Comparative Meat Qualities of Boston Butt Muscles (M. subscapularis) from Different Pig Breeds Available in Korean Market

Boston butts (M. subscapularis) were randomly selected from the slaughterhouses (Namwon and Suncheon), among the most popular varieties in the Korean pork market. Each five carcasses (Quality grade 1) of LYD barrows, Berkshire sows, or Berkshire barrows weighing about 110 kg were used in the study. Ibérico (bellota) butts were obtained from a direct pork trade company importing from Spain. After collecting available pork samples, the muscles were kept at 4°C until sampled and analyzed for their physicochemical meat quality traits. Muscle fiber samples for analysis were prepared by cutting each muscle into 1.0×1.0×1.5 cm pieces in a direction parallel to the muscle fiber. The specimens were immediately frozen in isopentane chilled with liquid nitrogen, and stored at –70°C until histological analysis.

Moisture contents of M. subscapularis muscle excised from the three different pig breeds were determined by drying the samples (3 g) at 102°C (AOAC, 2000). The crude protein content was measured by the method suggested by the (AOAC, 2000). Lipids were extracted from 5 g of muscle with chloroform/methanol (2:1), according to the (Folch and Lees, 1951). pH values of M. subscapularis were measured using a pH meter (Seven Excellence™, METTLER TOLEDO, Switzerland). The lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) of Boston butt samples from the different pigs were measured using a colorimeter (CR-410, Minota, Japan). All values of color were taken in triplicate for each sample. Shear force values were measured using a Warner-Bratzler shear attachment on a texture analyzer (TA-XT2, Stable Micro System, Surrey, UK).

The fatty acids composition of porcine M. subscapularis muscle was estimated by the method of (O’fallon et al., 2007), with a minor modification. The assay was performed using a Gas Chromatograph-Flame Ionization Detector (7890 series, Agilent, Santa Clara, CA, USA) under the following conditions: injector split mode with split ratio of 25:1, temperature 250°C. High purity air, high purity H₂, and high purity He were used as carrier gases. The flow rate was maintained at 40 mL/min for H₂ and 400 mL/min for air. An HP-88 column (60 m×250 μm×0.2 mm) was used for the analysis. Fatty acid composition is expressed as a percentage. The nutritional quality indexes of lipid for porcine M. subscapularis muscle were analyzed from the fatty acid composition data obtained for each group of pigs, according to the equations proposed by (Santos-Silva et al., 2002), as shown below:

The soluble amino acids composition of porcine M. subscapularis muscle was determined by using a slightly modified method described by (Hughes et al., 2002). High performance liquid chromatography (HPLC) analyses of free amino acids were obtained using an S433 auto analyzer, cation separation column (LCAK07/li; 4.6×150 mm), buffer change (A, pH 2.90; B, pH 4.20; C, pH 8.00), lithium citrate buffer solution having a buffer flow rate 0.45 mL/min, ninhydrin flow rate 0.25 mL/min, and column temperature 37°C.

Lipid oxidation rate of the Boston butt porcine muscle (M. subscapularis) was assessed according to the procedure described by Ahn et al. (1998), with a slight modification in the thiobarbituric acid-reactive substances assay (TBARS). Anti-oxidant capacity of M. subscapularis porcine muscle from the three different pig breeds was determined by applying the free radical scavenging assay, according to a method described by Blois (1958), and is expressed as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (%). The porcine samples were inspected on d 1 and 7 of refrigeration at 4°C.

Muscle fiber characteristics were determined by the method of Choi et al. (2012) with slight modifications. Frozen muscles were cut into 10 μm thick transverse sections using a cryomicrotome (CM1860, Leica Biosystems, USA) at –20°C. Each sample was mounted on 76×26×1 mm adhesive microscope slides (HistoBond^®, Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), coated with a drop of aqueous mounting medium (S3023, Dako, Carpinteria, CA, USA), and covered with a 22×22 mm coverslip (100 Deckglaser, Menzel-Glaser). All samples were viewed and photographed using a fluorescent microscope (BX51, Olympus, Tokyo, Japan) equipped with a DP72 digital camera (Olympus). Using a Photoshop CC (Adobe, California, USA), cross-sectional area (CSA; μm²) and muscle fiber density (fiber number/mm²) was determined from approximately 900 fibers per section.

Nucleotide contents were determined by the method of Lee et al. (2017) with slight modifications. Briefly, the M. subscapularis muscle (5 g) was homogenized with 20 mL of 0.6 M perchloric acid. The homogenate was centrifuged at 2,265×g for 15 min (Continent 512R, Hanil, Incheon, Korea) and filtered through a filter paper (Whatman No. 4, Whatman PLC., Brentford, UK). The filtrate was titrated to pH 5.5 using 0.6 N and 6 N KOH. After titration, the samples were transferred to a volumetric flask and the resultant solution was filtered through a membrane filter (0.2 μm) into a glass vial. Nucleotides were quantified using HPLC (Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA) with a Synergi^TM Hydro-RP 80 Å column (250×4.6 mm, 4 μm particles; Phenomenex, Seoul, Korea). The 20 mM phosphate buffer (pH 5.5) was eluted at a flow rate 1.0 mL/min, detection wavelength 254 nm, and column temperature 30°C. The meat freshness indicator, known as the K-index, was determined using the ATP degradation nucleotides formula suggested by Gil et al. (2011):

Data obtained were analyzed by multiple assay techniques, applying the Student-Newman-Keuls for significance test (p<0.05) using the general linear model of the SAS program (SAS, 2003). Significant differences were determined by applying the one-way ANOVA. Each treatment was performed in five replication (carcasses), and results are presented as the standard error of the means (SEM).

Proximate composition and meat quality characteristics (color, pH, cooking loss, and shear force value) of porcine M. subscapularis muscle were examined (Table 1). Results show that moisture contents ranged from 62.11% to 74.16%, with Ibérico pigs harboring significantly lower content than other breeds (p<0.05). However, previous studies have reported lower moisture contents ranging from 61.86% to 63.74% in pigs, which is similar to a study by Lim et al. (2014). It was demonstrated that the Ibérico pigs have a significantly higher lipid content as compared to other groups. The variation of fat content among groups is probably due to the breed effect (Stanišić et al., 2013). Moreover, the higher moisture loss in Ibérico pigs could be attributed to longer storage time under freezing conditions. The crude protein contents in Ibérico pigs were significantly higher than LYD pigs. Besides the ash content, no significant differences were found among the breeds. Our results indicate that except the yellowness value (CIE b*) of Ibérico pigs, the breed does not affect the overall meat color. The enhanced yellowness (CIE b*) for Ibérico pigs is associated with higher fat oxidation and pigment lability, as compared to other groups (Fernández‐Lopez et al., 2004). It is also noteworthy that Ibérico pigs significantly lost a higher amount of water during cooking than other breeds. It has previously been reported that lower cooking loss is associated with heavier pigs as compared to lighter animals (Magowan et al., 2011), and is also related to cooking processes, such as temperature and time at the heating phase (Madzimure et al., 2017). Considering the shear force value, M. subscapularis porcine muscle from Ibérico pigs show remarkably lower shear force value (kg.f) which is associated with more tender meat, as compared to other pig breeds (Table 1). Tenderer meat is related to proteolysis of muscles, specifically myofibrillar and cytoskeletal protein degradation including titin, desmin, nebulin, and troponin-T (Jeleníková et al., 2008). Meat tenderness is also affected by the origin of the animal as well as their age, breed, gender, and environmental conditions, and period of meat ageing (Ouali, 1990).

The fatty acid composition of muscles is an important factor in determining the nutritional quality of meat or adipose tissue subjected with special attention in human health. In our study, we determined the free fatty acid composition of porcine M. subscapularis muscle from different pig breeds (LYD, Berkshire, and Ibérico) (Table 2). Our results reveal that the SFA content for Berkshire sow is significantly lower than other breeds (p<0.05). Conversely, Berkshire sow harbored a higher content of UFA than the LYD, Berkshire barrow, and Ibérico pigs. The predominant fatty acids, (18:1) and (16:0), were significantly higher in Ibérico M. subscapularis porcine muscle. Moreover, significantly higher levels of (18:2) and (20:4) were obtained in Berkshire sow, as compared to the other tested breeds. The PUFA/SFA (P/S) ratio of meat contributes a favorable balance between the ω-6/ω-3 PUFA; the recommended P/S value is greater than 0.4, although meat from some natural sources has a value of around 0.1 (Wood et al., 2004). The P/S values obtained from the three different pig breeds were 0.38, 0.58, 0.41, and 0.20 for LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs, respectively. The ratio of ω-6/ω-3 unsaturated fatty acids of M. subscapularis porcine muscle was determined to be 14.32, 17.34, 14.45, and 7.71 for LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs, respectively. The significantly lower ratio of ω-6/ω-3 fatty acid noted for Ibérico M. subscapularis porcine muscle attributes as a desirable characteristic in terms of reducing chronic diseases, especially blood clot formations leading to heart attack, in humans (Wood et al., 2004). The recommended ω-6/ω-3 ratio is less than 4.00; however, some meats are higher than the recommended level which, however, can be manipulated by maintaining the P/S ratio in meat. The differences observed in the study were associated to breed effect and different genetic factors. In Berkshire barrow, the SFA level was higher than the Berkshire sow (p<0.05), which was in agreement with results reported by Kasprzyk et al. (2015). However, PUFA levels in the Berkshire sow were significantly higher than other breeds (p<0.05), mainly due to the high content of linoleic acid (Table 2). We infer that the higher ω-6/ω-3 ratio is due to the excessive saturated fat and lower levels or deficiency of ω-3 fatty acids in the diet, as well as the influence of breed attributes (Enser et al., 2000).

The nutritional quality indexes of lipid for M. subscapularis porcine muscle excised from three different pig breeds were evaluated (Table 2). Ulbricht and Southgate, (1991) reported that the atherogenic or hyperlipidemic SFAs are (C12:0), (C14:0), and (C16:0) acids, while (C14:0), (C16:0), and (C18:0) acids are thrombogenic SFAs. The long chain unsaturated fatty acids, especially ω-6 (linoleic) and ω-3 (linolenic) fatty acids, are thought to be anti-atherogenic and anti-thrombogenic, indicating that diets should contain lower index values to reduce human cardiovascular diseases (Cebulska et al., 2018). Lower atherogenicity index (AI) value represents a lower proportion of saturated to unsaturated fatty acids, which subsequently reduces the endothelial strength of blood vessels owing to collapsed lipids and plaque formation (Cebulska et al., 2018). Conversely, lower thrombogenicity index (TI) values determined from the proportion of other fatty acids, indicates a lower risk of disturbance to blood coagulation and clotting. In the current study, AI and TI index values obtained were 0.52, 0.46, 0.51, 0.52, and 1.24, 1.01, 1.14, 1.15, respectively, for LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs, respectively. These results indicate that Berkshire sow has significantly lower AI and TI values, as compared with other tested breeds (p<0.05); this could be attributed to the higher content of unsaturated fatty acids.

The free amino acids composition of porcine M. subscapularis muscle of the three different pig breeds was analyzed (Table 3). It has previously been classified that lysine, leucine, isoleucine, valine, phenylalanine, histidine, methionine, and threonine are the essential amino acids, whereas aspartic acid, serine, arginine, glutamic acid, tyrosine, glycine, and alanine are non-essential amino acids. Previous studies report that except lysine and histidine, all essential amino acid levels were significantly higher in Ibérico pigs as compared to other breeds (p<0.05). Taurine, the functional compound related to ATP production in muscles, was significantly higher in the Ibérico pigs. However, the glutamic amino acid which imparts the umami taste in meat, was significantly higher in LYD pigs as compared to Ibérico pigs (Wood et al., 2004). In the present study, amino acid levels were higher in Ibérico pigs than other breeds, and enriched with almost all essential amino acids, an important factor for eating quality and also imparting numerous health benefits for meat consumers (Subramaniyan et al., 2016). The increment of amino acids in Ibérico pigs can be attributed to breed effect, which depends on the amino peptidase and hydrolytic activity toward increased group with the proteolysis of muscle by enzyme known as calpain (Feidt et al., 1996; Nishimura et al., 1988). Moreover, Ibérico pigs harbor a higher amount of the total tasty and bitter amino acids, as compared with LYD, Berkshire sow, and Berkshire barrow pigs (p<0.05).

We examined the oxidative stability (lipid oxidation) of M. subscapularis muscle excised from LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs (Table 4). Lipid oxidation value was significantly higher for Ibérico pigs at both d 1 and 7 of storage, compared to other breeds, as determined by the TBARS value, which revealed a significantly increasing trend with increasing d of storage. Lipid oxidation is one of the primary causes for loss in meat quality, and generates the volatile compounds responsible for rancidity of fresh meat (Gray et al., 1996). Lipid oxidation results in deterioration of some meat quality traits like flavor, texture, and color, and also decreases the shelf life, along with the production of some toxic compounds (Mohamed et al., 2008). In addition to lipid oxidation, it is postulated that muscles with higher fat content in between or across the muscles, show a greater tendency to be oxidized via a continuous free-radical chain reaction (Ruban, 2009). The oxidation rate of post-mortem muscle depends on the antioxidant capacity existing in the muscle of the animal, which can be retarded by the action of endogenous antioxidant enzymes, and differ among different species, different breeds, and or even among animals of a single species (Ren et al., 2013).

Antioxidants are compounds which combat free radicals in the system by intervening in any of the three steps of lipid oxidation, viz., initiation, propagation, and termination (Cui et al., 2004). The total antioxidant capacity of the M. subscapularis porcine muscles collected from three different pig breeds was measured by performing the free radical scavenging assay (Table 4), and was found to be affected by pig breeds. Ibérico pigs had lower antioxidant capacity compared to LYD, Berkshire sow, and Berkshire barrow at d 1 and 7 of storage (p<0.05). Except the Ibérico pig meat, all other tested groups presented no significant differences between the two storage conditions. Free radical inhibition percentage ranged from 43.06% to 52.73% and 38.95% to 52.56% for d 1 and 7, respectively. In Ibérico pigs, the free radical scavenging activity showed a decreasing trend with increasing number of d. Higher MDA compounds in Ibérico pigs revealed a higher content of lipids that deteriorate the quality of meat, thereby indicating lower antioxidant capacity. Apart from the MDA compounds, the antioxidant activities are also affected by endogenous, non-enzymatic antioxidants, breed, diet, and muscle types (Králová, 2015).

Examination of the muscle fiber size and fiber density of porcine M. subscapularis from LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs showed no significant variation among the breeds (Table 5). Furthermore, different genders within breeds also had no significant impact on the muscle fiber size and fiber density. Moreover, the muscle fiber number is determined before birth genetically, and only the length and cross sectional area of the muscle fibers increases with age (Wigmore and Stickland, 1983). In addition, it is postulated that the total number, density, and size of different muscle fiber area as well as their composition are important histochemical attributes which impact the fresh meat or cooked meat during conversion from muscle to meat (Joo and Kim, 2011). Considering the muscle fiber density and fiber size, it has been determined that muscle fiber size positively correlates with the carcass weight, backfat thickness and loin eye area, whereas fiber density has a negative correlation in pork quality traits (Ryu et al., 2004). The density, compactness, and space of muscle fiber have been shown in Fig. 1. In our study, breed of the pigs imparted no effect on muscle fiber size and density. More research on muscle fiber types and composition is required to clarify the effect of breed on meat quality traits.

Fig. 1. Optical microscopic structure of porcine M. subscapularis muscle from LYD, Berkshire, and Ibérico pigs. LYD, Landrace×Yorkshire ×Duroc.

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The nucleotide contents of porcine M. subscapularis muscle from LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs were determined and are presented in Table 6. Ibérico pigs harbored significantly higher amounts of AMP and hypoxanthine than Berkshire sow and LYD pigs (p<0.05). AMP content differed depending on the animal age, sex, quality grade, and different cuts, possibly due to higher nucleotide contents. Regardless of the sex, Berkshire pigs had significantly higher IMP compared to Ibérico pigs (p<0.05). Nucleotides, AMP, GMP, and IMP are related to the umami and savory taste, while inosine and hypoxanthine impart the bitter taste (Dashdorj et al., 2015). Moreover, IMPs are important metabolites in meat flavor due to the synergistic effect with glutamic acid via the maillard reaction (Lee et al., 2017). The decomposition of ATP in different muscles is considered the most useful and reliable approach to evaluate the correct meat freshness. Owing to ATP decomposition, the analysis is based on the concept that after exemplification of pork, the ATP in meat decomposes in the following sequence: ATP-ADP-AMP-IMP-Ino-Hx (Hernández-Cázares et al., 2010). During these consequential changes, the smell and taste of meat also changes at different intervals. A similar autolytic process takes place in all animal, with variation among the different species. The concentration of ATP alone cannot be implied to measure the freshness index of meat, since it disappears within approximately 24 hours after post-mortem (Karube et al., 1984). In addition, a similar disappearing phenomenon is also observed for ADP and AMP. However, ATP degradation products generated have been suggested as indicators of meat freshness, and hence the concept of K-index was developed and introduced by Gil et al. (2011). Low K-index value is considered to indicate fresher meats than higher values. We therefore determined the K-index value of porcine M. subscapularis muscle from LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs (Table 6). The K-index value of LYD, Berkshire sow, Berkshire barrow, and Ibérico pigs were found to be 79.92, 70.55, 66.80, and 94.21 respectively. Table 6 shows that LYD and Berkshire pigs presented with significantly lower K-index value than Ibérico pigs (p<0.05). Thus, the significantly lower K-index and hypoxanthine content of Berkshire M. subscapularis muscle indicates meat quality close to freshness and superior to Ibérico pigs (Hernández-Cázares et al., 2010; Nishimura et al., 1988).