Effects of Sodium Nitrite and Tocopherol Incorporated Poly(Lactic Acid) Biodegradable Films on Dark-Cutting Beef Color

Film formulation

A twin-screw extruder (screw L/D ratio: 42; screw speed: 120 rpm; Century ZSK- 30; Traverse City, MI) was used to compound PLA resin (Ingeo^™ biopolymer 2003D poly [96% L-lactic acid]), sodium nitrite, and α-tocopherol. Two recipes were formulated with lower and higher concentrations of the active ingredients. Low-concentration films included 0.12% sodium nitrite and 0.5% α-tocopherol, and high-concentration films were created with 0.6% sodium nitrite and 2.5% α-tocopherol. The masterbatch packaging film formulation's temperature range was between 170 and 190 °C. A BT 25 pelletizer (Scheer Bay Co.; Bay City, MI) was used to form the extruded mass into pellets after being cooled in a water bath. The pellets were oven-dried for 24 h at 50°C before being used for later processing to remove moisture. A hot press (PHI QL438-C, City of Industry, CA) operated at 182°C with an applied force of 10 tons for 3 min was used to form film sheets from 5 g samples of each respective pellet concentration recipe. After compression, the film sheets were removed from the hot press, cooled at room temperature, and stored in a refrigerator until later application.

Oxygen permeability

A MOCON OX-TRAN^® (2/22 L model; MOCON Inc., Minneapolis, MN) was used to measure the oxygen permeability of the films. The instrument was set to 10°C at 34.3% ± 1% relative humidity based on the intended film application conditions and the instrument limitations. The analysis was completed with three different film sets separately.

Film transmittance analysis

A spectrophotometer (Thermo Scientific, USA) was used to collect the light transmittance of films within ultraviolet and visible light regions. Optical properties were measured in triplicate for each film recipe. Additionally, a digital micrometer (Testing Machines Inc.; Ronkonkoma, NY) with a sensitivity of 0.001 mm was used to determine film thicknesses. Film thickness was read in triplicate. The transparency value (TV) of the film samples was determined using TV = log (T₆₀₀)/x, where T₆₀₀ is the transmittance at 600 nm and x is the film thickness in mm (Han and Floros, 1997).

Thermogravimetric analysis

A Q50 TGA (TA Instruments; New Castle, DE) was used to evaluate the thermal stability of PLA films. A 10 mg sample of each film recipe was taken and tested from room temperature to 600°C with an increase of 10°C/min. Nitrogen gas was used to purge the system with a balance purge flow of 40 mL/min and a sample purge flow of 60 mL/min. Characteristic temperatures were noted during analysis in addition to residual ash content after heating. Initial decomposition temperature (T_onset) was determined when 5% of the sample weight was lost through testing. Decomposition peak temperature (T_peak) was noted by the peak of the derivative weight (%/°C) output curve during testing. Finally, residual ash was measured by the remaining weight percentage of the sample following testing.

Raw materials, processing, and packaging

Beef strip loins (longissimus lumborum) were collected from a commercial beef processing facility, 5 d postmortem. Seven USDA Low Choice (pH = 5.40 to 5.66) and seven dark-cutting (pH = 6.16 to 6.67) strip loins were selected, and vacuum-packaged loins were transported on ice to the Robert M. Kerr Food and Agricultural Products Center at Oklahoma State University (Stillwater, OK). Loins were repackaged (CRYOVAC; 16 × 24 pouches; 3 mil thickness) using a vacuum sealer (Sipromac 420A Double Chamber Vacuum Sealer; Sipromac; Drummondville, Canada) upon arrival and stored at 2°C in the dark for 2 d before being further processed.

pH and proximate composition analysis

A Hanna Instruments pH probe (Handheld HI 99163; probe FC232; Hanna Instruments, Woonsocket, RI) was used to measure the initial pH of normal-pH and dark-cutting loins during processing. Measurements were taken at 3 random locations of the longissimus lumborum muscle along the length of the loin.

The first and fifth steak from the anterior end of each loin was taken to determine the percentage protein, fat, and moisture for each strip loin. Longissimus lumborum muscle was ground with a tabletop grinder after trimming all visible external fat (Big Bite Grinder, coarse grind, LEM), pressed into a 140 mm sample cup, and analyzed with an AOAC-approved near-infrared spectrophotometer (FoodScan Lab Analyzer, Serial No. 91753206; Foss, NIRsystem Inc., Slangerupgrade, Denmark). Samples were read in triplicate and averaged.

Packaging and simulated retail display

Loins were sliced into 2.54-cm-thick steaks from the anterior end with a meat slicer (Bizerba USA INC., Piscataway, NJ). Normal-pH steaks were randomly selected for polyvinyl chloride (PVC) overwrap control. Dark-cutting steaks were randomly assigned to either a PVC overwrap control, a vacuum package control, a low-concentration PLA film in a vacuum package, or a high-concentration PLA film in a vacuum package. Steaks serving as PVC control steaks for normal-pH and dark-cutting loins were placed in Styrofoam trays and overwrapped with PVC (15,500 to 16,275 cm³ O₂/m² /24 h at 23°C, E-Z Wrap Crystal Clear Polyvinyl Chloride Wrapping Film; Koch Supplies; Kansas) using a film wrap machine (Winholt WHSS-1, 115V; Woodbury, NY). Vacuum package (Walton’s Vacuum Pouch; 10 × 10 pouches; 3 mil thickness) treatments were vacuum sealed using a vacuum sealer (Sipromac 420A Double Chamber Vacuum Sealer; Sipromac, Drummondville, Canada). Low- and high-concentration PLA treatments had the respective film placed directly on the steak cut surface and were vacuum packaged (Walton’s Vacuum Pouch; 10 × 10 pouches; 3 mil thickness) using a vacuum sealer (Sipromac 420A Double Chamber Vacuum Sealer; Sipromac, Drummondville, Canada). Packaged steaks were then placed in a simulated retail display using a white coffin-style display case at 2 ± 1°C under continuous LED lighting (Phillips LED lamps; 12 watts, 48 inches, color temperature = 3,500°K; Phillips, China) for either 5 or 6 d. Two steaks from each loin were taken for the respective packaging treatments; one steak was utilized for cooking analysis, and the other steak was used for microbial analysis and lipid oxidation. Cooking analysis took place on d 5 of the display to maximize nitric oxide myoglobin formation, whereas microbial growth and lipid oxidation were assessed on d 6.

Retail color analysis

A HunterLab 4500L MiniScan EZ Spectrophotometer (2.5-cm aperture, illuminant A, and 10° standard observer angle; HunterLab Associates, Reston, VA) was used to measure the instrumental color of steaks every 24 h where each packaged steak surface was read in triplicate. Both CIE L*, a*, and b* values and spectral reflectance from 400 to 700 were recorded. Chroma, hue angle, and nitric oxide myoglobin formation were calculated to determine surface color. Chroma $[\sqrt{(a^{*}{)}^2+(b^{*}{)}^2}]$ represents the red intensity of the steak color, with a larger value indicating a brighter red color (King et al., 2023). Nitric oxide myoglobin was calculated as the ratio of reflectance of 650 and 570 nm, where a larger number indicates more nitric oxide myoglobin formation (King et al., 2023).

Both raw and cooked visual colors were evaluated for all steaks by a trained panel (n = 6) every 24 h. The Oklahoma State University Institutional Review Board (approval number: AG-18-34) approved the visual color analysis. Before participating, panelists (n = 6) passed the Farnsworth Munsell 100-hue test (King et al., 2023). Panelists determined muscle color (1 = extremely bright cherry-red, 2 = bright cherry-red, 3 = moderately bright cherry-red, 4 = slightly bright cherry-red, 5 = slightly dark red, 6 = moderately dark red, 7 = dark red), surface discoloration (1 = no discoloration [0%], 2 = minimal discoloration [1–10%], 3 = slight discoloration [11–20%], 4 = small discoloration [21–40%], 5 = modest discoloration [41–60%], 6 = moderate discoloration [61–80%], 7 = extensive discoloration [81–100%]), and muscle darkening (1 = no darkening, 3 = slightly dark, 5 = moderately dark, 7 = very dark) of steaks using 7-point scales throughout display.

Microbiology

The total plate count was determined on d 0 and d 6 of storage. A 10 g muscle sample was aseptically collected from the surface of each loin (d 0) or steak (d 6) and placed in a sterile stomacher bag (VWR; Radnor, PA) in addition to 90 mL of 0.1% sterile peptone water (BactoTM Peptone Ref 211677 Becton; Dickinson and Company; Sparks, MD). Stomacher bags were placed in a stomacher (Seward; STOMACHER^® 400 Circulator; 3500 model) and mixed for 30 s. One mL of the stomacher bag mixture was removed and serially diluted into 9 mL of 0.1% sterile peptone water. Each dilution was aseptically plated in duplicate by 1 mL of the solution added to 3M Petrifilm Aerobic Count plates (3M Health Care; St. Paul, MN). Plates were incubated for 48 h at 37°C in a VWR Forced Air General Incubator (0.15 m³; VWR, Radnor, PA). After 48 h, plates were removed from the incubator and enumerated to determine the total plate count based on colony forming unit per g.

Thiobarbituric acid reactive substances assay

Lipid oxidation was evaluated on d 0 and 6 of storage. A 3 g muscle sample was taken from the surface and blended with 27 mL of trichloroacetic acid for 10 s using a Waring commercial blender (Model 33BL7; New Hartford, CT) before being filtered with a Whatman 42 filter paper. In a test tube, 1 mL of filtrate was combined with 1 mL of thiobarbituric acid and briefly vortexed before being placed in a 100°C water bath for 10 min. Samples were cooled for 5 min before obtaining the absorbance at 532 nm using a spectrophotometer (UV-2600, UV-VIS Spectrophotometer; Shimadzu, Columbia, MD) on the photometric setting. A blank was created by mixing 1 mL of trichloroacetic with 1 mL of thiobarbituric acid. The raw absorbance value of samples was taken and multiplied by a factor of 4.16 (for 1:9 dilution of the protocol) to convert the value to mg malondialdehyde per kg meat (King et al., 2023).

Cooked color analysis

After 5 d of retail display, steaks were cooked to an internal temperature of 71°C on a George Foreman Grill (Lean Mean Fat Grilling Machine George Foreman; Lake Forest, IL GRP99A). Steaks were placed on ice for 5 min after cooking to prevent post-temperature rise. Instrumental cooked color measurements were taken with a HunterLab 4500L MiniScan Spectrophotometer. The external cooked color was taken in triplicate across the cooked surface, while the internal color was taken in triplicate on bisected steak pieces cut perpendicular to the cooked surface near the center of the steak.

Six trained panelists determined visual cooked color using 7-point scales for both external and internal color. Visual external cooked color (1 = brown, 2 = light brown, 3 = slightly brownish-red, 4 = reddish-brown, 5 = pinkish-brown, 6 = slightly pinkish-red, 7 = pinkish-red) was observed on the cooked surface of each steak, while visual internal cooked color (1 = very red, 2 = slightly red, 3 = pink, 4 = slightly pink, 5 = pinkish gray, 6 = grayish-tan/brown, 7 = tan/brown) was noted based on the appearance of the bisected steak pieces used to determine instrumental internal cooked color.

Quantification of nitrite and nitrate in beef

Both nitrite and nitrate samples were quantified using a NOx Analyzer (ENO-30; Fushimi-ku Kyoto, Japan) after 5 d of storage in low- and high-concentration nitrite PLA films. The meat samples were taken from the entire thickness of the steak. The samples were frozen in liquid nitrogen and pulverized using a Waring blender. Frozen samples were shipped in dry ice. Five grams of powdered sample was weighed and homogenized with 30 mL of Dulbecco’s Phosphate Buffer Saline (2.5 mM EDTA and 10 mM N-ethylmaleimide) for 1 min at 10,000 rpm. Following homogenization, the slurry was centrifuged at 10,000 × g at 4°C for 8 min. The supernatant (400 μL) was transferred into a 2 mL centrifuge tube, and 400 μL of methanol was added. The samples were vortexed at high speed for 3 to 5 s and allowed to sit at 4°C for 10 min. The tubes were centrifuged for 10 min at 13,000 × g at 4°C. A 100 μL of the supernatant was placed into a 96-well plate and analyzed using an Eno-30 analyzer (high-performance liquid chromatography specific for nitrite and nitrate). The ENO-30’s high sensitivity is attained by the combination of a diazo coupling technique with the extract to be measured and the separation of nitrite and then nitrate using a reverse-phase column. To separate nitrite and nitrate, the nitrate is first reduced to nitrite through a reaction with cadmium and reduced copper inside a reduction column. The two resolved peaks are then mixed with Griess reagent (dinitrogen trioxide, N₂O₃, generated from acidified nitrite that reacts with sulfanilamide) in-line to form the classical diazo compound, which can be detected spectrophotometrically. This system allows for easy sample preparation, little if any cross-reactivity, and high throughput when coupled with an auto-sampler. The system is adaptable to a wide range of nitrite and nitrate concentrations regardless of their respective ratios and operates at a sensitivity level of 1 nM × 100-μL injections for each anion with no interference from protein or other colored species. The results from raw steak samples were reported in parts per million (ppm).

Statistical analysis

The effects of active PLA films incorporating sodium nitrite and α-tocopherol on dark-cutting beef color were determined based on a randomized complete block design. The treatments applied to dark-cutting loins consisted of either a control treatment in PVC overwrap, a control treatment in vacuum packaging, or the low- or high-concentration active PLA films within vacuum packages. Each loin represented a block, and the 7 replicates were served by the 7 strip loins. Packaging treatment, retail display time, and their interactions were the fixed effects. The random effect was each loin, and day was a repeated measure observed in the study. The repeated measures covariance-variance structure was determined by evaluating the AICC output. The GLIMMIX procedure of SAS (SAS 9.4; SAS Inst., Cary, NC) was used to determine the least-squares means, and significance was considered at P < 0.05. The least-squares means were separated using the PDIFF option (least-squares difference) and were significant at P < 0.05. Normal-pH steak values were reported as averages, and the significant differences were established based on standard error.

Oxygen transmission rate

There was no effect (P > 0.05) of PLA film type on the oxygen transmission rate (Table 1). Testing was completed by layering PLA films with the commercially available vacuum package. Thus, the PLA film layer was expected to have a limited effect on the oxygen transmission rate since the commercial vacuum package film created the main barrier with the adjusted testing approach. Therefore, low- and high-concentration PLA films exhibited similar (P > 0.05) oxygen transmission rate values.

Film fransparency

There was no difference (P > 0.05) between low- and high-concentration PLA film transparency values when sampled alone and when layered with the vacuum package. However, there were differences (P < 0.05) between vacuum and PLA films (Table 2). While transparency values between the PLA film recipes were not different (P > 0.05), there was a visual color difference (Figure 1). Previous work indicated increased yellowness in films incorporating α-tocopherol (Byun et al., 2010; Hwang et al., 2012; Manzanarez-López et al., 2011). The current work showed that the high-concentration PLA film recipe exhibited a more yellow appearance than the low-concentration PLA films because of the greater α-tocopherol content (Figure 1). In addition, increased transparency was previously noted with the addition of sodium nitrite in films (Chatkitanan and Harnkarnsujarit, 2020). However, in this study, sodium nitrite content within the PLA films did not (P > 0.05) affect transparency (Table 2).

Figure 1.

Images of color differences between poly(lactic acid) (PLA) film recipes¹. ¹Low-concentration PLA films were compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol, and high-concentration PLA films were compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol. PLA films were placed directly on display surface of steaks before being vacuum packaged (Walton’s Vacuum Pouch; 10 × 10 pouches; 3 mil thickness).

Film thermal stability

There was no effect (P > 0.05) of PLA film type on onset temperature, decomposition temperature, or residual ash percentage (Table 3), suggesting similar thermal stability properties were observed between low- and high-concentration PLA films. Both film recipes also noted a single-phase degradation (Figure 2). Previous research has shown that the addition of α-tocopherol (at 2.2% and 4.4% concentrations) did not impact the thermal stability of PLA films (Gonçalves et al., 2011). Alternatively, Chatkitanan and Harnkarnsujarit (2020) reported sodium nitrite within thermoplastic starch films significantly decreased the thermal stability of films. Further testing of film recipes containing PLA and PLA in combination with each active ingredient individually will help to determine the impact sodium nitrite and α-tocopherol have on thermal stability.

Figure 2.

Thermogravimetric response¹ of low²- and high³-concentration poly(lactic acid) (PLA) films. ¹Weight (%) results are given as a continuous line and derivative weight (%/°C) is shown as a dashed line. ²Low-concentration PLA films were compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol. ³High-concentration PLA films were compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol.

pH and proximate composition

In the current work, dark-cutting loins had greater pH (P < 0.05) than normal-pH loins (Table 4). Previous research also noted that dark-cutting beef has greater pH values than normal-pH beef (Mitacek et al., 2018; Ramanathan et al., 2018; Sawyer et al., 2009; Wills et al., 2017). Limited glycogen content postmortem leads to less pH decline in dark-cutting beef, resulting in greater pH values than normal-pH beef (Lawrie, 1958; Scanga et al., 1998). Both loin types showed similar (P > 0.05) protein content percentages (Table 4). However, there was a significant loin-type effect on the moisture and fat content of loins. Dark-cutting loins exhibited greater (P < 0.05) moisture and lower (P < 0.05) fat percentages than normal-pH loins (Table 4). The high-pH of dark-cutting beef results in a lack of muscle fiber shrinkage and increases water holding capacity (Hughes et al., 2017), which parallels the results shown within this study. Furthermore, the fat percentage difference noted between normal-pH and dark-cutting loins can be attributed to the dark-cutting loins being selected primarily for their pH value rather than their marbling content.

Residual nitrite and nitrate content due to contact with nitrite-containing PLA film

High-concentration PLA transferred more nitrite and nitrate to meat than low-concentration PLA film (Table 5). Previous study also noted the migration of active ingredients, such as sorbic acid, from PLA film to the food matrix. However, migration of sorbic acid was greater when 95% ethanol was used than 10% ethanol (Rodríguez-Martínez et al., 2016).

Retail display color

A packaging type × day interaction (P < 0.05) was observed for L* values during the display (Figure 3). Normal-pH steaks in PVC showed greater L* values (P < 0.05) compared with all other treatments throughout the display duration. Previous research showed greater deoxymyoglobin formation occurred in dark-cutting beef than in normal-pH beef, resulting in decreased L* values (English et al., 2016; Hughes et al., 2017; McKeith et al., 2016).

Figure 3.

Least-squares means of external L* values (packaging type¹ × day) of steaks during a 6 d retail display. Least-squares means with different letters (a–m) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 1.10). The experiment was replicated seven times (n = 7). ¹Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

A packaging type × day interaction (P < 0.05) was observed for a* and chroma values during the display (Figures 4 and 5). Normal-pH steaks in PVC overwrap exhibited a redder (P < 0.05) appearance than all other treatments from d 0 to d 5 of the simulated retail display. Previous work also observed greater a* and chroma values from normal-pH steaks than dark-cutting steaks in the aerobic display (Apple et al., 2011; Mitacek et al., 2018; Ramanathan et al., 2018; Stackhouse et al., 2016; Wills et al., 2017). Moreover, greater pH in dark-cutting steaks decreases light reflectance and increases oxygen consumption compared to normal-pH steaks (Hughes et al., 2017; McKeith et al., 2016); hence, lower a* and chroma values are reported in dark-cutting steaks (Hunt et al., 1999; Reesman et al., 2023). Redness increased (a* and chroma values) in both low- and high-concentration PLA film treatments within the first 24 h of the display. In support, a previous study using patented nitrite-film also reported increased redness in dark-cutting beef (Denzer et al., 2020, 2022). In addition, Ramanathan et al. (2018) noted that the addition of an antioxidant (rosemary) to nitrite film exhibited greater color stability through limiting oxidation of nitric oxide myoglobin in dark-cutting beef. Low- and high-concentration PLA film steaks possessed significantly greater a* values than dark-cutting steaks in vacuum packaging alone after d 2 of the display.

Figure 4.

Least-squares means of external a* values (packaging type¹ × day) of steaks during a 6-d retail display. Least-squares means with different letters (a–r) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 0.81). The experiment was replicated seven times (n = 7). ¹Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Figure 5.

Least-squares means of external chroma (packaging type¹ × day) of steaks during a 6-d retail display. Least-squares means with different letters (a–n) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 1.04). The experiment was replicated seven times (n = 7). ¹Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

There was a packaging type × day interaction (P < 0.05) for nitric oxide myoglobin formation (Table 6). Low- and high-concentration PLA film treatments showed significant increases in nitric oxide myoglobin formation throughout the display. Moreover, following d 1, high-concentration PLA film steaks consistently presented significantly greater (P < 0.05) levels of nitric oxide myoglobin formation than steaks packaged in low-concentration PLA films throughout the entire display period.

There was a packaging type × day interaction (P < 0.05) for muscle color (Figure 6), surface discoloration (Figure 7), and muscle darkening (Figures 8 and 9). A significantly brighter cherry-red appearance (P < 0.05) was noted for normal-pH steaks in PVC than all other treatments up to d 4 of retail display. Additionally, high-concentration film steaks exhibited brighter cherry red (P < 0.05) muscle color than dark-cutting steaks in PVC and vacuum packaging without nitrite. No discoloration was noted for all steaks in vacuum packaging during retail display, including low- and high-concentration PLA film steaks. However, both normal-pH and dark-cutting steaks in PVC exhibited discoloration during display. Normal-pH steaks in PVC demonstrated a more significant discoloration than dark-cutting steaks in PVC from d 4 to d 6. Moreover, high-concentration PLA film steaks showed decreased (P < 0.05) muscle darkness throughout the display than dark-cutting steaks packaged in PVC and vacuum package.

Figure 6.

Least-squares means of muscle color¹ evaluated by a trained panel (packaging type² × day) of steaks during a 6-d retail display. Least-squares means with different letters (a–t) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 0.22). The experiment was replicated seven times (n = 7). ¹Muscle color evaluated using a 7-point scale (1 = extremely bright cherry-red, 2 = bright cherry-red, 3 = moderately bright cherry-red, 4 = slightly bright cherry-red, 5 = slightly dark red, 6 = moderately dark red, 7 = dark red). ²Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Figure 7.

Least-squares means of surface discoloration¹ evaluated by a trained panel (packaging type² × day) of steaks during a 6-d retail display. Least-squares means with different letters (a–h) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 0.56). The experiment was replicated seven times (n = 7). ¹Surface discoloration evaluated using a 7-point scale (1 = no discoloration [0%], 2 = minimal discoloration [1–10%], 3 = slight discoloration [11–20%], 4 = small discoloration [21–40%], 5 = modest discoloration [41–60%], 6 = moderate discoloration [61–80%], 7 = extensive discoloration [81–100%]). ²Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Figure 8.

Least-squares means of muscle darkening¹ evaluated by a trained panel (packaging type² × day) of steaks during a 6-d retail display. Least-squares means with different letters (a–r) are significantly different (P < 0.05). Standard error of the mean indicated by error bars (SEM = 0.30). The experiment was replicated seven times (n = 7). ¹Muscle darkening evaluated using a 7-point scale (1 = no darkening, 3 = slightly dark, 5 = moderately dark, 7 = very dark). ²Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Figure 9.

Visual effect of packaging treatments¹ on steaks at the beginning and end of retail display. ¹Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Roberts et al. (2017) observed that bison steaks in nitrite-embedded packaging increased redness and experienced less discoloration than control packaged steaks. Furthermore, ribeye and round steaks sprayed with a nitrite solution showed improved red color (Song et al., 2015). Previous research also noted darker muscle color and less discoloration in dark-cutting steaks than normal-pH steaks during retail display (Apple et al., 2011; Mitacek et al., 2018; Sawyer et al., 2009; Stackhouse et al., 2016; Wills et al., 2017). Initial darkness of dark-cutting steaks in low-and high-concentration film packages can be caused by metmyoglobin formation during the interconversion to nitric oxide myoglobin (Fox Jr. and Ackerman, 1968; Siegel, 2011). The decreased muscle darkening observed by high-concentration PLA film steaks in the current work is due to the formation of bright red nitric oxide myoglobin (Ramanathan et al., 2018; Denzer et al., 2022).

Total plate counts and lipid oxidation

There were no differences (P > 0.05) in total plate count between normal-pH and dark-cutting loins on d 0 (Table 7). However, there was a significant packaging type effect for d 6 microbial growth analysis as dark-cutting steaks in PVC exhibited greater (P < 0.05) microbial growth than all other treatments (Table 7). Previous research reported a 1-log reduction for aerobic plate counts with nitrite-embedded packaging (Narváez-Bravo et al., 2017; Ramanathan et al., 2018). However, there was no difference (P > 0.05) in aerobic plate count between nitrite-embedded and control vacuum treatments. Thus, the differences in microbial growth in the current work may be attributed to the anaerobic environment rather than the active ingredients. Therefore, evaluating anaerobic plate counts for future studies may be beneficial to better understand the impact of nitrite and alpha-tocopherol addition within PLA films on total plate counts.

In addition, there was no difference (P > 0.05) between normal-pH and dark-cutting loin steaks for lipid oxidation on d 0 (Table 7). Nonetheless, a packaging type effect (P < 0.05) was noted on d 6, with normal-pH steaks in PVC overwrap possessing greater (P < 0.05) lipid oxidation values than all other treatments. Dark-cutting beef has lower lipid oxidation values than normal-pH beef (English et al., 2016; Wills et al., 2017). The addition of sodium nitrite in previous research reduced lipid oxidation compared to their respective control treatment (Roberts et al., 2017; Chatkitanan and Harnkarnsujarit, 2020). However, Denzer et al. (2022) observed that greater pH of dark-cutting beef minimizes packaging impact (PVC vs. vacuum) on lipid oxidation.

Cooked color

Cooked color is primarily due to myoglobin denaturation and Maillard reaction (Hunt et al., 1999; Trevisan et al., 2016). Denaturation of myoglobin leads to either red ferro- or brown ferrihemochorme. The pH of meat influences the denaturation of myoglobin. Deoxymyoglobin, carboxy myoglobin, and nitric oxide myoglobin are resistant to denaturation, while metmyoglobin and oxymyoglobin are very susceptible to heat (Denzer et al., 2020; Djimsa et al., 2017). Greater heat stability was noted with deoxymyoglobin at greater pH levels than metmyoglobin when heated to 70°C (Hunt et al., 1999). There was a packaging type effect (P < 0.05) on both external (Table 8) and internal (Table 9) cooked color a* values. Greater (P < 0.05) external cooked color redness (a* values) was exhibited by high-concentration PLA film steaks compared to all other treatments. In addition, high-concentration PLA film steaks possessed significantly greater (P < 0.05) internal cooked color a* values than normal-pH and dark-cutter steaks in PVC and dark-cutter steaks in vacuum packaging. Previous research also observed increases in a* values and redness for steaks packaged within nitrite packaging (Claus and Du, 2013; Song et al., 2015). Song et al. (2015) observed an increase in nitrosylhemochrome on the surface of cooked steaks that had previously been packaged in nitrite packaging. Nitrosylhemochrome is the distinctive pink pigment given by cured meats (MacDougall et al., 1975). Therefore, the higher cooked color redness of the high-concentration PLA film packaged steaks can be attributed to the greater formation of nitrosylhemochrome on the steak surface. There was no difference (P > 0.05) in external and internal cooked color redness between low-concentration PLA film steaks and dark-cutting steaks in vacuum packaging. Greater cooked color redness and cooked color a* values are noted with dark-cutting steaks compared to normal-pH steaks (Sawyer et al., 2009). Furthermore, there was a packaging type effect (P < 0.05) on both external (Table 8) and internal (Table 9) cooked color chroma values. High-concentration PLA film steaks exhibited greater (P < 0.05) external cooked color chroma values than all other treatments. Additionally, low-concentration PLA film and dark-cutting steaks in vacuum packages showed similar (P > 0.05) external and internal cooked color chroma values.

A packaging type effect (P < 0.05) was observed for external (Table 8) and internal (Table 9) cooked color hue angle values. Smaller (P < 0.05) external and internal cooked color hue angle values were expressed by high-concentration PLA film steaks compared to all other treatments. Furthermore, low-concentration PLA film steaks and dark-cutting steaks in vacuum packaging demonstrated similar (P > 0.05) external and internal cooked color hue angles.

There was a packaging type effect (P < 0.05) on the external cooked color panel evaluation (Table 8 and Figure 10). The trained panel determined the high-concentration PLA film steaks exhibited a redder pink (P < 0.05) external cooked color compared to all other treatments. Alternatively, panelists observed a similar (P > 0.05) external cooked color appearance between low-concentration PLA film steaks and dark-cutting steaks in vacuum packaging. Additionally, there was no significant packaging type effect on internal cooked color panel evaluation scores (Table 9). In the current research, dark-cutting cooked steaks had numerically greater redness than normal-pH cooked steaks. However, visual panelists were not able to observe visual differences in redness. A packaging type effect (P < 0.05) was shown for external cooked color L* values (Table 8). However, there was no packaging type effect (P > 0.05) on internal cooked color L* values (Table 9).

Figure 10.

Visual effect of packaging treatments¹ on steaks cooked to 71°C on a George Foreman grill after 5 d retail display. ¹Packaging treatments included normal-pH steaks in polyvinyl chloride (PVC) overwrap, dark-cutter steaks in PVC overwrap, dark-cutter steaks in vacuum package, dark-cutter steaks with low-concentration poly(lactic acid) (PLA) film (compounded with PLA, 0.12% sodium nitrite, and 0.5% α-tocopherol) in a vacuum package, and dark-cutter steaks with high-concentration PLA film (compounded with PLA, 0.6% sodium nitrite, and 2.5% α-tocopherol) in a vacuum package.

Although raw color was improved with low-concentration nitrite film, cooked color was not comparable to normal-pH steaks packaged in PVC. Hence, future studies will aim to decrease nitrite levels within packaging film and to decrease heat stability by lowering muscle pH with organic acid enhancement. In addition, including an oxygen-impermeable layer to PLA films will eliminate the need for vacuum packaging to create anaerobic conditions.