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Protein denaturation and water–protein interactions as affected by low temperature long time treatment of porcine Longissimus dorsi
Meat Science 88 (2011) 718–722
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Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Protein denaturation and water–protein interactions as affected by low temperature long time treatment of porcine Longissimus dorsi Line Christensen a, Hanne C. Bertram b,⁎, Margit D. Aaslyng c, Mette Christensen a a b c
Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark Danish Meat Research Institute, Technological Institute, Maglegaardsvej 2, DK-4000 Roskilde, Denmark
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 28 February 2011 Accepted 1 March 2011 Keywords: Diameter DSC Low field NMR Sarcomere length Cooking
a b s t r a c t The relationship between water–protein interactions and heat-induced protein denaturation in low temperature long time (LTLT) treated pork Longissimus dorsi was investigated by combining low-field NMR T2 relaxometry with DSC measurements and measures of shrinkage of porcine Longissimus dorsi heated to 53 °C, 55 °C, 57 °C and 59 °C for either 3 or 20 h. Water within the myofibrils, measured by NMR T21 relaxation times, was affected by both temperature and holding time during LTLT treatment between 53 °C and 59 °C. The changes in NMR T21 relaxation times were associated with decreased fiber diameter and increased cooking loss, revealing a relationship between transverse shrinkage, water–protein interactions and cooking loss. DSC measurements revealed a concomitant decrease in ΔH68 °C, which suggests impact of collagen denaturation on the retention of water within the meat during LTLT treatment. Furthermore, a decrease in ΔH75 °C suggested that prolonged cooking (20 h) resulted in actin denaturation leading to decreased T21 relaxation times and higher cooking loss. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Prolonged heat treatment at low temperature (LTLT) of meat is being increasingly used by the catering industry because the treatment improves tenderness and juiciness, and a more uniform color is retained in the meat. It has been shown that meat is more tender and has less cooking loss during LTLT treatment of both pork (Christensen, Ertbjerg, Aaslyng, & Christensen, 2011) and beef (Bramblett, Hostetler, Vail, & Draudt, 1959; Bouton & Harris, 1981; Beilken, Bouton, & Harris, 1986). Juiciness of meat is affected by both cooking rate and final temperature (Laakkonen, Wellington, & Sherbon, 1970; Aaslyng, Bejerholm, Ertbjerg, Bertram, & Andersen, 2003; Bejerholm & Aaslyng, 2004), and by using low-field nuclear magnetic resonance (NMR) relaxometry, Bertram, Aaslyng, and Andersen (2005) demonstrated that differences in juiciness as a result of different endpoint cooking temperatures could be ascribed to differences in water distribution within the meat. The majority of the water in the muscle (approximately 90–95%) is held within the myofibrils, and any large changes in the myofibrillar spacing will affect the mobility and distribution of water within the meat structure. Heat-induced transverse shrinkage of muscle fibers has been found to occur at temperatures between 45 °C and 60 °C, while longitudinal shrinkage has been found to occur at 60–90 °C (Offer,
⁎ Corresponding author. Tel.: +45 8999 3344. E-mail address: [email protected] (H.C. Bertram). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.03.002
Restall, & Trinick, 1984; Palka & Daun, 1999). In low temperature long time treatments where a temperature in the range of 50–60 °C typically is applied only transverse shrinkage of the muscle fibres will occur according to Palka and Daun (1999). However, to our knowledge, the effect of prolonged holding periods on shrinkage has not been explored. Low-field NMR has been widely used to study water–protein interactions in meat (Bertram & Andersen, 2004; Bertram, Meyer, & Andersen, 2009) and according to Bertram, Wu, van den Berg, and Andersen (2006) and Micklander, Peshlov, Purslow, and Engelsen (2002) changes in NMR T2 relaxation times upon cooking of porcine longissimus dorsi between 53 and 58 °C reflected myosin denaturation and longitudinal shrinkage of muscle fibres at 57 °C. Furthermore, Bertram, Purslow, and Andersen (2002) found the relaxation time of the major T2 component reflected the myofilamentous lattice spacing, which during heat treatment has been suggested to be a consequence of heat-induced shrinkage (Bertram et al., 2005). Nevertheless, studies on water–protein interactions as affected by prolonged heating at temperatures between 53 °C and 58 °C are lacking. Heat-induced denaturation of meat proteins has been studied for decades using differential scanning calorimetry (DSC) (Stabursvik & Martens, 1980; Martens, Stabursvik, & Martens, 1982; Bruggemann, Brewer, Risbo, & Bagatolli, 2010). However, to our knowledge, no studies have elucidated the effect of prolonged heating time on the denaturation of meat proteins. In a previous study (Christensen et al., 2011) it was found that cooking loss increased significantly when heating temperature was
L. Christensen et al. / Meat Science 88 (2011) 718–722
raised from 53 °C to 58 °C at heating times between 3 h and 20 h in porcine Longissimus dorsi. The aim of the present study was therefore to investigate the relationship between heat-induced protein denaturation and water–protein interactions in LTLT treated porcine Longissimus dorsi. Low-field NMR T2 relaxation measurements were combined with DSC measurements and measures of shrinkage of porcine Longissimus dorsi heated to temperatures between 53 °C and 59 °C for either 3 h or 20 h in order to obtain further understanding of how water–protein interactions within meat relate to structural changes occurring during LTLT treatment.
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Pro Plus (Media Cybernetics Inc., Silver Spring, USA). Sarcomere length and fiber diameter are the means of 10 sarcomeres/fibres. 2.5. Differential scanning calorimetric analysis
2. Materials and methods
DSC analyses were carried out on a MicroDSC III (Setaram, Caluire, France). Approximately 700 mg LTLT treated samples were placed into a DSC sample cell and placed in the DSC together with a reference cell containing water. DSC scans were recorded from 25 °C to 90 °C at a rate of 1 °C/min. The thermograms were analysed in SetSoft (Setaram, Caluire, France) where maximum temperatures and areas under the peaks (ΔH) were determined.
2.1. Raw material
2.6. Statistical analysis
M. longissimus dorsi (LD) were excised 24 h post mortem from both sides of 6 slaughter pigs (slaughter weight 77–83 kg). pH was measured and used as a selection criteria for the muscles (pH 5.55–5.68). After selection the muscles were vacuum packed and stored for 4 days at 4 °C. Then 3 samples of app. 4 × 12 × 10 cm were cut from each muscle, vacuum packed and frozen at −20 °C.
Analysis of variance was performed using SAS Software 9.2. As fixed effects the model included temperature, time and their interaction, while animal and sample location were included as random effects. In addition, principal component analysis (PCA) was carried out on the NMR T2 data using the Unscrambler software version 9.2 (Camo, Oslo, Norway). 3. Results
2.2. Heat treatment Muscle samples were thawed overnight at 4 °C and LTLT treatment was carried out in water baths (ICC “Roner”, Frinox Aps, Hillerød, Denmark) at 53 °C, 55 °C, 57 °C and 59 °C. Each water bath contained 3 samples at the same time and heat treatments were performed for 3 h and 20 h. LTLT treatments were arrested by immersing the samples in ice water for 10 min. Cooking loss was measured by weighing the samples before and after heat treatment and expressed as a percentage of the original weight. Mean values from 3 repetitions of each LTLT treatment were obtained. The LTLT treated samples for DSC, fiber diameter and sarcomere length were frozen at − 20 °C. The samples for NMR relaxometry were LTLT treated as described above however, cooking loss was not measured on these samples. 2.3. Low-field NMR The NMR relaxation measurements were performed on a Maran Benchtop Pulsed NMR Analyzer (Resonance Instruments, Witney, UK) with a resonance frequency for protons of 23.2 MHz. The NMR instrument was equipped with an 18 mm variable temperature probe. The LTLT treated meat strips were cut and placed in cylindrical glass tubes for the NMR measurements and tempered in a 25 °C water bath for 15–20 min prior to measurement. Proton transverse relaxation, T2, was measured using the Carr–Purcell–Meiboom–Gill sequence (CPMG). The T2 measurements were performed with a τ value (time between 90° pulse and 180° pulse) of 150 μs. The 90° and 180° pulses were 8.2 and 16.4, respectively. The repetition time between two scans was 3 s. Data from 4096 echoes were acquired as 16 scan repetitions, with one dummy scan. Only data from even-numbered echoes were used in data analysis to avoid the influence of imperfect pulse settings. The obtained T2 relaxation decays were analyzed using distributed exponential fitting analysis (Menon, Rusinko, & Allen, 1991). Mean time constants for the individual relaxation populations were calculated from the distributed T2 relaxation times, and values obtained for the major water population T21 (Bertram et al., 2001) are reported.
The overall effect of temperature on NMR T2 relaxation times is illustrated in the PCA scoreplot in Fig. 1, where T2 relaxation times measured in samples heated at 53 °C and 55 °C are clearly separated from samples heated at 57 °C and 59 °C. Fig. 2 shows examples of distributed NMR T2 relaxation times measured at 53 °C and 59 °C for 3 h (Fig. 2A) and 20 h (Fig. 2B). Two populations with relaxation times centered around 1–5 (T2B) and 40–120 (T21) ms, respectively, were observed. At 57 and 59 °C a tendency for splitting of the T21 population into two sub-populations was observed. A significant main effect of cooking temperature (P b 0.0001) on the distribution of T2 relaxation times was observed, as the T21 population moved towards faster relaxation times with increasing temperature (Fig. 2 and Table 1). In addition, when heating time was increased from 3 h to 20 h, T21 relaxation times decreased (P b 0.0001) at all temperatures (Table 1). The percentage cooking loss after LTLT treatment of pork LD is shown in Table 1. Significant main effects of cooking temperature (P b 0.0001) and time (P b 0.0001) on cooking loss were observed. Cooking loss increased both with increasing time and temperature, though no significant changes (P N 0.05) were observed in the cooking losses at 55 °C and 57 °C. A significant main effect of cooking temperature (P b 0.01) on fiber diameter was found. Samples heated for 3 h at 53 °C had larger (P b 0.001) fiber diameters (Table 1) compared with those heated at
2.4. Fiber diameter and sarcomere length Sarcomere length and fiber diameter were measured on frozen LTLT treated samples. Slices 10 μm thick were cut at −20 °C longitudinally to the fibre direction on a Frigocut (AusJena, Germany) and fixed on a glass slide. Sarcomeres and diameters were examined with a light microscope equipped with a camera, and were measured using the software Image-
Fig. 1. PCA scatterplot with principal component 1 (PC1) versus principal component 2 (PC2) based on NMR T2 relaxation times obtained on porcine Longissimus dorsi after LTLT treatments at 53 °C, 55 °C, 57 °C and 59 °C for 3 and 20 h (explained variance: 52%,and 21%, respectively).
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A
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with increasing temperature from 53 °C to 59 °C at both 3 h and 20 h. Meat proteins with a denaturation temperature around 75 °C did not seem to denature at 3 h of heating between 53 °C and 59 °C, but at 20 h the endothermic peak decreased (P b 0.05).
2,5
2,0
Amplitude
4. Discussion 1,5
1,0
0,5
0,0 1
10
100
1000
Relaxation time (ms)
B
2,5
Amplitude
2,0
1,5
1,0
0,5
0,0 1
10
100
1000
Relaxation time (ms) Fig. 2. Representative distribution of T2 relaxation times for porcine Longissimus dorsi heated at 53 °C (bold line) and 59 °C (dotted line) for 3 h (A) and 20 h (B).
59 °C. After 20 h samples heated at 55 °C had larger (P b 0.04) fiber diameters compared with those heated at 59 °C. The average sarcomere lengths were 1.93 μm and 1.86 μm in samples heated for 3 h and 20 h respectively, however, no significant changes were observed. DSC on LTLT treated samples revealed two detectable endothermic peaks at app. 68 °C and 75 °C (Table 1). Significant main effects of cooking temperature (P b 0.01) and time (P b 0.01) on the enthalpies for both peaks were found. As shown by a decrease in enthalpy for the peak at app. 68 °C, an increase in protein denaturation was observed
In the present study the effect of LTLT treatment on water–protein interactions and protein denaturation was investigated using a combination of low-field NMR relaxometry and DSC. NMR T21 relaxation time decreased markedly from 55 °C to 57 °C indicating that structural changes in the myofibrils affecting water–protein interactions occurred in this temperature region. The T21 relaxation population in porcine LD has been reported to reflect water located within the highly organized protein structures of the myofibrils (Bertram, Purslow and Andersen, 2002). During cooking of meat the T21 relaxation time has been reported to decrease with increasing temperature (Bertram et al., 2005; Shaarani, Nott, & Hall, 2006; Wu, Bertram, Bocker, Ofstad, & Kohler, 2007). This was ascribed to heatinduced shrinkage of the meat resulting in a reduction in myofibrillar spacing. Shrinkage of meat during cooking can occur both transverse and longitudinal to the fiber direction, and shrinkage results in cooking loss. In the current study only transverse shrinkage, measured as a decrease in fiber diameter, was observed. Micklander et al. (2002) found a relationship between longitudinal shrinkage and T2 relaxation times at 57 °C in porcine LD, although, Offer et al. (1984) and Palka and Daun (1999) observed that shrinkage of meat during cooking at 45–60 °C is primarily transverse and at 60–90 °C primarily longitudinal to the fiber direction. This observation is in agreement with the findings in the present study. Longitudinal shrinkage may be a consequence of both sarcomere shortening and collagen shrinkage, however, in the present study, no significant differences in sarcomere length were observed between the treatments. Denaturation of structural proteins within the meat has been suggested to be responsible for heat-induced shrinkage of the fiber, and the associated loss of water due to the decreased water-holding capacity of denatured proteins. In the current study DSC of LTLT treated meat revealed 2 peaks at 68 °C and 75 °C, respectively. The decrease in ΔH68°C with increasing temperature may be caused by breakage of hydrogen bonds in the collagen causing water to be expelled from the structure. T21 relaxation time decreased with increasing temperatures, and together with an increased cooking loss these findings suggest a reduced water-binding of the meat caused by changes in the collagen network. Hydroxyl groups stabilize the structure of collagen, and water forms hydrogen bonds between the hydroxyl groups and hydroxyproline, and consequently, thermal denaturation of collagen causes the breaking of bonds leading to shrinkage. Shrinkage of the collagen surrounding the myofibrils results in physical constraints on these structures and water is thereby forced out. In agreement with the present findings, Rochdi, Foucat, and Renou (2000) found that in bovine intramuscular connective tissue, a decrease in ΔH was associated with a decreased T21 relaxation time. Furthermore, it is noticeable that there is a tendency for splitting of the
Table 1 LS means and standard errors (SE) of NMR T21 relaxation times, cooking loss (%), fiber diameter, sarcomere length (μm), and DSC enthalpies (ΔH68°C and ΔH75°C) in low temperature–long time heat treated porcine Longissimus dorsi at 53 °C, 55 °C, 57 °C and 59 °C at 3 h and 20 h. Letters a–d refer to significance (P b 0.05) between treatments (n= 3). 3h
20 h
53 °C T21 (ms) Cooking loss (%) Fiber diameter (μm) Sarcomere length (μm) ΔH68°C (J/g) ΔH75°C (J/g)
55 °C a
112.6 8.0e 65.5a 1.94 0.280a 0.333b
ab
108.1 10.8de 54.8bc 1.89 0.149c 0.385a
57 °C
59 °C
c
c
81.1 14.0cd 57.6bc 1.94 0.126cd 0.324b
80.3 17.9bc 51.6c 1.96 0.024e 0.308b
53 °C bc
91.1 12.7d 59.6abc 1.81 0.214b 0.225c
55 °C
57 °C
59 °C
c
d
d
80.3 19.5b 61.7ab 1.86 0.059e 0.180d
51.4 18.0b 55.2bc 1.97 0.078de 0.096e
60.7 25.6a 53.5c 1.79 0.016e 0.037f
SE 9.6 1.3 3.1 0.1 0.02 0.02
L. Christensen et al. / Meat Science 88 (2011) 718–722
T21 population at 57 and 59 °C, which might be caused by structural constraints induced by collagen shrinkage at these temperatures. However, it is unclear which proteins the two endothermic DSC peaks at 68 °C and 75 °C arise from in the present study. Stabursvik and Martens (1980) found the denaturation temperature of isolated collagen to be 68 °C, however, Bruggemann et al. (2010) showed that connective tissue denaturation begins at 57 °C. In another study combining DSC and low field-NMR on pork (Bertram et al., 2006), 3 peaks were observed in the DSC thermogram at 54 °C, 65 °C and 77 °C, respectively. The authors tentatively ascribed the peak at 65 °C to sarcoplasmic proteins and the peak at 77 °C to actin denaturation. The consequences of denaturation of sarcoplasmic proteins on structural changes and water-binding in meat might be relatively small, since denatured sarcoplasmic proteins have been reported to participate little in the consistency of cooked meat (Tornberg, 2005). Therefore, the contribution from sarcoplasmic proteins on water distribution in LTLT treated meat is believed to be of minor importance. In the present study ΔH75°C decreased when the heating time was increased from 3 h to 20 h. If this peak corresponds to actin, it seems that actin denaturation was initiated after 20 h of heating at 53 °C and enhanced at increasing temperatures up to 59 °C. Decreasing ΔH75°C and the concomitant decrease in T21 relaxation times from 3 h to 20 h at all temperatures may therefore be a result of actin denaturation affecting the intrinsic waterbinding capacity within the myofibrils. Although actin has been reported to denature at around 77 °C (Stabursvik & Martens, 1980), the effect of prolonged heating has not previously been investigated. At 20 h of heating decreases in fiber diameter were observed with increasing temperature, which together with the decrease in ΔH75°C and T21 relaxation time, could suggest an influence of denaturation of actin on the transverse shrinkage of the muscle fiber at prolonged heating times. Myosin has been reported to denature at around 53–58 °C (Wright & Wilding, 1984) and from the present study it seems that myosin was already denatured at 53 °C since no peak was observed on the DSC thermograms around this temperature. It is thus believed that myosin denatured at lower heating temperature/times than investigated in the present study, which is in accordance with Bruggemann et al. (2010) who observed that the signal from the myofibers vanished at 53 °C (in Second Harmonic Generation Microscopy), suggesting complete denaturation of the myosin rod domain at 53 °C. This study is, to our knowledge, the first to demonstrate the effect of prolonged heat treatment at low temperatures on water–protein interactions and associated structural changes caused by protein denaturation in porcine muscle. In the current study water within the myofibrils, measured by NMR T21 relaxation times, was affected by both time and temperature during LTLT treatment between 53 °C and 59 °C. The changes observed in NMR T21 relaxation times were associated with increased cooking losses and decreases in fiber diameter. Furthermore, decreasing enthalpies at 75 °C (ΔH75°C), suggested actin denaturation, might be related to the transverse shrinkage of the muscle fiber leading to higher cooking losses and decreased T21 relaxation times. Also, a concomitant decrease in ΔH68°C, which may represent denaturation of collagen, suggested an influence of collagen on the distribution of water within the meat during LTLT treatment. 5. Conclusion The present study investigated the relationship between NMR T2 relaxation times and changes in cooking loss, fiber diameter, sarcomere length, and differential scanning calorimetric data as affected by LTLT treatment of porcine LD. The NMR T2 data revealed a significant change in water–protein interactions with increasing temperatures from 55 °C to 57 °C at 3 h and 20 h of heating. Increases in transverse shrinkage of the fiber were observed between 53 °C and 59 °C with a concomitant increase in cooking loss. Thus, the present
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study indicated a relationship between transverse shrinkage, water– protein interactions and cooking loss. DSC measurements showed a concomitant decrease in ΔH68°C, which suggests a negative contribution from collagen denaturation on the retention of water within the meat during LTLT treatment. Furthermore, a decrease in ΔH75°C suggested that prolonged cooking (20 h) caused denaturation of actin, which affected water–protein interactions and resulted in higher cooking losses.
Acknowledgement The authors would like to thank Peder Vithus Johansen (DMRI) for assistance on sampling, Nina Eggers (Aarhus University), Julie Cecilia Stefansen and Jonas T. Sprehn (UC-LIFE) for laboratory assistance. The authors thank The Danish Ministry of Food, Agriculture and Fisheries for funding the projects entitled “Safety and gastronomic quality of new LTLT treatments of meat” and “Integrated characterization of food quality and microbial safety”.
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Contents lists available at ScienceDirect
Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Protein denaturation and water–protein interactions as affected by low temperature long time treatment of porcine Longissimus dorsi Line Christensen a, Hanne C. Bertram b,⁎, Margit D. Aaslyng c, Mette Christensen a a b c
Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark Danish Meat Research Institute, Technological Institute, Maglegaardsvej 2, DK-4000 Roskilde, Denmark
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 28 February 2011 Accepted 1 March 2011 Keywords: Diameter DSC Low field NMR Sarcomere length Cooking
a b s t r a c t The relationship between water–protein interactions and heat-induced protein denaturation in low temperature long time (LTLT) treated pork Longissimus dorsi was investigated by combining low-field NMR T2 relaxometry with DSC measurements and measures of shrinkage of porcine Longissimus dorsi heated to 53 °C, 55 °C, 57 °C and 59 °C for either 3 or 20 h. Water within the myofibrils, measured by NMR T21 relaxation times, was affected by both temperature and holding time during LTLT treatment between 53 °C and 59 °C. The changes in NMR T21 relaxation times were associated with decreased fiber diameter and increased cooking loss, revealing a relationship between transverse shrinkage, water–protein interactions and cooking loss. DSC measurements revealed a concomitant decrease in ΔH68 °C, which suggests impact of collagen denaturation on the retention of water within the meat during LTLT treatment. Furthermore, a decrease in ΔH75 °C suggested that prolonged cooking (20 h) resulted in actin denaturation leading to decreased T21 relaxation times and higher cooking loss. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Prolonged heat treatment at low temperature (LTLT) of meat is being increasingly used by the catering industry because the treatment improves tenderness and juiciness, and a more uniform color is retained in the meat. It has been shown that meat is more tender and has less cooking loss during LTLT treatment of both pork (Christensen, Ertbjerg, Aaslyng, & Christensen, 2011) and beef (Bramblett, Hostetler, Vail, & Draudt, 1959; Bouton & Harris, 1981; Beilken, Bouton, & Harris, 1986). Juiciness of meat is affected by both cooking rate and final temperature (Laakkonen, Wellington, & Sherbon, 1970; Aaslyng, Bejerholm, Ertbjerg, Bertram, & Andersen, 2003; Bejerholm & Aaslyng, 2004), and by using low-field nuclear magnetic resonance (NMR) relaxometry, Bertram, Aaslyng, and Andersen (2005) demonstrated that differences in juiciness as a result of different endpoint cooking temperatures could be ascribed to differences in water distribution within the meat. The majority of the water in the muscle (approximately 90–95%) is held within the myofibrils, and any large changes in the myofibrillar spacing will affect the mobility and distribution of water within the meat structure. Heat-induced transverse shrinkage of muscle fibers has been found to occur at temperatures between 45 °C and 60 °C, while longitudinal shrinkage has been found to occur at 60–90 °C (Offer,
⁎ Corresponding author. Tel.: +45 8999 3344. E-mail address: [email protected] (H.C. Bertram). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.03.002
Restall, & Trinick, 1984; Palka & Daun, 1999). In low temperature long time treatments where a temperature in the range of 50–60 °C typically is applied only transverse shrinkage of the muscle fibres will occur according to Palka and Daun (1999). However, to our knowledge, the effect of prolonged holding periods on shrinkage has not been explored. Low-field NMR has been widely used to study water–protein interactions in meat (Bertram & Andersen, 2004; Bertram, Meyer, & Andersen, 2009) and according to Bertram, Wu, van den Berg, and Andersen (2006) and Micklander, Peshlov, Purslow, and Engelsen (2002) changes in NMR T2 relaxation times upon cooking of porcine longissimus dorsi between 53 and 58 °C reflected myosin denaturation and longitudinal shrinkage of muscle fibres at 57 °C. Furthermore, Bertram, Purslow, and Andersen (2002) found the relaxation time of the major T2 component reflected the myofilamentous lattice spacing, which during heat treatment has been suggested to be a consequence of heat-induced shrinkage (Bertram et al., 2005). Nevertheless, studies on water–protein interactions as affected by prolonged heating at temperatures between 53 °C and 58 °C are lacking. Heat-induced denaturation of meat proteins has been studied for decades using differential scanning calorimetry (DSC) (Stabursvik & Martens, 1980; Martens, Stabursvik, & Martens, 1982; Bruggemann, Brewer, Risbo, & Bagatolli, 2010). However, to our knowledge, no studies have elucidated the effect of prolonged heating time on the denaturation of meat proteins. In a previous study (Christensen et al., 2011) it was found that cooking loss increased significantly when heating temperature was
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raised from 53 °C to 58 °C at heating times between 3 h and 20 h in porcine Longissimus dorsi. The aim of the present study was therefore to investigate the relationship between heat-induced protein denaturation and water–protein interactions in LTLT treated porcine Longissimus dorsi. Low-field NMR T2 relaxation measurements were combined with DSC measurements and measures of shrinkage of porcine Longissimus dorsi heated to temperatures between 53 °C and 59 °C for either 3 h or 20 h in order to obtain further understanding of how water–protein interactions within meat relate to structural changes occurring during LTLT treatment.
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Pro Plus (Media Cybernetics Inc., Silver Spring, USA). Sarcomere length and fiber diameter are the means of 10 sarcomeres/fibres. 2.5. Differential scanning calorimetric analysis
2. Materials and methods
DSC analyses were carried out on a MicroDSC III (Setaram, Caluire, France). Approximately 700 mg LTLT treated samples were placed into a DSC sample cell and placed in the DSC together with a reference cell containing water. DSC scans were recorded from 25 °C to 90 °C at a rate of 1 °C/min. The thermograms were analysed in SetSoft (Setaram, Caluire, France) where maximum temperatures and areas under the peaks (ΔH) were determined.
2.1. Raw material
2.6. Statistical analysis
M. longissimus dorsi (LD) were excised 24 h post mortem from both sides of 6 slaughter pigs (slaughter weight 77–83 kg). pH was measured and used as a selection criteria for the muscles (pH 5.55–5.68). After selection the muscles were vacuum packed and stored for 4 days at 4 °C. Then 3 samples of app. 4 × 12 × 10 cm were cut from each muscle, vacuum packed and frozen at −20 °C.
Analysis of variance was performed using SAS Software 9.2. As fixed effects the model included temperature, time and their interaction, while animal and sample location were included as random effects. In addition, principal component analysis (PCA) was carried out on the NMR T2 data using the Unscrambler software version 9.2 (Camo, Oslo, Norway). 3. Results
2.2. Heat treatment Muscle samples were thawed overnight at 4 °C and LTLT treatment was carried out in water baths (ICC “Roner”, Frinox Aps, Hillerød, Denmark) at 53 °C, 55 °C, 57 °C and 59 °C. Each water bath contained 3 samples at the same time and heat treatments were performed for 3 h and 20 h. LTLT treatments were arrested by immersing the samples in ice water for 10 min. Cooking loss was measured by weighing the samples before and after heat treatment and expressed as a percentage of the original weight. Mean values from 3 repetitions of each LTLT treatment were obtained. The LTLT treated samples for DSC, fiber diameter and sarcomere length were frozen at − 20 °C. The samples for NMR relaxometry were LTLT treated as described above however, cooking loss was not measured on these samples. 2.3. Low-field NMR The NMR relaxation measurements were performed on a Maran Benchtop Pulsed NMR Analyzer (Resonance Instruments, Witney, UK) with a resonance frequency for protons of 23.2 MHz. The NMR instrument was equipped with an 18 mm variable temperature probe. The LTLT treated meat strips were cut and placed in cylindrical glass tubes for the NMR measurements and tempered in a 25 °C water bath for 15–20 min prior to measurement. Proton transverse relaxation, T2, was measured using the Carr–Purcell–Meiboom–Gill sequence (CPMG). The T2 measurements were performed with a τ value (time between 90° pulse and 180° pulse) of 150 μs. The 90° and 180° pulses were 8.2 and 16.4, respectively. The repetition time between two scans was 3 s. Data from 4096 echoes were acquired as 16 scan repetitions, with one dummy scan. Only data from even-numbered echoes were used in data analysis to avoid the influence of imperfect pulse settings. The obtained T2 relaxation decays were analyzed using distributed exponential fitting analysis (Menon, Rusinko, & Allen, 1991). Mean time constants for the individual relaxation populations were calculated from the distributed T2 relaxation times, and values obtained for the major water population T21 (Bertram et al., 2001) are reported.
The overall effect of temperature on NMR T2 relaxation times is illustrated in the PCA scoreplot in Fig. 1, where T2 relaxation times measured in samples heated at 53 °C and 55 °C are clearly separated from samples heated at 57 °C and 59 °C. Fig. 2 shows examples of distributed NMR T2 relaxation times measured at 53 °C and 59 °C for 3 h (Fig. 2A) and 20 h (Fig. 2B). Two populations with relaxation times centered around 1–5 (T2B) and 40–120 (T21) ms, respectively, were observed. At 57 and 59 °C a tendency for splitting of the T21 population into two sub-populations was observed. A significant main effect of cooking temperature (P b 0.0001) on the distribution of T2 relaxation times was observed, as the T21 population moved towards faster relaxation times with increasing temperature (Fig. 2 and Table 1). In addition, when heating time was increased from 3 h to 20 h, T21 relaxation times decreased (P b 0.0001) at all temperatures (Table 1). The percentage cooking loss after LTLT treatment of pork LD is shown in Table 1. Significant main effects of cooking temperature (P b 0.0001) and time (P b 0.0001) on cooking loss were observed. Cooking loss increased both with increasing time and temperature, though no significant changes (P N 0.05) were observed in the cooking losses at 55 °C and 57 °C. A significant main effect of cooking temperature (P b 0.01) on fiber diameter was found. Samples heated for 3 h at 53 °C had larger (P b 0.001) fiber diameters (Table 1) compared with those heated at
2.4. Fiber diameter and sarcomere length Sarcomere length and fiber diameter were measured on frozen LTLT treated samples. Slices 10 μm thick were cut at −20 °C longitudinally to the fibre direction on a Frigocut (AusJena, Germany) and fixed on a glass slide. Sarcomeres and diameters were examined with a light microscope equipped with a camera, and were measured using the software Image-
Fig. 1. PCA scatterplot with principal component 1 (PC1) versus principal component 2 (PC2) based on NMR T2 relaxation times obtained on porcine Longissimus dorsi after LTLT treatments at 53 °C, 55 °C, 57 °C and 59 °C for 3 and 20 h (explained variance: 52%,and 21%, respectively).
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A
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with increasing temperature from 53 °C to 59 °C at both 3 h and 20 h. Meat proteins with a denaturation temperature around 75 °C did not seem to denature at 3 h of heating between 53 °C and 59 °C, but at 20 h the endothermic peak decreased (P b 0.05).
2,5
2,0
Amplitude
4. Discussion 1,5
1,0
0,5
0,0 1
10
100
1000
Relaxation time (ms)
B
2,5
Amplitude
2,0
1,5
1,0
0,5
0,0 1
10
100
1000
Relaxation time (ms) Fig. 2. Representative distribution of T2 relaxation times for porcine Longissimus dorsi heated at 53 °C (bold line) and 59 °C (dotted line) for 3 h (A) and 20 h (B).
59 °C. After 20 h samples heated at 55 °C had larger (P b 0.04) fiber diameters compared with those heated at 59 °C. The average sarcomere lengths were 1.93 μm and 1.86 μm in samples heated for 3 h and 20 h respectively, however, no significant changes were observed. DSC on LTLT treated samples revealed two detectable endothermic peaks at app. 68 °C and 75 °C (Table 1). Significant main effects of cooking temperature (P b 0.01) and time (P b 0.01) on the enthalpies for both peaks were found. As shown by a decrease in enthalpy for the peak at app. 68 °C, an increase in protein denaturation was observed
In the present study the effect of LTLT treatment on water–protein interactions and protein denaturation was investigated using a combination of low-field NMR relaxometry and DSC. NMR T21 relaxation time decreased markedly from 55 °C to 57 °C indicating that structural changes in the myofibrils affecting water–protein interactions occurred in this temperature region. The T21 relaxation population in porcine LD has been reported to reflect water located within the highly organized protein structures of the myofibrils (Bertram, Purslow and Andersen, 2002). During cooking of meat the T21 relaxation time has been reported to decrease with increasing temperature (Bertram et al., 2005; Shaarani, Nott, & Hall, 2006; Wu, Bertram, Bocker, Ofstad, & Kohler, 2007). This was ascribed to heatinduced shrinkage of the meat resulting in a reduction in myofibrillar spacing. Shrinkage of meat during cooking can occur both transverse and longitudinal to the fiber direction, and shrinkage results in cooking loss. In the current study only transverse shrinkage, measured as a decrease in fiber diameter, was observed. Micklander et al. (2002) found a relationship between longitudinal shrinkage and T2 relaxation times at 57 °C in porcine LD, although, Offer et al. (1984) and Palka and Daun (1999) observed that shrinkage of meat during cooking at 45–60 °C is primarily transverse and at 60–90 °C primarily longitudinal to the fiber direction. This observation is in agreement with the findings in the present study. Longitudinal shrinkage may be a consequence of both sarcomere shortening and collagen shrinkage, however, in the present study, no significant differences in sarcomere length were observed between the treatments. Denaturation of structural proteins within the meat has been suggested to be responsible for heat-induced shrinkage of the fiber, and the associated loss of water due to the decreased water-holding capacity of denatured proteins. In the current study DSC of LTLT treated meat revealed 2 peaks at 68 °C and 75 °C, respectively. The decrease in ΔH68°C with increasing temperature may be caused by breakage of hydrogen bonds in the collagen causing water to be expelled from the structure. T21 relaxation time decreased with increasing temperatures, and together with an increased cooking loss these findings suggest a reduced water-binding of the meat caused by changes in the collagen network. Hydroxyl groups stabilize the structure of collagen, and water forms hydrogen bonds between the hydroxyl groups and hydroxyproline, and consequently, thermal denaturation of collagen causes the breaking of bonds leading to shrinkage. Shrinkage of the collagen surrounding the myofibrils results in physical constraints on these structures and water is thereby forced out. In agreement with the present findings, Rochdi, Foucat, and Renou (2000) found that in bovine intramuscular connective tissue, a decrease in ΔH was associated with a decreased T21 relaxation time. Furthermore, it is noticeable that there is a tendency for splitting of the
Table 1 LS means and standard errors (SE) of NMR T21 relaxation times, cooking loss (%), fiber diameter, sarcomere length (μm), and DSC enthalpies (ΔH68°C and ΔH75°C) in low temperature–long time heat treated porcine Longissimus dorsi at 53 °C, 55 °C, 57 °C and 59 °C at 3 h and 20 h. Letters a–d refer to significance (P b 0.05) between treatments (n= 3). 3h
20 h
53 °C T21 (ms) Cooking loss (%) Fiber diameter (μm) Sarcomere length (μm) ΔH68°C (J/g) ΔH75°C (J/g)
55 °C a
112.6 8.0e 65.5a 1.94 0.280a 0.333b
ab
108.1 10.8de 54.8bc 1.89 0.149c 0.385a
57 °C
59 °C
c
c
81.1 14.0cd 57.6bc 1.94 0.126cd 0.324b
80.3 17.9bc 51.6c 1.96 0.024e 0.308b
53 °C bc
91.1 12.7d 59.6abc 1.81 0.214b 0.225c
55 °C
57 °C
59 °C
c
d
d
80.3 19.5b 61.7ab 1.86 0.059e 0.180d
51.4 18.0b 55.2bc 1.97 0.078de 0.096e
60.7 25.6a 53.5c 1.79 0.016e 0.037f
SE 9.6 1.3 3.1 0.1 0.02 0.02
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T21 population at 57 and 59 °C, which might be caused by structural constraints induced by collagen shrinkage at these temperatures. However, it is unclear which proteins the two endothermic DSC peaks at 68 °C and 75 °C arise from in the present study. Stabursvik and Martens (1980) found the denaturation temperature of isolated collagen to be 68 °C, however, Bruggemann et al. (2010) showed that connective tissue denaturation begins at 57 °C. In another study combining DSC and low field-NMR on pork (Bertram et al., 2006), 3 peaks were observed in the DSC thermogram at 54 °C, 65 °C and 77 °C, respectively. The authors tentatively ascribed the peak at 65 °C to sarcoplasmic proteins and the peak at 77 °C to actin denaturation. The consequences of denaturation of sarcoplasmic proteins on structural changes and water-binding in meat might be relatively small, since denatured sarcoplasmic proteins have been reported to participate little in the consistency of cooked meat (Tornberg, 2005). Therefore, the contribution from sarcoplasmic proteins on water distribution in LTLT treated meat is believed to be of minor importance. In the present study ΔH75°C decreased when the heating time was increased from 3 h to 20 h. If this peak corresponds to actin, it seems that actin denaturation was initiated after 20 h of heating at 53 °C and enhanced at increasing temperatures up to 59 °C. Decreasing ΔH75°C and the concomitant decrease in T21 relaxation times from 3 h to 20 h at all temperatures may therefore be a result of actin denaturation affecting the intrinsic waterbinding capacity within the myofibrils. Although actin has been reported to denature at around 77 °C (Stabursvik & Martens, 1980), the effect of prolonged heating has not previously been investigated. At 20 h of heating decreases in fiber diameter were observed with increasing temperature, which together with the decrease in ΔH75°C and T21 relaxation time, could suggest an influence of denaturation of actin on the transverse shrinkage of the muscle fiber at prolonged heating times. Myosin has been reported to denature at around 53–58 °C (Wright & Wilding, 1984) and from the present study it seems that myosin was already denatured at 53 °C since no peak was observed on the DSC thermograms around this temperature. It is thus believed that myosin denatured at lower heating temperature/times than investigated in the present study, which is in accordance with Bruggemann et al. (2010) who observed that the signal from the myofibers vanished at 53 °C (in Second Harmonic Generation Microscopy), suggesting complete denaturation of the myosin rod domain at 53 °C. This study is, to our knowledge, the first to demonstrate the effect of prolonged heat treatment at low temperatures on water–protein interactions and associated structural changes caused by protein denaturation in porcine muscle. In the current study water within the myofibrils, measured by NMR T21 relaxation times, was affected by both time and temperature during LTLT treatment between 53 °C and 59 °C. The changes observed in NMR T21 relaxation times were associated with increased cooking losses and decreases in fiber diameter. Furthermore, decreasing enthalpies at 75 °C (ΔH75°C), suggested actin denaturation, might be related to the transverse shrinkage of the muscle fiber leading to higher cooking losses and decreased T21 relaxation times. Also, a concomitant decrease in ΔH68°C, which may represent denaturation of collagen, suggested an influence of collagen on the distribution of water within the meat during LTLT treatment. 5. Conclusion The present study investigated the relationship between NMR T2 relaxation times and changes in cooking loss, fiber diameter, sarcomere length, and differential scanning calorimetric data as affected by LTLT treatment of porcine LD. The NMR T2 data revealed a significant change in water–protein interactions with increasing temperatures from 55 °C to 57 °C at 3 h and 20 h of heating. Increases in transverse shrinkage of the fiber were observed between 53 °C and 59 °C with a concomitant increase in cooking loss. Thus, the present
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study indicated a relationship between transverse shrinkage, water– protein interactions and cooking loss. DSC measurements showed a concomitant decrease in ΔH68°C, which suggests a negative contribution from collagen denaturation on the retention of water within the meat during LTLT treatment. Furthermore, a decrease in ΔH75°C suggested that prolonged cooking (20 h) caused denaturation of actin, which affected water–protein interactions and resulted in higher cooking losses.
Acknowledgement The authors would like to thank Peder Vithus Johansen (DMRI) for assistance on sampling, Nina Eggers (Aarhus University), Julie Cecilia Stefansen and Jonas T. Sprehn (UC-LIFE) for laboratory assistance. The authors thank The Danish Ministry of Food, Agriculture and Fisheries for funding the projects entitled “Safety and gastronomic quality of new LTLT treatments of meat” and “Integrated characterization of food quality and microbial safety”.
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