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unpalatable by rigor mortis. It is well known that during postmortem aging meat toughness decreases and its texture is improved. In general, chilled storage of ...
Proceeding of BIOATLAS 2012 Conference

PROTEIN CHANGES OF CHICKEN LIGHT AND DARK MUSCLES DURING CHILLED STORAGE K. VASSILEV *, G. IVANOV*, D. BALEV **, G. DOBREV *** Abstract: Chicken breast and leg meat samples (representing light and dark muscles, respectively) were stored at 0-2 ºC for seven days. Evaluation of the protein changes in light and dark muscles during chilled storage was performed by SDS-PAGE electrophoresis and free amino groups determination. The general aspects of chicken breast and leg meat proteolysis were associated with degradation of proteins with molecular weight about 100 kDa and 20-40 kDa, and with the appearance of new protein bands in the range of 110 to 150 kDa, 95 kDa and 30-40 kDa. Some differences in the proteolytic changes of light and dark muscles were also found. The higher pH values and free amino groups content, and lower total protein bands area of chilled stored leg (dark) muscles indicated for pronounced proteolysis associated with greater protein changes in comparison with the breast meat. Keywords: protein, chicken, dark and light muscles, proteolysis, chilled storage 1. Introduction Chicken meat is one of the most nutritious foods. Immediately after slaughtering, chicken meat is soft, but it soon becomes very tough and unpalatable by rigor mortis. It is well known that during postmortem aging meat toughness decreases and its texture is improved. In general, chilled storage of chicken is performed at 0 to 4 ºC for about 5 days. Meat flavor as well as texture is improved during storage at low temperatures. Postmortem biochemical changes transforming muscle into meat plays important role in determining the quality of meat (Sinku et al., 2003). The most important functional components of the muscle are proteins. They determined many of the desirable physicochemical and sensory properties of muscle foods (Xiong, 1997). Muscle proteins include 15–22% of the total muscle weight and can be divided into three major groups on the basis of their solubility characteristics: stroma proteins (insoluble), sarcoplasmic proteins (water soluble) and myofibrillar proteins (salt soluble) (VallejoCordoba et al., 2010). Proteolysis occurring in meat is responsible to a large extent for the development of meat flavor and tenderness,

which are the most important criteria for the consumer acceptability. The improvement of meat taste and flavor is associated with the increase in free amino acids and peptides in meats during postmortem aging (Nishimura, 1998). The increase in peptides is caused by the action of cathepsins B and L, and calpains on muscle proteins, while the increase in free amino acids is caused by the action of aminopeptidases C, H and P on the peptides during postmortem aging. In the studies on cytoskeletal proteins the electrophoretic technique using polyacrylamide gel with SDS (SDS-PAGE) has been used so far and has facilitated the qualitative analysis of changes in meat proteins (Tomaszewska-Gras et al., 2002). The changes observed were most frequently evaluated by electrophoretic separation (Huff-Lonergan et al., 1995; Lusby et al., 1983; Paxhia & Parrish, 1988). Cytoskeletal proteins are of labile nature, they are sparingly soluble in buffers and show high molecular weight that make their examination and quantitative determination even more difficult (Tomaszewska-Gras et al., 2002). Chicken muscles from different part of carcasses have different quantitative composition. Chicken breast muscle (light muscles) has higher

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Department of Food Preservation and Refrigeration Technology, Technological Faculty, University of Food Technologies, Plovdiv, Bulgaria. Corresponding author: Kiril Vassilev [email protected] ** Department of Meat and Fish Technology, Technological Faculty, University of Food Technologies, Plovdiv, Bulgaria. ***

Department of Department of Biochemistry and Molecular Biology, Technological Faculty, University of Food Technologies, Plovdiv, Bulgaria.

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protein content (about 20%) than chicken leg muscles (dark muscles) (about 16%). In addition the stroma protein content of breast muscles is more than 2 times lower than leg muscles (Khan and Van den Berg, 1964). It is well known that, the proteolysis of different types of muscles is not universal because different cytoskeletal proteins are subject to different rates and degrees of degradation (Hwan and Bandman, 1989; Taylor et al., 1995; Morrison et al., 1998; Van Laack et al., 2000). The knowledge for the protein changes of chicken light and dark muscles during postmortem storage would be of crucial importance for the quality assurance of chilled stored poultry products. The aim of the present study was to evaluate the protein changes of chicken light and dark muscles during chilled storage by using of SDSPAGE. 2. Materials and methods Meat samples: The chicken samples were delivered from local market. Breast and leg meat samples were on second day postmortem. The samples were stored at 0-2 ºC for seven days. At the day of analyze meat was double minced in laboratory meat mincer. Chemicals: Bovine serum albumin, ammonium persulphate, ninhydrin, fructose were purchased from Merck. Protein standard was delivered by Bio-Rad. Moisture: Moisture content was determined by sample heating at 105oC to constant weight according to the standard methods (AOAC, 1980). Ash: Ash content was determined by mineralization of meat as described in AOAC (1980). Protein: Protein content in meat sample was determined according to the standard method of Kieldahl (AOAC, 1980) with automatic analysis on Keltec Auto, model 1030 (Tecator, Sweden). Lipids: Determination of total lipids content was performed by method of Bligh and Dyer (1959). pH: value was determined potentiometrically according to Korkeala et al.

(1986) by using of pH meter pH211 (HANNA instruments). Extraction of muscle proteins: Extraction was conducted as described by Khan (1962), with some modifications. Meat sample with 2,5 g weight was homogenized with 48,5 cm3 PBS (phosphate buffered saline) buffer (49 mM Na2HPO4.7H20, 4,5 mM NaH2PO4.H20, KCl to obtain I=0,55). Homogenate was conditioned for 12 h in refrigerator and after that was centrifuged at 1000 g for 15 min. Protein content of supernatant was determined by Lowry method (Lowry, 1951). Free amino groups: Free amino groups are determined as follow: 2cm3 of supernatant were transferred in test tube and 1cm3 solution of ninhydrin reagent (0,5% ninhydrin; 10% Na2HPO2.12H2O; 6%KH2PO4; 0,3% fructose) was added. The mixture was heated in boiling water for 16 min. Sample was cooled at room temperature for 20 min and was diluted with 5 cm3 water-ethanol (3:2) solution of KIO3 (2%). Absorbance was read at 570 nm against blank sample using a UV-Vis spectrophotometer Helios Omega, furnished with software VISIONlite (Thermo Fisher Scientific, Madison, WI, USA). Concentration of free amino groups was calculated on standard curve made by leucine. Results were presented as mgLeucin/g meat. Electrophoresis: The preparation and running of the SDS-PAGE electrophoresis were done according Laemli (1970). For separation of proteins in protein extracts 10% SDS-PAGE was used. Electrophoresis was performed with Omni PAGA Electrophoresis system and CVS10D (Cleaver Scientific Ltd.) at 20mA per gel. Gels were stained with solution of Coomassie Brilliant Blue (0,2% coomassie brilliant blue, 40% ethanol, 7% CH3COOH) for 20 min and destained by solution without coomassie blue. Gels were scanned and analyzed with software GelAnalyzer 2010. Band area of each protein was determined and band area of each protein/total proteins band area ratio was calculated. Molecular weight of the studied proteins was determined by using of protein standard (Bio-Rad). Statistical analysis. Statistical analyses were carried out on the averages of the triplicate results. Data were analyzed by the analysis of variance (one-way ANOVA) method with a

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significant level of P 0.05 (Draper and Smith, 1998). The Duncan’s multiple comparison test (SPSS) with a significant difference set at P 0.05 was used to compare sample means. Significant differences between means less than 0.05 were considered statistically significant (Kenward, 1987). All statistical procedures were computed using the Microsoft Excel 5.0 software. 3.Results and discussion Physicochemical analysis: The physicochemical characteristics of studied chicken breast and leg samples are shown in Table 1. It is evident that leg and breast meat had similar moisture and ash contents. Protein content of breast muscles was significantly (P 0.05) higher and fat content was significantly (P 0.05) lower than the leg muscles. The pH values of breast meat were slightly lower in comparison with leg meat. For both types of

muscles pH increased significantly (P 0.05) during chilled storage, thus indicating for proteolysis development. Greater increase of pH values was found in leg meat compared to the breast meat. Electrophoresis: Electrophoresis of extracted salt soluble proteins from chicken breast and leg muscles showed from 18 to 24 protein bands. Fig. 1 presents electrophoregrams of salt soluble proteins of chicken breast muscles (A) and leg muscles (B) after 1st and 7th day of chilled storage (2nd and 9th day postmortem, respectively). Identification of some typical meat proteins was performed by using of standard proteins. This way myosin, -actinin, actin, troponin-T and tropomyosin were identified. It is evident (Fig. 1) that during postmortem storage of chicken light and dark muscles some proteins were partially or completely degraded. Significant decrease of myofibrillar proteins with molecular weight Table 1

Breast muscles (BM) Chilled storage Moisture, Protein, Lipid, Ash, % % % % 1 day 72,05 19,31 1,63 1,07 7 day 71,87 19,35 1,71 1,10

Moisture, pH % 6,15 68,35 6,25 68,66

Leg muscles (LM) Protein, Lipid, Ash, pH % % % 15,65 8,22 0,88 6,45 15,36 7,96 0,97 6,95

Fig. 1. Electrophoregrams of salt soluble proteins of chicken: A - breast muscles (BM1 and BM7 at the1st and 7th day of chilled storage, respectively) and B - leg muscles (LM1 and LM7 at the1st and 7th day of chilled storage respectively).

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about 100 kDa (bands 6, 7 and 8 for breast muscle and bands 7 and 8 for leg muscle) was found. For both muscle types bands 2 and 8 completely disappeared after 7 days of storage, whereas the -actinin was partially degraded. Several differences in the protein changes of light and dark muscles were found. Results obtained showed that at the 7th day of chilled storage, protein with molecular weight 50-60 kDa (band 14) was accumulated in breast meat, whereas in thigh muscles the same protein totally disappeared. For both muscles, the proteins with molecular weight about 40 and 20-30 kDa (bands 17 and 21, respectively) decreased significantly (P 0.05). The rate of degradation of these proteins in chicken light and dark muscles is different. Greater decrease of troponin-T (band

17) content was established in leg meat in comparison with breast meat. In contradiction, more rapid degradation of the protein presented with band 21 was found in breast meat. No significant changes of the main muscle proteins myosin and actin were observed in the present study (Fig. 1). That statement was in agreement with the findings of Kolczak et al. (2003). The relative area representing band area of each protein to total protein bands area ratio was calculated for chicken breast and leg meat, respectively (Fig. 2 and Fig. 3). It was established, that the total protein bands area of both muscle types decreased significantly (P 0.05) during chilled storage, indicating for proteolysis development.

Fig. 2 Band area of salt soluble chicken breast proteins to total band area ratio: BM1 and BM7 at the1st and 7th day of chilled storage respectively.

Fig. 3 Band area of salt soluble chicken leg proteins to total band area ratio: LM1 and LM7 at the1st and 7th day of chilled storage respectively

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The total protein bands area of breast meat was higher compared to the leg meat (Fig. 1). Therefore, the proteolysis during postmortem storage of chicken dark muscles could be assumed as more intensive in comparison with that in the light muscles. Four new bands were detected after 7 days of chicken breast and leg meat storage at 0-2ºC. For both types of chicken muscles two of these bands were localized at the same place. First one (band 9) has molecular weight about 95 kDa, and second (band 18) about 40 kDa. Our findings were in agreement with the results obtained for calf and cow meat (Kolczak et al., 2003), and with suggestion of Taylor, et al. (1995) and Koohmaraie (1994). Greater (P 0.05) accumulation of protein with molecular weight 95 kDa (band 9) was established in light muscles compared to the dark muscles. The rest two new bands, found after 7 days of postmortem storage were different for the both types of muscles. In the range of 110 kDa to 150 kDa appeared one new protein band 5 for the breast meat and two (bands 4 and 6) for the leg meat. Kolczak et al. (2003) also observed accumulation of new proteins with similar molecular weight in bovines aged muscles. According to these authors, the new proteins found, probably are a product of high molecular weight myofibrillar proteins hydrolysis. A new low molecular weight protein (about 15-20 kDa) (band 23) was detected in breast meat after 7 days of chilled storage, which was absent in leg meat. Pronounced degradation of troponin-T and tropomyosin and accumulation of protein bands in range of 30 to 40 kDa during leg muscles storage was found (Fig. 3). A significant difference in protein changes between chicken light and dark muscles was observed in the range of 50 to 60 kDa (band 14). The content of this protein increased significantly in chicken breast muscles and decreased in chicken leg muscles during chilled storage. Generally the proteolytic changes of chicken dark muscles after 2th day post mortem seem to be more deeply. This is probably due to the higher activity of µ/mcalpain in ictio tibialis than pectoralis superficialis from chicken (Lee et al, 2007). Results obtained, showed that the pathway of chicken light and dark muscle proteolysis is not universal. Hwan and Bandman (1989), Morrison et al. (1998), Taylor et al. (1995) and Van Laack et al. (2000) also found that the rate and degree of protein degradation of meat proteins from different animal and muscle types are different.

Taylor et al. (1995), Koohmaraie (1994) suggested, that titin, nebulin, desmin, troponin-T and vinculin are most hydrolyzed proteins. Free amino groups. Increasing of free amino groups is associated with accumulation of end products of proteolysis. Free amino groups content of chicken breast and leg meat is shown in Table 2. Table 2. Free amino groups, mg Leucin/g meat Sample 1day 7day BM 20,81±0,27 23,75±0,66 LM 15,74±0,52 20,04±0,24 During the 7 days chilled storage of chicken light and dark muscles, the free amino groups content significantly (P 0.05) increased. Similar results were reported for bovine meat (Parrish et al., 1969) and chicken breast and leg meat (Khan and van den Berg, 1964). Free amino groups values reported by these authors were lower than ours, probably because of the differences in the extraction procedure. Higher increase of free amino groups content was found in chicken leg muscles (21,4%) in comparison with breast muscles (12,4%). 4. Conclusion Proteolysis of chicken light and dark muscles had similar general aspects. Most substantial was degradation of proteins about 100 kDa and from 20 kDa to 40 kDa, and appearance of proteins in the range of 110 to 150, 95 and 30-40 kDa. Some differences in the proteolytic changes of light and dark muscles were also found. Although, the breast as well as leg high molecular weight proteins were degraded by accumulation of polypeptides about 110-150 kDa, in breast muscles is appeared one protein (band 5), whereas in leg muscles appeared two new protein bands, which were clearly indicated (bands 4 and 6). On the other hand, the accumulation of proteins with molecular weight about 95 kDa was more intensive in leg muscles than in breast muscles. The higher pH values and free amino groups content, and lower total protein bands area of chilled stored leg (dark) muscles indicated for more intensive proteolysis associated with greater protein changes in comparison with the breast (light) muscles.

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