the 3 treatments when cooked to an internal temperature of 63°C; however, when cooked to an internal temperature of 71°C, there were greater (P < 0.05) levels of cooking loss for the A21-F2 treatment compared with the A21-NF and A21-F1 treatments.
Interactive effects of aging/freezing treatment and endpoint cooking temperature on (A) cooking loss and (B) Warner-Bratzler shear force of pork loins; cooking loss was evaluated for pork chops cooked to an internal endpoint temperature of either 63°C or 71°C using a precision water bath and calculated using the following equation: cooking loss = [(uncooked weight – cooked weight)/uncooked weight)] × 100. a–eLeast-squares means with different letters are significantly different (P < 0.05); error bars show standard error of the means. 1Loins were randomly assigned to 4 aging/freezing treatments (A14-NF, aged 14 d and never frozen; A21-NF, aged 21 d and never frozen; A21-F1, aged 14 d followed by one freeze-thaw event and 7 additional d of aging; A21-F2: aged 14 d followed by one freeze-thaw event, 7 additional d of aging followed by a second freeze-thaw event).
Greater levels of purge loss are attributed to changes in the water-holding capacity of pork upon freezing and subsequent thawing events. In agreement, previous research studies have documented greater purge loss and/or drip loss in pork loins (Kim et al., 2018; Zhang et al., 2018; Tippala et al., 2021) subjected to freezing and subsequent thawing events compared with never-frozen counterparts. Freezing leads to the formation of ice crystals, which initially develop in the extracellular space of muscle fibers, causing water to move osmotically from the interior to the exterior of muscle fibers. This process further results in the transverse shrinkage of muscle fibers and subsequent dehydration. Ice crystals forming within the intracellular space can cause damage to cell membranes, resulting in leakage of intracellular fluids, while the growth of ice crystals in the extracellular space exerts pressure on muscle fibers, leading to structural deformation and reduced water-holding capacity. Tippala et al. (2021) reported that freezing and subsequent thawing events resulted in larger gaps between muscle fibers and more widely opened extracellular drip channels, which led to greater levels of purge loss compared with aging alone. Cooking loss is heavily influenced by endpoint cooking temperature, as observed in this study, but the aforementioned damage to cell membrane space could have been influential when observing the greater levels of cooking loss for A21-F2 compared with the A21-NF and A21-F1 treatments. Additional research observing the microstructure and muscle fiber characteristics could help pinpoint the mechanisms associated with these differences.
Warner-Bratzler shear force was significantly influenced (P < 0.01) by the interaction between aging/freezing treatment and endpoint cooking temperature (Figure 2B). Similar to the cooking loss data, the interaction may best be described by the relationship among the 3 treatments aged for 21 d. When cooked to an internal temperature of 63°C, treatments A21-NF and A21-F1 had greater (P < 0.05) WBSF compared with treatment A21-F2 (indicating that multiple freeze-thaw events improved WBSF). When cooked to an internal temperature of 71°C, treatment A21-NF had greater (P < 0.05) WBSF compared with treatments A21-F1 and A21-F2 (indicating that freezing once or multiple times improved WBSF). The advantages in instrumental tenderness following freezing and subsequent thawing events were likely the result of a combination of factors but are primarily attributed to the loss of structural integrity caused by ice crystal formation (Leygonie et al., 2012). This has been reported in several other studies (Xia et al., 2009; Kim et al., 2018; Tippala et al., 2021) and is not a new finding within the discipline of meat science.
The release of proteinases into extracellular spaces during freeze-thaw events may contribute to the hydrolysis of muscle proteins, further enhancing tenderization and making extended aging following a freeze-thaw event an interesting proposition (Leygonie et al., 2012). Previous research has reported greater levels of proteolysis and degradation of Z-line proteins in meat samples subjected to freezing and subsequent thawing events (Yu et al., 2010; Grayson et al., 2014; Setyabrata and Kim, 2019). However, the limited improvements in tenderness observed in the current study suggest that proteolytic potential might be diminished in pork loins aged for 14 d postmortem and beyond. Therefore, the differences in WBSF among A21-NF, A21-F1, and A21-F2 treatments were likely attributed to the physical changes in muscle fibers caused by ice crystal formation during freeze-thaw events, rather than additional levels of proteolysis.
There has been significant interest from the pork industry, particularly as it pertains to pork loin chops, to recommend lowering the endpoint cooking temperature to 63°C. The suggestion has been supported with several research efforts over the past 20 years, where consumers preferred pork cooked to an endpoint cooking temperature of 63°C compared with higher temperatures (Moeller et al., 2010; Honegger et al., 2019; Douglas et al., 2025). Lowering the endpoint cooking temperature in this study from 71°C to 63°C resulted in a greater reduction in WBSF compared with the effects of extended postmortem aging and freezing, and subsequent thawing events. In agreement with our results, numerous studies have reported that increasing endpoint cooking temperatures increased shear force values for pork loin chops (Bryan et al., 2019; Honneger et al., 2019). A greater endpoint cooking temperature can induce a greater level of structural and conformational changes in muscle proteins, including the disruption of cell membranes, denaturation of myofibrillar proteins, aggregation of sarcoplasmic proteins, and shrinkage of connective tissue, which can collectively contribute to a reduction in meat tenderness.
Conclusion
Endpoint cooking temperature had a more pronounced impact on cooking loss and WBSF compared with aging/freezing treatments; however, the significant interactions between aging/freezing treatments and endpoint cooking temperature highlight the potential interplay between freezing events and endpoint cooking temperature. Future research should build upon these preliminary results and address muscle fiber characteristics and sensory attributes of pork undergoing different aging and freezing sequences and cooked to different temperatures and with different methods.
Conflict of Interest
The authors declare no conflicts of interest.
Acknowledgments
This study was partially funded by the Internal Grant Program of the College of Food, Agricultural, and Environmental Sciences at The Ohio State University.
Author Contribution
Yifei Wang: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing – original draft, Writing – review & editing.
Rebecca A. Brown: Investigation
Milena Conte: Investigation
Benjamin M. Bohrer: Conceptualization, Data curation, Methodology, Investigation, Supervision, Funding acquisition, Project administration, Writing – review & editing.
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