Effect of Aging and Freezing Conditions on Meat Quality and Storage Stability of 1⁺⁺ Grade Hanwoo Steer Beef: Implications for Shelf Life

Fresh 1⁺⁺ grade loin, striploin, tenderloin and top round muscles from 10 Hanwoo steers were purchased on the day after slaughter, from the Nonghyup Pyeongchang branch of Kangwondo. The meat was vacuum-packaged and aged for 0, 7, and 14 d at 2°C, and continuously stored for 0, 3, 6, or 9 mon at −18°C. For composition and quality analysis, samples were collected on each day of storage and analyzed.

Protein, fat, moisture, and collagen contents were analyzed using the Food Scan^TM Lab 78810 (Foss Tecator Co., Ltd., Denmark), according to the methods of the Association of Official Analytical Chemists (AOAC, 2016).

Color values were determined using a CR-400 Chroma Meter (Konica Minolta Sensing, Inc., Japan) on a freshly cut surface. The standard for lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) was calibrated after a 30-min blooming period at 2°C (Commission Internationale de I’Eclairage, 1986). WHC was determined by a centrifugation-based method (Kristensen and Purslow, 2001). The homogenized tissue (0.5 g) was placed in a 2-mL centrifugal filter unit with 5 μm of microporous mem brane (Biofil^®, Millipore, Japan), and then placed in a 50-mL centrifugation tube, heated in a 70°C water bath for 30 min, and centrifuged at 100 × g (Hitachi, SCR20BA, Japan) for 10 min at room temperature (ca. 18°C). WHC was expressed as the percentage of weight loss in sample tissue during centrifugation. For determination of Warner-Bratzler shear force (WBSF), the meat blocks (50 × 50 × 25 mm) were cooked in a pre-heated water bath for 40 min until the core temperature reached 80°C, and then cooled under running water (ca. 18°C) for 30 min to achieve a core temperature below 30°C. Eight cores of 1.27-cm diameter were prepared for each sample, and peak force was determined using a V-shaped shear blade of an Instron Universal Testing Machine (Model 5543, USA) with a cross-head speed of 400 mm/min (Wheeler et al., 2000). Cooking loss (CL, %) was calculated as the percentage of weight change during cooking for WBSF measurement. The samples were freshly cut into blocks and weighed (initial weight). Individual cooked meat block samples were removed from the water bath, cooled in cold water, and weighed. CL was then expressed as the percentage of the initial sample weight (Honikel, 1998).

TBARS content was measured according to the method described by Sinnhuber and Yu (1977). The beef samples (0.5 g) were mixed with ca. 0.1 g of antioxidant mixture [54% (w/w) propylene glycol-40% (w/w) Tween 20-3% (w/w) butylated hydroxytoluene-3% (w/w) butylated hydroxyanisole], 3 mL of 1% (w/v) TBA-0.3% (w/v) NaOH, and 17 mL of 2.5% (w/v) TCA-36 mM HCl, boiled at 100°C for 30 min, and ice-cooled for 10 min. Then, 5 mL of the upper solution was transferred to a conical tube and 3 mL of chloroform was added before centrifugation at 4°C, 3000 × g (Avanti J-E Centrifuge, Beckman Coulter, Inc., CA) for 30 min. Absorbance values of supernatants were measured at 532 nm and converted into mg malonaldehyde (MA)/kg meat.

VBN content was analyzed according to the Korea Food Standards Codex (2017) of MFDS. The sample (10 g) was homogenized in 20 mL of deionized water (DW), using a Polytron device (PT-MR 2100, Kinematica AG, Switzerland) with the speed set at 10,000 rpm, for 1 min. The suspension volume was brought up to 50 mL with DW, and then filtered through Whatman filter paper number 1. Borate buffer [1 mL, 1% (w/v] containing 6.6 ppm of methyl red and 3.3 ppm of bromocresol green was placed in an inner section of a Conway dish, and the filtrate was then transferred to the outer section of the same dish. Saturated K₂CO₃ (1 mL) was poured into the outer section and the dish was incubated at 37°C for 120 min. The green-colored solution in the inner section was then titrated with 0.01 N H₂SO₄. VBN content was calculated as mg% of ammonia, using the following formula: 0.14 × titration volume of sample (mL) × factor of 0.01 N H₂SO₄ × dilution factor × [100/sample weight (g)].

Each animal within the same slaughtering age group was treated as a replicate. Data were analyzed by the Student-Newman-Keuls’ multiple comparison test, using the GLM procedure of the SAS program (2015). The significance level was set at p<0.05.

The protein, moisture, fat, and collagen contents of the four Hanwoo steer beef muscles are shown in Table 1. The protein content (%) in the loin, striploin, tenderloin, and top round muscles was between 17.72 and 19.26%, with no significant difference between the four muscles (p>0.05). The moisture contents of the tenderloin (63.41%) and top round (62.96%) muscles were significantly higher than that of striploin muscle (57.27%) (p<0.05). Intramuscular fat (IMF) content was highest in the striploin muscle (22.64%), and lowest in the top round muscle (12.42%) (p<0.05). The collagen contents of the loin (2.04%) and striploin (2.01%) muscles were higher than that in the top round muscle (1.66%) (p<0.05) (Table 1).

Canto et al. (2016) showed that muscle source did not affect the protein or ash content. Similarly, Mitterer-Deltoe et al. (2012) reported that the protein content of beef muscles generally exhibits low variability. Hwang et al. (2010) demonstrated that the moisture content of Hanwoo steer muscle was inversely related to lipid content. Several groups have reported that IMF is important because of its positive effect on eating quality traits such as tenderness, juiciness, and flavor (Hocquette et al., 2010). Jeremiah et al. (2003a) also suggested that fat and moisture contents are correlated with the juiciness of beef. IMF deposited between the fasciculi disrupts the honeycomb structure of the endomysium and separates the perimysial fibers, thereby increasing tenderness. To ensure consumer acceptability, a minimum level of 5% IMF in grilled cuts of red meat, such as beef and lamb, was recommended by Hopkins et al. (2006).

Collagen content affects beef tenderness. In the study by insoluble collagen was more closely associated with toughness in palatability traits than total or soluble collagen when bovine muscle is cooked (Dutson, 1974; Herring et al., 1967; Jeremiah et al. 2003b). Collagen with few intermolecular cross-likages is converted to gelatin, which improves tenderness, but when more intermolecular cross-links are present, moisture is squeezed out of the muscle, decreasing tenderness (Ledward, 1984). In this study, the insoluble collagen levels of the tenderloin, striploin and loin in this study might be relatively lower than top round muscle, although their total collagen levels in raw meat are higher than that of top round muscles. In addition, the top round has been reported to contain heavy deposits of coarse elastin fibers and they also contribute to meat toughness to about the same extent as denatured collagen (Bendall, 1967).

Most consumers prefer high-quality meat for sensory satisfaction. The quality of raw meat is generally evaluated by assessing the color, WHC, texture, and palatability (Harris et al., 2012; Muhlisin et al., 2014). The meat color (CIE L*, a*, b*) of the loin, striploin, tenderloin, and top round muscles did not significantly change during the aging period; the lightness (CIE L*) values of the tenderloin muscles decreased as the freezer storage period increased (p<0.05) (Table 2). However, most muscles showed no significant changes in redness (CIE a*) and yellowness (CIE b*) values during aging and freezer storage. Color is the most critical quality trait considered during the purchase of fresh beef (Suman et al., 2014). The bright cherry-red color that is attractive to a consumer has been often associated with freshness of meat, whereas brownish discoloration is perceived as indicative of spoilage, leading to product rejection (Suman et al., 2014). Several studies demonstrate that beef color stability is a highly muscle-specific trait (Jeong et al., 2009; Joseph et al., 2012). Joseph et al. (2012) reported that psoas major muscle is a color-labile beef muscle, whereas longissimus thoracis et lumborum muscle is color-stable. These two muscles differ with respect to surface color, metmyoglobin reducing activity, oxygen consumption rate, and lipid oxidation.

WHC, CL, and WBSF of the loin, striploin, tenderloin, and top round muscles during aging and freezer storage are compared in Table 3. The WHC values were increased when four muscle were aged for 14 d in tenderloin, striploin and top round muscles at 3 mon of freezer storage and loin muscle at 3 and 6 mon of freezer storage (p<0.05). However, the WHC values of the four muscles from each aging day did not significantly differ after the 0 to 9 mon of freezer storage.

CL (%) values after 14 d of aging and 9 mon of freezer storage were as follows: 22.68-28.42% for the loin, 20.49-27.56% for the striploin, 24.99-30.49% for the tenderloin, and 23.32-30.20% for the top round muscle. In the four muscles, these values were significantly increased as the freezer storage period increased (p<0.05); however, they were not significantly different after 0, 7, or 14 d of aging (p>0.05). Only in the case of the striploin muscle, CL values were higher after 6 mon freezer storage with 7 and 14 d of aging than with 0 d of aging (p<0.05).

With the exception of the tenderloin, the WBSF values of the other muscles in this study decreased significantly as the aging period increased (p<0.05). The WBSF value of the tenderloin muscle changed very little (2.21-2.60 kg) during the 14 d aging period. There were no significant differences in WBSF values during the 9-mon freezer-storage period after 0, 7, and 14 d of aging.

Tenderness is one of the most important quality attributes of beef (Devitt et al., 2002). Fernandez et al. (1999) reported that the high IMF levels are favorable effect on tenderness. Wheeler et al. (2005) determined that a significant negative correlation exists between IMF vs. WBSF, and positive correlation exists between IMF vs. tenderness, juiciness, and flavor scores, respectively. In addition, Rhee et al. (2004) reported that protein patterns and degradation rates of structural proteins such as titin, nebulin, and filamin, vary during refrigerated storage for 28 d. Fast degradation of these myosin family proteins is a key factor for the improvement of meat tenderness.

Freezer storage has many benefits; it inhibits oxidation, delays bacterial replication, retains meat flavor, and is particularly useful for long-distance transport (Lawrie and Ledward, 2006). However, ice crystals formed by freezing lead to desiccation of cell membrane proteins in animal muscles (Love, 1966). This increases the effluence of water-soluble nutritional components and textural deterioration, and decreases WHC (Cho et al., 2014; Lagerstedt et al., 2008). In other words, freezing causes decline of the quality of meat. Kim et al. (2011) reported that quality defects in frozen/thawed meat may be minimized by aging meat prior to freezing. Several studies have supported the notion that employing aging prior to freezing results in meat quality attributes, such as tenderness, WHC, drip loss, and/or color stability, that are better than, or equivalent to, those of aged-only beef and lamb meat (Choe et al., 2016; Kim et al., 2015; Wiklund et al., 2009). However, in this study, CL (%) increased as the freezer-storage period increased. According to Rhee et al. (2004), CL of longissimus dorsi (LD) is lower than that of semitendinosus (ST). These results were, in general, consistent with those of the current study.

The changes in VBN and TBARS values significantly increased with the increasing duration of aging and freezer storage (p<0.05) (Fig. 1). The VBN values for the four muscles were between 16.67 and 18.49 mg% after 14 d of aging and 9 mon of freezer storage. These values were close to the limit for packaged meat suggested by MFDS (2014), i.e., VBN less than 20 mg%. The TBARS values of the four muscles were between 0.75 and 0.82 mg MA/ kg meat after 14 d aging and 9 mon freezer storage (Fig. 2). When defining the shelf life of QG 1⁺⁺ Hanwoo beef, the distance and facility of distribution for export to China and Hong Kong should be considered.

Fig. 1. Changes of volatile basic nitrogen (VBN, mg%) values for 1⁺⁺ grade Hanwoo beef during aging at 2°C for 14 d and freezer storage at −18°C for 9 mon.

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Fig. 2. Changes of thiobarbituic acid reactive substances (TBARS, mg malonaldehyde/kg) values for 1⁺⁺ grade Hanwoo beef during aging at 2°C for 14 d and freezer storage at −18°C for 9 mon.

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Lipid oxidation in stored meat, which is associated with undesirable off-flavor, toxic substances, and discoloration, is a principal contributing factor to the decline in meat freshness and quality (Gray et al., 1996). Protein oxidation is associated with protein carbonylation, fragmentation, or polymerization (Xiong, 2000). This process decreases WHC and sensory qualities, e.g. juiciness and tenderness, of meat. Lipid oxidation-induced myoglobin oxidation leads to accumulation of metmyoglobin and subsequent surface discoloration (Faustman et al., 2010). Insani et al. (2008) reported that beef discoloration is accelerated by the oxidation of lipids and proteins. Furthermore, several other studies have demonstrated that lipid oxidation and protein oxidation compromise the color stability of fresh meat (Faustman et al., 2010; Lund et al., 2011). In postmortem oxidative muscle, mitochondrial metabolism surpasses the myoglobin’s ability to bind oxygen, leading to accelerated metmyoglobin formation and surface discoloration (King et al., 2010). The myoglobin content (Hwang et al., 2010; King et al., 2011) and mitochondrial concentration are greater in oxidative muscles, such as the tenderloin (PM), than in glycolytic muscles such as the striploin (LL). PM contains a greater proportion of fiber type I than II (Hwang et al., 2010), which is positively correlated with myoglobin content. Furthermore, an increased level of myoglobin in the muscle is reportedly associated with low color stability (King et al., 2010). In general, color-stable muscles, such as LL, exhibited lower TBARS than their color-labile counterparts, such as PM, during 5 d of display. In contrast, Jeong et al. (2009) reported that there was no difference in TBARS between LL and PM muscles from Hanwoo bulls during 7 d storage. The results of the current study are similar to the findings of Jeong et al. (2009).