Chicken muscle homogenate gelation properties: effect of pH and muscle fiber type

Meat Science 64 (2003) 399–403 www.elsevier.com/locate/meatsci

Chicken muscle homogenate gelation properties: effect of pH and muscle fiber type Tomasz Lesio´wa,*, Youling L. Xiongb a

Quality Analysis Department, University of Economics, ul. Komandorska 118/120, 53-345 Wroc•aw, Poland b Department of Animal Sciences, University of Kentucky, Lexington, KY 40546, USA Received 23 April 2002; received in revised form 28 July 2002; accepted 2 August 2002

Abstract Gelation properties of chicken breast and thigh muscle homogenates at a protein concentration of 4.5% under different pH conditions (5.80–6.60) and those of myofibrillar proteins at a protein concentration of 2% were compared to determine the influence of muscle fibre types on gelation. The optimal gelling pH for breast muscle homogenates (pH 6.30) was slightly higher than that for thigh muscle homogenates (pH 5.80–6.30), a similar trend was found for the isolated chicken myofibrillar proteins (pH 6.00 for breast and 5.50 for leg). Similarly, the pH values at which breast muscle homogenate gels were weaker (pH < 6.20) or stronger (pH56.20) than thigh muscle homogenate gels were higher when compared with chicken breast and leg myofibrillar protein gels (pH <5.80 and pH> 5.90, respectively). # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Chicken; Muscle homogenate; Myofibrils; Salt soluble proteins; Gelation; pH

1. Introduction Myofibrillar protein functionality, particularly gelation, in relation to muscle food quality, has been extensively researched, and reviewed (Lefevre, Culioli, Joandel-Monier, & Ouali, 1999; Wang & Smith, 1994; Xiong, 1994). It has been suggested that the gelation properties of myofibrillar proteins are influenced by the distribution of specific fibre types in the muscle samples from which myofibrillar proteins are extracted. The latest literature concerning unfolding, aggregation and gelation of poultry muscle myofibrillar proteins with respect to muscle type, pH and heating conditions has been reviewed by Lesio´w and Xiong (2001b). It is stressed that under dynamic conditions aggregation plays a major role in producing gel elasticity differences between white and red myofibrillar proteins. In another review Lesio´w and Xiong (2001a) noted considerable concentration-dependent differences in the gelation properties of chicken and turkey white and red muscle myofibrils, at low (< 2.5%), intermediate (5–7%) and * Corresponding author. Tel.: +48-71-3680-427; fax: +48-713672-778. E-mail address: [email protected] (T. Lesio´w).

high (10%) protein concentrations, as well as that the gelation properties of myofibrillar proteins and comminuted meats are pH dependent. The object of this study was to compare the gelation properties of chicken breast and thigh muscle homogenates at an intermediate protein concentration (4.5%) and varying pH values (from 5.80 to 6.60), as well as those of myofibrillar proteins at a low protein concentration (2%).

2. Materials and methods 2.1. Muscles samples Investigations conducted in two different laboratories, were performed on chicken broilers stored at 2–4
C for 24 h post mortem. In each of the three to five replicated experiments, the combined breast or thigh/leg muscle from two broiler carcasses were used for the preparation of meat homogenates or myofibrillar proteins, i.e. myofibrils and salt-soluble proteins (SSP). After trimming off connective tissue and fat, muscles were separately ground through a 3 mm plate. The pH was measured with a pH-meter and electrode (type

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OSH-10–10) or with a combination probe connected to a Model 220 Corning pH meter (Xiong & Blanchard, 1994) inserted into the ground meat or muscle homogenates. Meat homogenates were obtained by homogenisation of 15 g ground breast (B) or 17.67 g ground thigh (T) muscles with 60 ml 0.67 M NaCl (cold) (at the pHs specified below) for 1 min at 4000 rpm. Depending on the natural pH of the muscle, which was 5.82  0.13 for breast muscle and 6.19  0.06 for thigh muscle (Lesio´w, 2001), solutions of NaCl of pH: 8.00; 6.50; 6.00 or 5.60 for breast muscle, and 6.85; 6.10; 5.40 and 3.50 for thigh muscle were added during homogenisation to give the final pH’s of homogenates close to specific values (if necessary re-adjusted with 0.1 M HCl or 0.1 M NaOH) i.e. 6.60; 6.30; 6.00 and 5.80 (Table 1, Fig. 1a). The protein content in the meat homogenates was 45.5 mg/g. The extractability of proteins from breast and thigh muscles (0.67 M NaCl, pH from 5.80 to 6.60) was determined according to Richardson and Jones (1987) with slight modifications, i.e. 0.67M NaCl solutions of pH 6.6; 6.3; 6.0 and 5.8 were used and centrifugation was at 5000 rpm for 30 min (Lesio´w, 2001). The protein content in both muscles and extracts was determined using the Kjeltec System (1026 Distilling Unit, Tecator, Sweden). Myofibrils and SSP were prepared as previously described (Xiong & Brekke, 1989). The isolation buffer for myofibrils (pH 7.00 or pH 7.40) was composed of 0.1 M NaCl, 50 mM phosphate, 1 mM NaN3 and 5 mM EDTA. After isolation and purification, the myofibrils were suspended in 8 volumes (w/v) of 0.1 M NaCl and adjusted to specific pH values, i.e. 5.87 and 6.38 (Xiong & Blanchard, 1994); 5.50, 5.80, 6.00, 6.50, 7.00 and 7.5 (Xiong, 1992) or 5.50–6.50 at 0.25 intervals (Xiong & Brekke, 1991) using 0.1 M HCl prior to centrifugation. Salt-soluble protein (SSP) was extracted from myofibrils in 0.6 M NaCl, 50 mM piperazine-N, N bis (PIPES) (pH 6.00) and 1mM NaN3, and separated from nonsoluble protein by centrifugation at 5.000g for 15 min. Protein concentration was determined by the biuret

method using bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as a standard. 2.2. Gelation properties For each pH value, two parallel samples from a meat homogenate were placed in the glass tubes (2540 mm; Dia.L) and were heated at 70
C for 30 min in a water bath. After heating, the gels were cooled in an ice slurry for 1 h and stored overnight at 2–4
C. Before textural analysis, gels were equilibrated at room temperature (20
C) for 30 min. Deformation of the gels upon compression was measured on a modified ‘‘Ho¨ppler’’ RheoViskometer (VEB Pru¨fgera¨te-Werk, Medingen, Sitz Freital, Germany) equipped with a flat 14.8 mm diameter plunger. Stepwise changed stress, in multiples of 0.285 kPa, was measured after each 30 s. The stress at failure, i.e., the stress required to rupture the gel, was taken as the gel strength (kPa). Myofibrils and salt-soluble protein were suspended (20 mg/ml) in 50 mM piperazine-N, N bis at pH 5.5–7.5 containing 0.6 M NaCl and gels were formed in 16.580 mm (Dia.L) glass vials by heating in a water bath from 20 to 70
C at 0.8
C/min (Xiong, 1992) or at 1
C/min (Xiong & Brekke, 1991). After 3-h storage at 2
C, gels were equilibrated at room temperature for 45 min prior to gel strength measurement. Gels in the glass tubes were penetrated with a 12.5 mm diameter stainless steel probe in an Instron Universal Testing instrument (Instron Corp., Canton, MA). The gels were penetrated for 15 mm at a constant cross-head speed of 20 mm/ min. The penetration force, defined as the force required to rupture the gels (first peak), was taken as the gel strength. Moreover, nondestructive, oscillatory measurements of the muscle homogenates during gelation (heated from 27 to 90
C at 3
C/min) were performed using a TMA/SS 150U (Seiko) as described (Lesio´w, 2001). The dynamic oscillatory measurements of myofibril and SSP suspensions during gelation (heated from 20 to 74
C at 1
C/min) were performed using a Bohlin

Table 1 Transition temperatures (T0 max and T0 min) and storage modulus at peak (G0 max) and at the end of heating (G0 74 thigh (T) salt soluble proteins (SSP), myofibrils and muscle homogenates at different pH Attribute

Myofibrils (Pa)

BpH 6.00 TpH 6.00 BpH 5.87


of chicken breast (B) and

Type of sample/pH SSP (Pa)

0

or 75
C)

T max ( C) 50.0a T0 min (
C) 55.0a 300.0a G0 max G0 74/75
C 461.0a

51.5b 58.0b 125.0b 106.0b

Muscle homogenate (kPa) TpH 5.87

BpH 6.38

49.4a (0.3) 51.0b (0.0) 50.3ab (0.6) 53.9a 54.7ab 55.7b 305.0a (33.0) 111.0b (25.0) 365.0a (42.0) 629.0a (102.0) 198.0b (24.0) 355.0c (95.0)

TpH 6.38

BpH 6.00

TpH 6.00

51.5b (0.5) 52.7a (1.2) 52.5a 58.3c 58.6a (0.2) 55.7b 82.0b (35.0) 6.0a (1.4) 47.3b 67.0d (10.0) 72.4a (8.5) 144.8b

BpH 6.30

TpH 6.30

(0.7) 55.6b (0.5) 59.0a (7.1) 71.4c (12.6) 163.4cd

(1.0) 54.8b (0.9) 58.5a (3.4) 47.4b (2.8) 152.5bd

(0.9) (0.9) (9.2) (2.3)

Means with different letters in the same row (separately for each type of sample) are significantly different at P<0.05. Values in parenthesis are standard errors for the means.

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Fig. 1. (a) Effect of pH on stress at failure (kPA) of chicken breast (1, closed symbols) and thigh (10 , open symbols) muscle homogenate gels. Total protein concentration in the gels was 45.5 mg/ml. (b) Effect of pH on gel rupture force (N) of chicken breast (2) and leg (20 ) myofibrils. Total protein concentration in the gels was 20 mg/ml (with permission from Xiong, 1992). (c) Effect of pH on gel rupture force (N) of hen breast (3) and leg (30 ) myofibrils. Total protein concentration in the gels was 20 mg/ml (with permission from Xiong & Brekke, 1991).

VOR rheometer (Bohlin Instruments, Inc., Cranbury, NJ) as described by Xiong (1993). Rheological properties of the meat homogenates and myofibrillar gelling systems (sol and gel) were described in terms of transition temperature (T 0 max and T 0 min) and storage modulus at peak (G0 max) and at the end of heating (G0 74 or 75
C). 2.3. Statistics Within each of three to five replications, for each pH value and muscle type, two parallel measurements of gelation properties were made. Reported data are means of 8–10 measurements (Fig. 1a); three replicates with duplicate measurements (Fig. 1b and c) or 4–6 measurements (Table 1). The analysis of variance and the Duncan’s multiple range test method (Oktaba, 1980) or the LSD procedure (Snedecor & Cochran, 1989) were used to identify significant differences at P < 0.05.

3. Results and discussion 3.1. Gelation determined by compression testing The gel strength of breast muscle homogenates increased with increasing pH up to 6.30 where a maximum strength value was obtained (Fig. 1a) (Lesio´w, 2000). A further increase in the pH to 6.60 slightly (but nonsignificantly, P > 0.05) decreased the gel strength when compared with the value at pH 6.30. Gelation of thigh muscle homogenates was not as sensitive to pH as that of the breast muscle homogenate which is in accordance with results on hen muscle myofibrils obtained by Xiong and Brekke (1991). The strength of thigh muscle homogenate gels was highest at pH 6.00. However, it was not significantly different from the values at pH 5.80 and 6.30. At pH 6.60 thigh muscle homogenate gels were weaker than at pH 6.00. Slightly higher values for the optimal gelation pH of breast compared with thigh muscle homogenates (pH 6.30

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versus pH 5.80–6.30), and weaker gel formation at higher pH values (Fig. 1b) agree with results reported by Xiong (1992) and Xiong and Brekke (1991) for chicken/hen breast and leg myofibrils. The gel strength of thigh muscle homogenates was significantly higher than that of breast muscle homogenates at pH 5.80–6.00 (Fig. 1a). This accords with the observation of Lan, Novakofski, McCusker, Brewer, Carr, and McKeith (1995) who found that at 5% protein concentration and pH 6.00, chicken breast myofibrils produced weaker gels than thigh myofibrils. However, the gel strength of breast muscle homogenates was significantly higher than that of thigh muscle homogenates at pH 6.30 (by 14.63%) and at pH 6.60 (by 18.14%). Xiong (1992) found that chicken leg myofibril gels formed at pH 5.50–5.80 were more rigid than the corresponding breast gels, but above pH 6.00, this was reversed (Fig. 1b). It was shown in another study (Xiong & Blanchard, 1994) that myofibrillar proteins isolated from chicken Pectoralis major and Pectoralis minor muscles formed stronger gels than those from thigh and drumstick muscles at pH from 5.87 to 6.53. This finding agrees with our results for muscle homogenates; however, the pH at which breast muscle homogenate gels were stronger than those from thigh muscles was shifted to a higher pH value, i.e. pH 6.30. In contrast, hen breast myofibrillar gels were stronger than those made from leg myofibrils over the entire pH range studied, i.e. pH > 5.50–6.50 (Fig. 1c). It thus appears that pH-dependent interactions between protein molecules at an intermediate concentration in a muscle homogenate (mixture of sarcoplasmic and saltsoluble proteins, insoluble fraction of myofibrillar proteins, myofibrillar fragments and connective tissue proteins) may differ from those occurring in a dilute suspension of myofibrils (salt-soluble proteins and non soluble myofibrils). Extractability of proteins from breast and thigh muscles decreased with pH from 5.80 to 6.60. One exception was found for the extractability of proteins from thigh muscles at pH 6.00 and 6.30 for which the values were not significantly different (Lesio´w, 2001). An inverse relationship was found between the strength of breast muscle homogenate gels and protein extractability over the pH range studied, while for thigh muscle homogenate gels no such relationship was evident (Lesio´w, 2001). The respective equations between stress at failure of gel homogenates (SF) and extractability (E) of proteins from breast (B) and thigh (T) muscles are: SFB ¼ 0:289EB þ 34:050 SFT ¼ 0:100ET  0:056

r ¼ 0:883ðrk ¼ 0:878 at P < 0:05Þ and r ¼ 0:767

Thus, variations in gel strength between breast and thigh muscle homogenates can not be fully explained by

differences in protein extractability; but may be ascribed to the isoforms of myosin (Asghar, Morita, Samejima, & Yasui, 1984; Morita, Choe, Yamamoto, Samejima, & Yasui, 1987) and different protein-protein and other meat component interactions. Xiong and Brekke (1991) have shown that solubility (SSP from myofibrils) differences between chicken breast and leg myofibrils only partially contributed to the variation in gel strength, because such differences could only alter the volume of the effective gelling components without affecting the specific protein bonds in the gel matrix. This hypothesis is supported by the observation that at pH values above 6.00, where changes in solubility of myofibrillar proteins of breast muscle were very small but yet, the proteins formed substantially weaker gels as the pH was raised from 6.00 to 7.00 (Xiong, 1994). In the case of myofibrils from leg muscles, their gelation properties are even less directly related to protein solubility because their optimal pH was 5.50 at which protein solubility was low compared with the solubility at higher pH values. 3.2. Gelation determined by dynamic elasticity testing Results of dynamic testing of gelled chicken muscle homogenates are presented in Table 1. Rheological properties of muscle homogenates were clearly pHdependent. At pH 6.00 the storage modulus at peak (G0 max) and at the end of heating (G0 75
C) of breast muscle homogenates was lower than for thigh muscle homogenates. However, at pH 6.30 the reverse was seen but only for the storage modulus at peak (G0 max) with breast muscle homogenates producing more elastic gels. The values of G0 max and G0 75
C for breast meat homogenate at pH 6.30 were higher than at pH 6.00, but for thigh meat homogenates the values were not significantly different. Therefore, optimum gelation of breast meat homogenates was at pH 6.30 and for thigh muscle homogenates was over a slightly wider pH range (6.00–6.30). Chicken breast (white) SSP and myofibrils developed a more elastic gel structure (greater G0 max and G0 74
C) than the corresponding thigh proteins at all pH values studied (Table 1), indicating that the thigh proteins were less able to form a cross-linked gel network. However, differing from muscle homogenates, other workers have found that for isolated breast muscle proteins when a pH of 5.50–6.50 favors the formation of an elastic gel network compared with other pH values (G0 80 or 74
C) (Wang, Smith, & Steffe, 1990; Xiong & Blanchard, 1994). In addition, thigh muscle myofibrils formed more elastic gels (G0 74
C) at pH 5.87 than at 6.38 while changes of G0 max and G0 75
C for thigh muscle homogenates at pH 6.00 and 6.30 were not significant (Table 1). In spite of a similar pattern of changes in storage modulus of chicken breast and thigh SSP at pH 6.00 (Xiong, 1994), myofibrils at pH 5.87 and 6.38 (Xiong & Blanchard, 1994) and muscle homogenates at pH 6.00

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and 6.30 (Lesio´w, 2001), i.e. after reaching a maximum, the G0 declined rapidly, muscle homogenates attained peak values at higher temperatures (T 0 max) than SSP or myofibrils (Table 1). This difference was more pronounced at pH 6.30 for breast and thigh at pH 6.30/6.38 compared with breast and thigh at pH 6.00/5.87, probably reflecting the contribution of muscle constituents other than myofibrillar proteins. The T 0 max values for breast myofibrils at pH 6.38 and breast muscle homogenates at pH 6.00 and 6.30 were not significantly different from the corresponding values for thigh myofibrils and muscle homogenates (Table 1). However, the T0 max values for breast SSP at pH 6.00 and breast myofibrils at pH 5.87 were significantly different from their thigh counterparts. The T 0 min values for both thigh SSP and myofibrils were shifted to higher temperatures when compared with breast proteins. Such a dependence was not found in the case of muscle homogenates (Table 1).

4. Conclusions Optimal gelation of breast muscle homogenates, as determined by gel strength or dynamic elasticity was at pH 6.30, while for thigh muscle homogenates, it was at pH 5.80–6.30. Similarly, isolated chicken breast proteins (SSP, myofibrils) had a higher pH optimum than their leg counterpart (pH 6.00 versus 5.50) for optimal gelation, although for both muscle types, the pH optima was shifted to a lower pH when compared with muscle homogenates. Breast muscle homogenate gels were weaker at pH 5.80 and 6.00 than thigh muscle homogenates and the reverse relation was found at pH 6.30 and 6.60. Breast myofibrils and salt-soluble proteins generally formed stronger gels than thigh proteins except at pH< 5.80. In light of the critical importance of gelation in comminuted and whole muscle manufactured poultry products, the results from the present study indicate the necessity to process chicken light (breast) and dark (thigh or leg) meat homogenates with different formulations and pH and thermal conditions for optimal product quality.

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