Quality Improvement of Pork Loin by Dry Aging

Five pork carcasses (ten half carcasses) were purchased from a large commercial processor on the day of slaughter, and transported to SD Food Co. (Korea). Pork loins (Longissimus lumborum muscles: 6^th dorsal vertebra to 6^th lumbar vertebra) were dissected from the right half of five pork carcasses, and used as control sample. The left half of the pork carcasses were suspended by the Achilles tendon in aging room and aged at 2±1℃ and a relative humidity of 80% for 40 d. After the completion of dry aging, pork loins were dissected from the dry aged carcasses with the removal of dried surface, and used as dry aged samples. The muscle samples were individually vacuumpackaged in 50×30 cm low-density polyethylene/nylon vacuum bags, and transported to the Chungnam National University meat laboratory in insulated icebox with reusable ice packs on the day of dissection.

Each pork loin of control and dry-aged sample was divided into dorsal vertebra and lumbar vertebra parts. The dorsal vertebra parts were individually vacuum-packaged in a low-density polyethylene/nylon vacuum bags (50×30 cm) and stored at −70℃ until analysis of sensory parameters. The lumbar vertebra part was cut into four sections. Three of the four sections were used as three repetition samples (observations) of each carcass. Microbial count, proximate composition, pH, cooking loss, and texture of pork loin were conducted with fresh meat (unfrozen meat) on the day of dissection. The meat samples for analysis of free amino acid content, carnosine and anserine content, and nucleotides were weiged in conical tube on the day of dissection and stored at −70℃ until analysis.

Meat samples (10 g) were blended with sterile saline (90 mL) for 2 min by using a stomacher (BagMixer^® 400; Interscience Ind., France). A series of decimal dilutions was prepared using sterile saline. Each diluent (0.1 mL) was spread on tryptic soy agar plates (Difco Laboratories, USA) for count of total aerobic bacteria and MRS agar plates (Difco Laboratories, USA) for count of lactic-acid bacteria. The plates were incubated at 37℃ for 48 h, and the microbial counts were expressed as log colony-forming units/g (Log CFU/g).

The moisture, crude protein, crude fat and crude ash contents of each sample were determined as outlined by AOAC (2005). Briefly, the moisture content was measured by drying the samples (2 g) at 102℃ for 15 h. The crude protein content was analyzed by the Kjeldahl method (VAPO45, Gerhardt Ltd., Germany). The crude fat content was determined using the Soxhlet extraction system (TT 12/A, Gerhardt Ltd., Germany). The crude ash content was measured by igniting the sample (2 g) in a furnace at 600℃ for 6 h.

Control and dry-aged pork loins (5 g) were mixed with 45 mL of distilled water and homogenized at 1,130 g for 1 min using a homogenizer (T25 basic, IKA GmbH & Co. KG, Germany). The homogenate was filtered using filter paper (No. 4 filter paper, Whatman) and the filtrate was measured pH using a pH meter (SevenEasy, Mettler-Toledo, Korea).

About 30 g of pork loins were individually vacuum-packaged in a low-density polyethylene/nylon vacuum bags (20×15 cm). The packaged pork loins were cooked in a 75℃ water bath for 20 min and then cooled in tab water for 10 min. Cooking loss of pork loins was examined by comparing weight of before and after cooking.

The meat sample (1.0 g) was homogenized in 10 mL of 5% TCA at 1,130 g for 1 min using a homogenizer (T25 basic). The homogenate was kept at 4℃ for 1 h for precipitation of protein. After centrifugation at 10,000 g for 10 min at 4℃ (HM-150IV, Hanil), the supernatant was filtered through a 0.2 μm PTFE syringe filter (Whatman, England) and the filtrate was used for the following experiments. The extraction and derivatization of amino acids were conducted by using AccQ-Tag Reagent kit (AccQ-Tag Reagent kit, Waters, USA). The 1 mL of AccQ-Fluor Reagent Diluent was transferred to a vial containing AccQ-Fluor Reagent powder. This closed vial was mixed by vortexing for 10 s and heated on a water bath at 55℃ until is dissolved (reconstituted AccQ-Fluor reagent). The 10 mL of filtered sample was mixed with 70 mL of AccQ-Fluor borate buffer, and then the mixed solution was vortexed briefly. The 20 mL of reconstituted AccQ-Fluor reagent added to the mixed solution and then mixed by vortexing for several seconds. This mixed solution was heated on a water bath at 55℃ for 10 min (derivatives). The derivatives were analyzed using HPLC (Ultimate 3000RS, Thermo Fisher Scientific Inc., USA). With regard to the analytical conditions for HPLC, a Waters-AccQ-Tag Ultra C18 column (2.1×100 mm, 1.7 μm particles, Waters Co., USA) was utilized. Mobile phase consisted of solvent A (10% AccQ-Tag Eluent A concentrate) and solvent B (100% AccQ-Tag Eluent B concentrate) with gradient mode; 0-0.54 min: 99.9% solvent A and 0.1% solvent B, 0.54-5.74 min: 90.9% solvent A and 9.1% solvent B, 5.74-7.74 min: 78.8% solvent A and 21.2% solvent B, 7.74-9.04 min: 40.4% solvent A and 59.6% solvent B, 9.04-10.0 min; 10% solvent A and 90% solvent B, 10.0-13.0 min: 99.9% solvent A and 0.1% solvent B. The flow rate of the mobile phase was 0.7 mL/min and the injection volume was 10 μL. The column temperature was maintained at 35℃ and the detection was monitored at a wavelength of 260 nm. The peaks of the individual amino acid were identified using the retention times for standards: Amino acid standard, L-glutamin, L-asparaging, L-tryptophan, and GABA (Sigma), and the concentration was calculated using the area for each peak.

The contents of carnosine and anserine were determined using the method described by Mora et al. (2007). Meat sample (2.5 g) was homogenized with 7.5 mL of 0.01 N HCl at 1,130 g for 1 min (T25b). The homogenate was centrifuged at 17,030 g for 15 min (HM-150IV, Hanil Co., Ltd., Korea), and the supernatant (250 mL) was mixed with 750 mL acetonitrile. After storage at 4℃ for 20 min and centrifugation at 17,030 g for 10 min (HM-150IV, Hanil), the supernatant was filtered through a 0.2 μm PVDF syringe filter (Whatman) and injected into a highpressure liquid chromatography (HPLC) column equipped with a Waters 1525 pump and a Waters 717 plus autosampler (Millipore Co-Operative, USA). An Atlantis HILIC silica column (4.6×150 mm, 3 μm, Waters) was used. A diode array detector (Waters 2487, Millipore Co-Operative, USA) was used at 214 nm to measure creatine, carnosine, and anserine contents. The mobile phase A contained 0.65 mM ammonium acetate in water/acetonitrile (25:75, v/v, pH 5.5) and the B contained 4.55 mM ammonium acetate in water/acetonitrile (70:30, v/v, pH 5.5). The mobile phase B was supplied at 1.2 mL/min for 16 min with a linear gradient (0% to 100%). The content of compounds was calculated by using standard curve of each compound. Standards (carnosine and anserine) were obtained from Sigma-Aldrich (USA).

The meat samples (5 g) were mixed with 25 mL of 0.7 M perchloric acid and centrifuged for 1 min at 1,130 g to extract nucleic acids. The extracted nucleic acids were then centrifuged for 15 min at 2,090 g and filtered through a Whatman No.4 filter paper (Whatman). The supernatant was then adjusted to pH 7 with 5 N KOH. The pH-adjusted supernatant was placed in a volumetric flask and made up to a volume of 100 mL with 0.7 M perchloric acid (pH 7). After 30 min of cooling, it was centrifuged at 1,130 g (4℃) and the supernatant was filtered through a 0.2 μm PVDF syringe filter (Whatman). The filtrate (5 mL) was analyzed using HPLC (ACME 9000, Younglin Instruments Inc., Korea). With regard to the analytical conditions for HPLC, a Waters-Atlantis dC18 RP column (4.6×250 mm, 5 μm particles, Waters Co., USA) was utilized, with a mobile phase of 0.1 M trimethylamine in 0.15 M acetonitrile (pH 7.0). The flow rate of the mobile phase was 1.0 mL/min and the injection volume was 10 μL. The column temperature was maintained at 35℃ and the detection was monitored at a wavelength of 260 nm. The peaks of the individual nucleotides were identified using the retention times for standards: hypoxanthine, inosine, inosine- 5'-phosphate (IMP), guanosine-5'-phosphate (GMP) (Sigma), and the concentration was calculated using the area for each peak.

Pork loins were individually vacuum-packaged in a low-density polyethylene/nylon vacuum bags (20×15 cm). The packaged pork loins were cooked in a 75℃ water bath for 20 min and then cooled in tab water for 10 min.

The analysis of texture properties of cooked pork loin were conducted by two bite system using a Texture Analyzer (Model A-XT2, Stable Micro Systems Ltd., UK) with a 70 mm compression probe attachment. The cooked pork loin (2×2×1.5 cm) underwent two cycles of 70% compression with a test speed of 2 mm/s. Texture characteristics of cooked pork loin were expressed as hardness, springiness, cohesiveness, chewiness, gumminess, and resilience.

Shear force of cooked pork loin was determined using a Texture Analyzer (Model TA-XT2) with a Warner-Bratzler-blade attachment. Cooked pork loin was cut into 1 cm of diameter with 3 cm of length. Cross-sections of pork lone were placed at middle to the blade and sheared perpendicularly to the muscle fiber orientation. Crosshead speed was 2 mm/s and full-scale load was 50 kg.

Sensory evaluation of normal and dry aged meats were implemented using a group consisting of thirty two panels who have experience in sensory test of meat and meat product. Each meat sample was cooked at 180℃ for 3 min using electric steam oven (EON-C305CSM, Tongyang Magic Co., Korea) and cut regularly. The scoring of each sample was done on a single sheet using a 9-point hedonic scale (1 = extremely dislike, 9 = extremely like). The sensory parameters scored were color, flavor, taste, texture, and acceptability.

Data were analyzed using the PROC GLM procedure of SAS software (version 9.3, SAS Institute Inc., USA) in a complete randomized design. The experimental unit was the Longissimus lumborum muscle from half carcass. The statistical model for parameters of meat quality included the effect of dry aging as a main effect, and carcasses and panelists for sensory analysis as covariates. Specific comparisons were performed by Tukey’s multiple range test when the main effect was significant. Results are reported as least square mean values and standard error of the least square means (SEM). Statistical significance was considered at p<0.05.

The total number of aerobic bacteria and lactic-acid bacteria in each pork loin is shown in Table 1. There was no significant difference in total aerobic bacterial count between control and dry aged pork loin (p>0.05). The dried surface of dry-aged meats along with low temperature in aging room retard the proliferation of aerobic bacteria (Campbell et al., 2001); dry aging did not affect total aerobic bacteria in pork loin. Lactic-acid bacteria were not detected in both the controls and the dry-aged pork loins.

The proximate composition of control and dry-aged pork loin is presented in Table 2. The moisture content of dry-aged pork loin was significantly lower than that of the control (p<0.05). Campbell et al. (2001) reported that the moisture of meat evaporated after dry aging because of exposure to air. The dry-aged pork loin had significantly high protein and ash content compared to that of the control, because of the relatively low moisture content (p<0.05). However, there was no significant difference in fat content between the dry-aged pork loin and the control. These results were in accordance with those of a previous study (Juárez et al., 2011; Lee et al., 2015). Juárez et al. (2011) reported that dry aged pork loin for 14 d resulted in the decrease of moisture content, increase of protein content, and no difference in fat content. Lee et al. (2015) also found that dry aging for 21 d results in decrease of moisture content and increase in protein and ash contents in strip loin of beef.

The pH and cooking loss of pork loin were measured (Table 2). The pH of dry-aged pork loin was higher than that of the control (p<0.05). Generally, pH of meat increases after storage because of protein degradation caused by proteinase released from microorganisms with increased proliferation during storage period (Agunbiade et al., 2010). However, in the present study, the number of total aerobic bacteria of pork loin was not changed after dry aging. Therefore, it is considered that the increase of pH in dry-aged pork loin may be due to the increase of basic free amino acids that were generated during the degradation of proteins by endogenous proteinases in the meat. Previous studies reported that the pH of beef strip loin was increased with dry ageing period (Ahnström et al., 2006; Lee et al., 2015).

Dry-aged pork loin had significantly low cooking loss compared to the control (p<0.05). The low water content in the dry-aged pork loin compared with that of control might be one of reasons for low cooking loss of dry-aged pork loin. The higher pH of the dry-aged pork loin than control was found in the present study. Lee et al. (2015) reported that dry-aged strip loin of beef had low cooking loss with high pH compared to that of the unaged strip loin. Once the pH decreases to the isoelectric point of myofibrillar proteins, the net charge between myofibrillar proteins was decreased and consequently, the water-holding capacity of meat was decreased (Huff-Lonergan and Lonergan, 2005). In addition, Kristensen and Purslow (2001) suggested that the degradation of cytoskeletal proteins while aging decreased the rigor-induced shrinkage of myofibrils, which enabled extracellular water to flow into the muscle cell and consequently increased water-holding capacity of meat. Therefore, dry-aged pork loin showed low cooking loss in the present study.

After dry aging of pork loin, there was a significant increase in most free amino acids such as Ala, Arg, Asn, GABA, Gln His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Try, Tyr, and Val contents (p<0.05, Table 3). However, there was no significant difference in Cys and Gly content between the control and dry-aged pork loins, and Asp and Lys content were significantly lower in the dry-aged pork loin than in the control (p<0.05). Although the contents of some free amino acids are maintained or decreased in pork loin after dry aging, the amount of total free amino acid was increased in dry-aged pork loin by as much as 73% (p<0.05). Moya et al. (2001) found that free amino acid content increases in pork loin after aging. Koutsidis et al. (2008) reported the increase of free amino acid content in beef loin by as much as 80% after aging for 21 d. The muscle contains various proteinase and peptidases that degrade myofibrillar and cytoskeletal proteins in meat to peptides and free amino acids during postmortem aging (Laville et al., 2009; Toldrá et al., 1995). Free amino acid is an important flavor precursor that has specific taste and produces various aroma compounds through the Maillard reaction and Strecker degradation in cooked meat (Koutsidis et al., 2008; Mottram, 1998). The free amino acid contents that increased in the present study after dry aging have taste characteristics such as sweet (Ala, Ser, and Thr), bitter (Arg, His, Ile, Leu, Met, Phe, and Try), sweet with after bitter taste (Val and Pro), and salty-bitter (Gln) (Fukunaga et al., 1989; Mau and Tseng, 1998). Although some amino acids generate an unpleasant taste, free amino acids are important to generate pleasant aroma compounds in cooked meat (Koutsidis et al., 2008).

Carnosine and anserine are histidyl dipeptides that are widely distributed in the skeletal muscle and are known as bioactive compounds that function as antioxidants and neurotransmitters (Peiretti et al., 2011; Tian et al., 2007). However, they also act as precursors for bitter taste (Macleod, 1994). In the present study, there is no significant difference in carnosine and anserine contents between control and dry-aged pork loin. Moya et al. (2001) found no change in carnosine and anserine content in pork loin after aging, and reported that no proteases could hydrolyze them in postmortem meat.

GMP and IMP in meat contribute to umami taste of meat as flavor precursors (Koutsidis et al., 2008). Hypoxanthine is closely associated with the bitter taste of meat (Tikk et al., 2006). However, the GMP and IMP content in pork loin were significantly decreased, whereas the inosine and hypoxanthine content in pork loin were significantly increased after dry aging in the present study (p<0.05, Table 4). This result was because of the enzymatic degradation of nucleotides such as GMP and IMP to nucleosides, followed by further degradation to purine and ribose. Yano et al. (1995) reported that the degradation of IMP resulted in the accumulation of its enzymatic breakdown products, such as inosine, hypoxanthine, and ribose in meat. Ribose is a type of reducing sugar that plays an important role in producing aromas by the Maillard reaction and Strecker degradation with free amino acids (Mottram, 1998). However, the reducing sugar content of dry-aged pork loin was not significantly different from that of control in the present study (data not shown). The results suggest that there was no improvement in flavor caused by nucleotides and their degraded products in dry-aged pork loin.

The hardness, cohesiveness, gumminess, chewiness, and resilience were significantly decreased in dry-aged pork loin compared to that in control (p<0.05, Table 5). In contrast, springiness of dry-aged pork was significantly increased (p<0.05). In addition, dry-aged pork loin showed lower shear-force than the control (p<0.05). These results suggested that dry-aged pork loin was tender than control. Meat tenderness is improved with the structural degradation of myofibrils caused by degradation of myofibrillar and cytoskeletal proteins after aging (Nowak, 2011). A previous study reported the degradation of desmin and integrin in pork loin after postmortem aging (Yin et al., 2014). In addition, Cruzen et al. (2014) found the troponin-T and titin degradation in beef during the aging period.

The scores for sensory parameter of cooked pork loin are shown in Table 6. Dry-aged pork loin received higher scores in color, taste, flavor, and texture than control (p<0.05). Consequently, the score for overall acceptability was significantly higher in dry-aged pork loin than control (p<0.05), suggesting that dry aging improved palatability of pork loin. Juárez et al. (2011) found improvement in tenderness, juiciness, and flavor in pork after dry aging. Campbell et al. (2001) reported that dry aging increased the tenderness and juiciness of beef with the increase of aged flavor.