Characteristics of selected pork muscles 45 min and 24 h post mortem

The muscles of the pork topside – musculus adductor (AD) and m. semimembranosus (SM), and of the silverside – m. biceps femoris (BF) and m. semitendinosus (ST) were analysed and their properties compared with the m. longissimus thoracis (LT) and m. supraspinatus (SS) muscles. Colour (CIE L*a*b*), D/L-lactic acid content, and pH values were measured 45 min and 24 h post mortem. The basic chemical composition of muscles was analysed 24 h after slaughtering. A significant correlation (r = -0.61, P < 0.001) was found between the pH values and the level of lactic acid 45 min post mortem, though not 24 h after slaughter (r = -0.25, P > 0.05). The results confirmed that a direct comparison cannot be made between the fall in pH values in meat and the increase in the level of lactic acid. The lightness L* 24 h post mortem was higher (P < 0.05) in AD, BF, ST and LT muscles than those in samples measured 45 min after slaughter. The toughest muscle was biceps femoris, with a mean value of shear force of 90.5 N. The differences in shear force between the individual analysed muscles were significant (P < 0.05). There were significant differences in the intramuscular fat content between the topside and silverside muscles (P < 0.05). The results of the present study are of value to meat producers who might intend to substitute these parts of the leg with one another during the production, particularly in the case of wholemuscle meat products. Lactic acid, pH value, colour, shear force, protein content, intramuscular fat Colour, tenderness, and water-binding capacity are considered the most important characteristics of fresh meat (Hughes et al. 2014). England et al. (2015) believe the quality of pork meat to be determined by two basic factors – the speed at which the pH falls in the muscles and the final pH value attained post mortem. Joo et al. (2013) defined three groups of meat traits that are of interest to the consumer –appearance quality traits, eating quality traits, and reliance quality traits. These characteristics are particularly important in the retail during the selection and purchase of meat cuts by consumer. In production, producers prioritise properties that guarantee the uniformity of the products they produce. Colour, or more accurately the balance of colour tone of contiguous muscles (meat) in whole-muscle meat products (and hams in particular), is also important, in addition to the fore-mentioned features such as the water binding capacity and tenderness (McKeith and Pringle 2013). The proportion of fat and, in particular, protein, is also important. The legislation of many countries stipulates limits defining the minimum content of pure muscle protein in selected meat products (DLB 2010) or minimum values for the water : protein ratio (ÖLB 2012). The chemical composition of meat is important in these cases with regard to the balance between the content of total (crude) protein, collagen and fat. The speed and extent of the fall in pH values and the final pH value can, on the other hand, influence the colour of the meat (Ruusunen et al. 2012). ACTA VET. BRNO 2018, 87: 173-180; https://doi.org/10.2754/avb201887020173 Address for correspondence: Alena Saláková Department of Meat Hygiene and Technology Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackého tř. 1946/1, 61242 Brno, Czech Republic Phone: +420 541 562 749 E-mail: salakovaa@vfu.cz http://actavet.vfu.cz/ Meat from the legs, particularly the topside and silverside, is used in the production of premium cooked hams. The topside is composed largely of two muscles, m. adductor (AD) and m. semimembranosus (SM), and the silverside of m. biceps femoris (BF) and m. semitendinosus (ST). Each muscle has a specific role to play in the body. The unique characteristic of skeletal muscle is its diversity resulting from the formation of the individual muscles, the type of muscle fibres, and the composition and heterogeneity of individual fibres. No two muscles in the body are identical (Karlsson et al. 1999). What is, on one hand, crucial to the life of the individual, i.e. the heterogeneity of muscle tissue, is, on the other hand, disadvantageous to producers in their effort to ensure the standard production of particular types of meat products, and whole-muscle products in particular. A great many studies considering selected properties of pork meat (differences in fibre type distribution, myofibrillar degradation, colour, tenderness, pH value etc.) have been published in the scientific literature over the last twenty years (e.g. Christensen et al. 2004; Melody et al. 2004; Gil et al. 2008; Choe et al. 2009; Purchas et al. 2009; Choi et al. 2013; Realini et al. 2013). The majority of these works have studied in detail just a small number of muscles, such as the m. longissimus thoracis, m. semimembranosus or m. semitendinosus. To date, there has been no detailed analysis of the technologically important muscles in the leg. The aim of this study was to analyse and compare the topside (AD and SM) and silverside (BF and ST) muscles of slaughter pigs. Materials and Methods

Colour, tenderness, and water-binding capacity are considered the most important characteristics of fresh meat (Hughes et al. 2014).England et al. (2015) believe the quality of pork meat to be determined by two basic factors -the speed at which the pH falls in the muscles and the final pH value attained post mortem.Joo et al. (2013) defined three groups of meat traits that are of interest to the consumer -appearance quality traits, eating quality traits, and reliance quality traits.These characteristics are particularly important in the retail during the selection and purchase of meat cuts by consumer.In production, producers prioritise properties that guarantee the uniformity of the products they produce.Colour, or more accurately the balance of colour tone of contiguous muscles (meat) in whole-muscle meat products (and hams in particular), is also important, in addition to the fore-mentioned features such as the water binding capacity and tenderness (McKeith and Pringle 2013).
The proportion of fat and, in particular, protein, is also important.The legislation of many countries stipulates limits defining the minimum content of pure muscle protein in selected meat products (DLB 2010) or minimum values for the water : protein ratio (ÖLB 2012).The chemical composition of meat is important in these cases with regard to the balance between the content of total (crude) protein, collagen and fat.The speed and extent of the fall in pH values and the final pH value can, on the other hand, influence the colour of the meat (Ruusunen et al. 2012).
Meat from the legs, particularly the topside and silverside, is used in the production of premium cooked hams.The topside is composed largely of two muscles, m. adductor (AD) and m. semimembranosus (SM), and the silverside of m. biceps femoris (BF) and m. semitendinosus (ST).Each muscle has a specific role to play in the body.The unique characteristic of skeletal muscle is its diversity resulting from the formation of the individual muscles, the type of muscle fibres, and the composition and heterogeneity of individual fibres.No two muscles in the body are identical (Karlsson et al. 1999).What is, on one hand, crucial to the life of the individual, i.e. the heterogeneity of muscle tissue, is, on the other hand, disadvantageous to producers in their effort to ensure the standard production of particular types of meat products, and whole-muscle products in particular.A great many studies considering selected properties of pork meat (differences in fibre type distribution, myofibrillar degradation, colour, tenderness, pH value etc.) have been published in the scientific literature over the last twenty years (e.g.Christensen et al. 2004;Melody et al. 2004;Gil et al. 2008;Choe et al. 2009;Purchas et al. 2009;Choi et al. 2013;Realini et al. 2013).The majority of these works have studied in detail just a small number of muscles, such as the m.longissimus thoracis, m. semimembranosus or m. semitendinosus.To date, there has been no detailed analysis of the technologically important muscles in the leg.
The aim of this study was to analyse and compare the topside (AD and SM) and silverside (BF and ST) muscles of slaughter pigs.

Meat samples
Samples of pork meat were obtained from the slaughterhouse of the University of Veterinary and Pharmaceutical Sciences in Brno.Gilts of the Danish-Norwegian breed Topigs were used for the purposes of assessment (n = 9), at a slaughter weight of 100 ± 10 kg.The pigs came from a farm within 50 km of the place of slaughter.The animals were transported to the slaughterhouse 2-3 h before slaughter.Stunning was performed with an electric current (head-only stunning), bleeding in the hanging position.Following halving, the left side was cut into individual parts immediately; the right side was chilled at a temperature of 2 °C and cut up the following day 24 h post mortem.
The following muscles were selected for evaluation: AD, SM, ST, BF, SS and LT.Colour in the CIE L*a*b system, the D/L-lactic acid content and pH values were measured 45 min and 24 h post mortem.Other properties were evaluated 24 h post mortem.A total of 54 samples of muscles were analysed.

The pH value and lactic acid analysis
The pH values were measured with a Double Pore needle probe (Hamilton, Switzerland) on a 340i WTW pH-meter (WTW, Germany).D/L-lactic acid was determined using an enzymatic test kit (MEGAZYME, Ireland).The quantification of D-lactic acid required two enzyme reactions.In the first reaction catalysed by D-lactate dehydrogenase (D-LDH), D-lactic acid (D lactate) was oxidised to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD + ), and the product, pyruvate, trapped by the conversion of pyruvate to D-alanine and 2-oxoglutarate with the enzyme D-glutamate-pyruvate transaminase (D-GPT) in the presence of a large excess of D-glutamate, while the NADH formed was quantified by measuring the absorbance at 340 nm.
In a similar set of reactions, L-lactic acid (L-lactate) was oxidised to pyruvate by L-lactate dehydrogenase (L-LDH) in the presence of nicotinamide adenine dinucleotide (NAD + ), and the product, pyruvate, trapped by the conversion of pyruvate to D-alanine and 2-oxoglutarate with the enzyme D-glutamate-pyruvate transaminase (D-GPT) in the presence of a large excess of D-glutamate.The NADH formed was quantified by measuring the absorbance at 340 nm (MEGAZYME 2011).All samples were measured in triplicate.

Instrumental analysis
Colour was measured by the CIE L*a*b* system using a Minolta CM 2600d (Konica Minolta, Japan).A measuring area of 8 mm, illuminant D65 and 10 ° standard observer were used.The instrument was standardised using a standard white plate.CIE L* -lightness, a* -redness, b* -yellowness were measured.Five independent subsamples were measured for each sample.
Raw samples were tested by Warner-Bratzler test (WB) using the Instron Universal Testing Machine (model 5544) (Instron Corporation, England).Samples of 2.5 × 2.5 × 2.0 cm and a perpendicular arrangement were used for the WB test.Force time curves were recorded at a crosshead speed of 50 mm•min -1 .Maximum shear force was evaluated (WBSF) from five replicates from each sample.

Chemical analysis
A drying method (ISO 1442:1997) at 103 ± 2 °C for a period of 24 h was used for the determination of the dry matter content.The samples were weighed after cooling and the dry matter content was calculated.The fat content was determined using a SOXTEC instrument (TECATOR, Sweden).Samples were left in the drier for 3 h at 135 ± 2 °C and extracted by the extraction agent (diethyl ether) in the instrument.The collagen content was determined spectrophotometrically at a wavelength of 550 nm in a GENESYS TM 6 spectrophotometer (Thermo Electron Corporation, USA) as the quantity of 4-hydroxyproline.The content of hydroxyproline was obtained from the calibration curve and converted into the collagen content (f = 8).Crude proteins were determined after subsequent conversion of organic nitrogen to inorganic nitrogen in a KJELTEC instrument (TECATOR, Sweden) by the Kjeldahl method.A factor of 6.25 was used for the conversion of the nitrogen content into the protein content.Pure muscle proteins were determined following the precipitation of non-protein N-substances by hot tannin and subsequent conversion of organic nitrogen into inorganic nitrogen in a KJELTEC instrument (TECATOR, Sweden) by the Kjeldahl method.A factor of 6.25 was used for the conversion of the nitrogen content into the protein content.Two independent subsamples of each muscle were used also for determining the dry matter, fat content, and protein content.

Statistical analysis
The results of the instrumental and chemical analyses are reported as mean values ± standard deviation (SD).Statistical data analyses were conducted using the statistical program STATISTICA 7 CZ (StatSoft, Prague, Czech Republic).ANOVA (Tukey's test) was used for the determination of significant differences between the groups of samples.Correlation analyses among indicators were performed using Pearson correlation coefficients.Significance levels of P < 0.05, P < 0.01 and P < 0.001 were used.

The pH value and lactic acid content
Mean pH 45 values ranging from 6.35 (AD) to 6.03 (ST) were found in this study (Table 1).A level of D/L-lactic acid ranging from 82 (AD) to 302 (SS) µmol•g -1 of dry matter, with the mean values between 141 (BF) and 209 (SS) µmol•g -1 of dry matter, was found in the tested muscles 45 min after slaughter (Table 1).Over the next 24 h, the D/L-lactic acid content increased (P < 0.05) in AD, BF, ST and LT muscles.There were no significant differences in the lactic acid level between individual muscles 24 h post mortem.

Instrumental indicators
The instrumental analysis of meat colour showed that the L * values increased with the falling pH in the muscles.The L * value increased (P < 0.05) in LT, AD, BF and ST muscles during the 24 h post mortem, but not in SS and SM (P > 0.05).Significant correlations were found between pH values and the value of lightness L * 45 min post mortem (r = -0.33,P < 0.05) and 24 h post mortem (r = -0.56,P < 0.001).
Significant correlations were found 24 h after slaughter between the values of pH and a * (r = 0.45; P < 0.05), and also between the levels of lactic acid and indicator a * (r = -0.49;P < 0.001).A lower value of significant correlations was demonstrated 45 min post mortem between the values of pH and a * (r = -0.33,P < 0.05) and also between the values of pH and indicator b * (r = -0.32;P < 0.01).
The highest mean values of a * were measured after slaughter in SS, the lowest in LT (P < 0.05).In the legs muscles, the values 45 min after slaughter were lowest in AD and highest in ST (P < 0.05).The mean a * values 24 h post mortem had risen in the LT muscle (P < 0.05), but not in the muscles AD SM and BF (P > 0.05).The increase in the b * values after 24 h was significant (P < 0.05) in muscles with the exception of SS (P > 0.05).The shear force value (WBSF) expresses the reciprocal value of The same superscripts within rows with the same indicators mean a significant difference (P < 0.05) LA -DL lactic acid, WBSF -Warner-Bratzler shear force, L* -lightness, a* -redness, b* -yellowness, SD -standard deviation tenderness of the meat.In this regard, the toughest muscle 24 h post mortem was the BF, with a mean value of 90.5 N, followed by AD (73.0 N) and SS (69.9 N).
The differences in the value of WBSF between the individual analysed muscles were significant (P < 0.05; Table 1).

Chemical composition of pork muscles
The basic chemical composition of the analysed pork muscles is given in Table 2.The samples did not differ in the dry matter content (P > 0.05).highest content of crude proteins was measured in the AD (mean value 23.6 %) and LT (23.0 %) muscles.In the leg muscles, significant differences (P < 0.05) were found between AD and BF, and between AD and ST in the crude protein content.There were no significant differences (P > 0.05) in the proportions of pure muscle proteins (myofibrillar and sarcoplasmic proteins) or collagen content between leg muscles (Table 2).
The leanest muscles with the lowest proportion of intramuscular fat were both of the muscles of the topside (AD, 2.4%; SM, 2.3%).The differences in the proportion of fat between the two topside muscles and the ST were significant (P < 0.05).

Discussion
Values of pH 45 of less than 5.90 were measured in a number of individual samples of all four leg muscles which can be considered a manifestation of the PSE (pale, soft, exudative meat) type defect (Feiner 2006).pH values < 5.90 were found 45 min post mortem in one case in the AD muscle, in two cases in the BF muscle, and in The same superscripts within rows with the same indicators mean a significant difference (P < 0.05) SD -standard deviation three animals in the case of the SM and ST muscles (data not shown).The lowest pH 45 value of 5.69 was measured in the AD muscle, with the lowest pH value in the SM muscle also being detected in the same carcass.Values of pH 45 < 5.90 were also found in the BF and ST muscles from the same animal (BF: 5.88; ST: 5.77), though these were not the lowest values recorded.The same animal also had the lowest measured pH 45 value of all the LT muscles (6.07).
The mean pH values ranging from 5.37 (AD) to 5.45 (ST) were found in the leg muscles 24 h post mortem.These values are lower than those reported in literature.Warner et al. (1993) found pH u values of 5.76 (SM), 5.82 (BF), and 5.86 (ST) in the muscles of 19 carcasses.The mean pH u value in the SS shoulder muscle was 5.97.Christensen et al. (2004) measured pH values of 5.77 in the ST and 5.61 in the SM 24 h after slaughter.Gil et al. (2008) determined a pH u value of 5.49-5.55 in the SM, whereas Tomović et al. (2008) found a mean pH 24 value of 5.70 in the same muscle.The results closest to the values found in our study were those of the authors Voutila et al. (2007) who measured a mean pH u value of 5.45 in a population of 14 pigs in Finland, though 13 gilts from Ireland had a pH u value of 5.70 in the same muscle.
The accumulation of lactate is a good measure of the extent and level of glycolysis.It is, however, deceptive to use the level of lactate directly to determine the fall in the pH value in the muscles (Ferguson and Gerrard 2014).A significant correlation (r = -0.61,P < 0.001) was found between pH values and the level of lactic acid 45 min post mortem, though not 24 h after slaughter (r = -0.25,P > 0.05).The results of this study confirmed that a direct comparison cannot be made between the fall in pH values in the meat and the increase in the level of lactic acid.
Biochemical processes in the muscle tissue post mortem have an effect on the dispersion of light and, thereby, on the indicators of meat colour.The relocation of liquid occurs in the muscle fibres post mortem as a result of the fall in pH values.The dispersion and reflection of light also increases.The result is an increased value of lightness (Hughes et al. 2014).
In the leg muscles 24 h after slaughter, mean L * values ranging between 57.9 and 49.9 were found.Similar values were also found in the SM by Purchas et al. (2009) and Voutila et al. (2007).Warner et al. (1993), however, found lower L * values 24 h after slaughter in selected leg muscles (ST: 52.6; SM: 46.4; BF: 45.6).Tomović et al. (2008) found mean a * values of 9.21 and b * of 4.68 in SM muscles 24 h post mortem with the use of a conventional method of chilling.Ruusunen et al. (2012) measured mean a * values of 7.0-8.4 in LT, whereas Realini et al. (2013) found an a * value of 7.53 in LT.
Meat tenderness is determined by the amount and solubility of the connective tissue, shortening of sarcomeres during rigor development, post mortem proteolysis of myofibrillar and myofibrillar-associated proteins and the amount of intramuscular fat (Warner et al. 2010).The pH of muscles is reduced early post mortem and it affects the shrinkage of myofibrils (Huff-Lonergan and Lonergan, 2005).In the present study, the toughest muscle was the BF muscle, the most tender was the SM muscle.Comparing the pH values between the two above mentioned muscles, there were no significant differences in pH 45 and the values of pH 24 were the same for both muscles.In the intramuscular fat content the differences between the BF and SM muscles were not significant.Likewise, there were no significant differences in the collagen content.The WBFS values of the assessed muscles were apparently influenced by several factors depending on the functional status and chemical composition of the individual muscles.Kim et al. (2008) published a detailed analysis of the chemical composition of 21 pork muscles.They studied muscles obtained from 10 hybrids (5 gilts and 5 barrows) with a mean carcass weight of 86.00 ± 5.68 kg.The proportion of dry matter determined in the muscles was lower than that found in the present study.Nevertheless, the topside muscles still showed a lower proportion of dry matter (AD 23.9%; SM 24.5%) than the BF (25.1%) and ST (25.7%) muscles.The silverside muscles contained a lower proportion of protein (BF 19.8%; ST 18.8%) compared to the topside muscles (AD 21.3%; SM 20.9%).The fattiest muscle in the leg was, in agreement with the present study, the ST (6.1%).The muscles of the topside had a low proportion of fat (AD 1.7%; SM 3.1%).Voutila et al. (2007) found a practically similar proportion of collagen in the SM (0.4%), though a lower content in the LT (0.25-0.31%) compared to the present study (0.51%).A proportion of fat of 2.31% and 2.52% and a total protein content of 22.4% and 23.6% were found in the SM of pigs fattened extensively and intensively in New Zealand (Purchas et al. 2009).The given values correspond to the proportions found in the present study.Ruusunen et al. (2012) measured a mean proportion of total proteins in the LT between 21.8 and 22.9% a mean proportion of dry matter ranging from 25.9 to 26.7% in various breeds in Scandinavia.Realini et al. (2013) found a mean percentage of dry matter of 24.0%, and proportions of total proteins of 21.0%, collagen 0.9% and fat 2.3% in ST.The mean values found in the LT were 25.9% (dry matter), 24.4% (total proteins), 0.5% (collagen) and 1.0% (fat).
Although the differences in the chemical composition of the muscles of the topside and silverside in present study are not particularly large, they should be given due consideration by producers if they intend to substitute these parts of the leg with one another during the production of meat products, as this could affect the proportion of fat or pure muscle proteins in final products, particularly in the case of whole-muscle meat products.

Table 1 .
pH values, lactic acid levels (µmol•g -1 of dry matter), meat colour indicators and WBSF shear force values (N) in selected pork muscles 45 min and 24 h post mortem.Values are shown as a mean ± standard deviation along with minimum and maximum measured values.

Table 2 .
Results (%) for the basic chemical composition of selected pork muscles 45 min and 24 h post mortem.Values are shown as a mean ± standard deviation along with minimum and maximum measured values.