Physical Aspects of Meat Cooking

Physical Aspects of Meat Cooking: Time Dependent Thermal Protein Denaturation and Water Loss B. I. Zielbauer, J. Franz, B. Viezens & T. A. Vilgis

Food Biophysics ISSN 1557-1858 Food Biophysics DOI 10.1007/s11483-015-9410-7

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Author's personal copy Food Biophysics DOI 10.1007/s11483-015-9410-7

ORIGINAL ARTICLE

Physical Aspects of Meat Cooking: Time Dependent Thermal Protein Denaturation and Water Loss B. I. Zielbauer 1 & J. Franz 1 & B. Viezens 1 & T. A. Vilgis 1

Received: 23 March 2015 / Accepted: 22 July 2015 # Springer Science+Business Media New York 2015

Abstract Selective denaturation of meat proteins - essential to reach desired textures - requires cooking temperatures corresponding to their different structure and interactions. Sousvide cooking allows precise control over the denaturation state of meat proteins (and thus the cooking state of meat products) due to the possibility to cook at very well defined temperatures. Additionally, kinetic effects also play an important role. Differential scanning calorimetry (DSC) has been used here to follow the denaturation state of proteins in pork filet (Musculus psoas major), which had been heat treated at different time (10–2880 min) and temperature (45–74 °C) combinations. Additionally, the water loss (cooking loss) occurring during heat treatments has been determined. Four endothermic peaks have been observed in the DSC curves. Their individual time and temperature dependent enthalpies show that proteins become denatured at temperatures well below the peak temperatures if kept there for long times. This observation is underlined by statistical arguments. Cooking loss increases with time and temperature, while the main water loss occurs during the first 240 min and at temperatures above 60 °C. Due to the different kinetics found for protein denaturation and cooking loss, it is not possible to directly correlate the two quantities.

Keywords Sous-vide cooking . Pork . Thermal denaturation . Denaturation kinetics . Cooking loss . Differential scanning calorimetry

* B. I. Zielbauer [email protected] 1

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

Introduction During recent years, sous-vide cooking has become a standard method in gastronomy, allowing to precisely control the cooking state and thus the texture of meat, fish or vegetables [1]. This is achieved by cooking the vacuum packed product in a water bath at a well-defined temperature. The fact that cooking takes place at a specific temperature is of particular interest for fish and meat, which owe their textural properties mainly to the complex structural arrangement and water binding capacity of muscle proteins. Upon heating, these proteins denature, losing their native conformation and causing a change in the texture of the product. Most relevant to sousvide cooking is the fact that different proteins, being responsible for different properties of the final product (tenderness, juiciness, etc.), denature at different temperatures. This allows tailoring the properties of the food by selectively denaturing some proteins while leaving others intact. The muscle is a highly hierarchical structure [2], which owes its stability to the fibrous nature of its main proteins, the myofibrillar and connective tissue proteins. Myofibrils have a striped appearance resulting from the structure of its repeating unit, the sarcomere. It contains interdigitating thin and thick filaments, consisting primarily of actin and myosin, respectively. The main protein in connective tissue is collagen. Apart from these structural aspects, fresh meat contains about 75 % of water, which dissolves so-called sarcoplasmic proteins of globular structure. Cooking meat is a complex process involving several physical and chemical processes on different time and length scales. Thermal denaturation of proteins leads to changes in protein-water interactions as well as geometrical changes such as longitudinal and transverse shrinking of fibers, leading to altered distances between muscles fibers as well as the occurrence of pressure gradients. All this is resulting in changes in

Author's personal copy Food Biophysics

the water holding capacity of meat as well as ultimate water loss at high cooking temperatures. Current hypotheses for the basis of water binding in meat are reviewed e.g. in [3–5]. Besides tenderness, juiciness is usually a desired quality in a piece of meat and it is related to the protein denaturation stage. However, depending on the protein, its denaturation may have a positive or negative impact on the water content of the meat. Shrinkage of fibrous proteins such as actin due to entropic reasons results in water squeezed out from the product. On the other hand, unfolding and subsequent association of globular proteins results in a gel under the right conditions. This is the case for the sarcoplasmic proteins as well as for collagen, once it has been thermally solubilized under denaturation into gelatin and cooled down. Such a gel formation will lead to improved water binding. The well-known long time/low temperature cooking of collagen rich meat takes advantage of this effect. Since the denaturation temperature of actin is higher than that of collagen, cooking is perfomed at a temperature low enough to keep the actin intact while denaturing the collagen, thus maximizing the water content of the piece. However, the denaturation of collagen, which has a complex triple helical structure [6], needs a certain time to complete, thus long cooking times are necessary. At the same time, activity of proteolytic enzymes in meat has been shown to persist at moderate cooking temperatures up to around 60 °C [7, 8] and may play a role in tenderization of meat during low temperature long time cooking. For a long time it has been assumed for thermodynamic reasons, that when cooked meat is held at a constant temperature until serving, no further molecular changes of proteins and thus changes in cooking state take place as long as the temperature is not increased. The aim of this study was to investigate systematically the impact of time (10 min–48 h) at different cooking temperatures (45–74 °C) on the state of protein denaturation and water content of the cooked piece of meat. Although a number of studies have addressed this problem [8–14] they usually investigated a narrower range of times and temperatures or fewer measurement points within this range. Pork filet was chosen as an example due to its regular muscle fiber structure, which allows a reproducible preparation of samples. Differential scanning calorimetry (DSC) offers the possibility to measure denaturation of meat proteins in their native environment, i.e., the muscle, and was therefore used to determine the amount of protein denaturation occurring for the different cooking conditions. In this method, the sample and a reference are heated at a constant rate, and endo- or exothermic reactions occurring in the sample are registered as differences in heat flow to sample and reference. The denaturation of meat proteins during heating results in several endothermic peaks that can be assigned to the different proteins present in meat. Since this assignment is sometimes controversial in literature and not many studies exist about pork, sarcoplasmic proteins and

connective tissue were also isolated from raw meat and analyzed by DSC. In order to relate the water holding capacity of the meat to the protein denaturation as measured by DSC, the water loss occurring during cooking (cooking loss) was also determined for the same time-temperature combinations. Because frozen samples had to be used due to logistic reasons, the loss of water after thawing (thawing loss) was also determined.

Materials and Methods Sample Preparation Commercially available pork filet (M. psoas major) was kept at 4 °C after purchase and processed further on the same day. The pH of each filet was measured with a portable pH meter for meat (HI 99163, HANNA Instruments) and was between 5.4 and 6.1 for all filets. Slices of 1.25 cm thickness were cut perpendicular to the muscle fiber direction, and the outer layer of each slice, containing visible connective tissue and fat, was removed. The weight of each slice was recorded before it was vacuum packed individually and stored at −20 °C until further usage. Before cooking, samples were thawed inside their bags for 10 min in water of room temperature, followed by 15 min on the lab bench. Then they were removed from their bags, carefully dried with paper without pressing and weighted anew. Afterwards they were vacuum packed again in heat stable plastic bags (allfo Vakuumverpackungen) and immediately used for cooking. Sous-Vide Cooking Samples were placed into preheated water baths (D5, DocDeli) of 45, 51, 60 or 74 °C and cooked therein for times ranging from 10 min to 48 h. After that the samples were cooled down quickly by placing them in ice water for 10 min and resting them for 15 min at room temperature afterwards. As a reference, thawed and vacuum packed samples were also kept at room temperature for the same times, followed by the cooling step as well. In order to estimate the influence of vacuum packing, samples were also rested at room temperature in tightly sealed but not evacuated bags. Cooking and Thawing Loss After cooking and chilling, samples were removed from their bags, dried and weighted again. Thawing loss was the difference in weight before freezing and after thawing and was expressed in percent of the initial weight. Similarly, cooking loss was determined as the difference in weight before and after cooking, expressed in percent of the weight before cooking.


Sarcoplasmic Proteins (in the centrifugal drip) and Connective Tissue Preparation To obtain sarcoplasmic proteins, the centrifugation method was used, because the liquid obtained from fresh meat by centrifugation (centrifugal drip) has been shown to be comparable in composition to sarcoplasmic extracts [15]. For each of the preparations 20 g of fresh meat was cut manually into pieces of about 5×5 mm. For the preparation of sarcoplasmic proteins those pieces were placed into 50 ml centrifuge tubes and centrifuged at 4 °C and 6000×g for 2 h. The supernatant was collected as centrifugal drip and frozen at −20 °C until use. It has been shown that centrifugal drip is similar in composition to sarcoplasmic protein extracts [15]. In order to extract the connective tissue, Milli-Q water was added to the chopped meat (1:1 w/w) and the mixture was homogenized using an Ultra-Turrax (T18 basic, IKA) for 2 min at setting 3 (11 000 rpm in water) while keeping the beaker on ice. The homogenate was then sieved through a strainer with mesh size 0.5 mm and the remaining material was washed with 200 ml of Milli-Q water. Afterwards the residue was placed into a 1.5 ml Eppendorf tube filled with Milli-Q water and centrifuged at 10,000×g and 4 °C for 30 min. The pellet was frozen at −20 °C until further use.

Differential Scanning Calorimetry (DSC) After determination of the cooking loss, 1–2 cm3 of each sample were cut up manually into pieces as small as possible while trying to avoid water loss. Visible pieces of fat or connective tissue were removed. The minced meat was put into 1.5 ml Eppendorf tubes, sealed with Parafilm and stored at −20 °C until measurement. It was thawed at room temperature directly before the measurement. The measurements were perfomed using a Mettler Toledo DSC-822. 33–55 μg of meat/connective tissue or 50 μl of centrifugal loss were placed into 100 μl aluminum pans and tightly sealed. An empty pan was used as a reference. The weighted sample as well as the total weight of the pan was recorded. Measurements were run from 10 to 90 °C (10–75 °C for centrifugal loss) at a heating rate of b=1 K/min. After the measurement, the total pan was weighted again, to make sure that no water loss occurred during the measurement. In order to determine the dry matter content of the weighted sample, the pans were punctured with a needle, dried at 105 °C in an oven for 24 h and weighted immediately after they were removed from the oven. The dry matter content of the sample was used to normalize the measured heat flow by weight. The dry weights of all samples were between 9 and 17 μg. In order to calculate the total specific enthalpy of the transitions observed, a baseline connecting beginning and end of the peak by a straight line was defined manually (Fig. 1a).

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Fig. 1 Representative DSC measurement of raw pork filet recorded at a heating rate of 1 K/min, showing 4 endothermic peaks a Original data including baseline (dashed line) and integration limits (dotted lines) b Data after baseline subtraction. The peaks I to IV correspond to the denaturation of groups of proteins and are discussed in the text

The curves were then integrated from 40 to 80 °C, taking into account T=bt to obtain the correct unit. For better graphical comparability, curves illustrating the time and temperature effect are presented with baselines subtracted as shown in Fig. 1b. The areas under the different peaks were also integrated individually in the following way: peak I: 40–53.6 °C; peak II: 53.6–64.9 °C; peak III: 64.9–69.6 °C and peak IV: 69.6– 80 °C. These ranges were determined according to the minima in specific heat flow of raw meat separating the different peaks as visible in Fig. 1b. Statistics Cooking experiments for all time-temperature combinations were repeated at least in triplicate. Values reported are arithmetic means and the standard deviation was taken as error.

Results Thawing Loss The thawing loss varied between 4 and 14 % of the fresh sample weight. The thawing loss was not correlated to the initial sample weight, which varied due to the different


diameters of the muscles from different carcasses and because slices where cut all along the muscle which does not has a constant diameter.

Cooking Loss The cooking loss versus the cooking time for the different temperatures is shown in Fig. 2. In general, both, higher cooking temperatures and longer cooking times lead to increased water losses (Fig. 2a). However, for 74 °C, cooking times longer than 240 min did not lead to a further increase in water loss. Interestingly, for 45 °C the cooking loss at intermediate times exceeded that of the higher temperature 51 °C. The reference sample that had been rested in an evacuated bag at 24 °C also showed a significant increase in cooking loss up to 1440 min and then a decrease for the longest cooking time of 2880 min. The samples rested at room temperature in a

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Fig. 2 Cooking loss as percentage of initial weight for different cooking times and temperatures. a Cooking loss vs. time parametric in temperature. b Cooking loss vs. temperature parametric in time

sealed but not evacuated bag exhibited a much lower loss of water than those packed under vacuum. This results most likely from the pressure the deformed plastic bag exerts on the packed product [13]. The strongest increase in cooking loss occurred when the temperature was raised from 60 to 74 °C, especially for times up to 120 min (Fig. 2b).

DSC Measurements A typical result of a DSC measurement of raw pork filet is shown in Fig. 1a as well as in Fig. 3 (top curve: whole meat). Between 40 and 80 °C, four endothermic transitions at about 51, 60, 68 and 74 °C can be observed. Figure 3 additionally shows the normalized results of the DSC measurements of centrifugal loss and connective tissue, obtained from the same filet as the whole meat sample. For a better comparability the curves have been shifted along the yaxis. To illustrate the effect of time and temperature, Fig. 4 compares representative measurements for samples cooked at different temperatures for the same time (Fig. 4a) or at the same temperature for different times (Fig. 4b, c and d). The cooking temperatures were chosen according to the protein denaturation peaks as visible in the DSC measurements. For higher temperatures as well as for longer times the total peak area is decreasing. For a cooking temperature of 74 °C, even cooking times as short as 10 min result in a complete disappearance of the endothermic transitions (Fig. 4a). The total specific enthalpy versus the cooking time is shown in Fig. 5. For 74 °C only a few samples were measured at random, all showing a flat line without any visible transitions. Therefore the enthalpy for all samples cooked at 74 °C was taken as zero. No changes occurred with time for the samples rested at room temperature. It is clearly visible that the total specific enthalpy is reduced at a higher rate for higher cooking temperatures.

Fig. 3 Normalized DSC measurements for whole meat, centrifugal drip and connective tissue. Curves have been shifted along the y-axis for comparability reasons

Author's personal copy Food Biophysics Fig. 4 Representative DSC measurements for a) different cooking temperatures for a constant time of 10 min, b), c), d) different cooking times for the temperatures 45, 51 and 60 °C

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In order to describe in more detail the denaturation kinetics of the different proteins, the enthalpies for the individual peaks were determined as described in Materials and Methods. These remaining areas can be seen as proportional to the amount of protein that is still in the native state [14] and have been plotted in Fig. 6 as percentages of the signal for nontreated meat. In general, for higher treatment temperatures denaturation proceeds faster and even for cooking temperatures Tb well below the actual peak temperature (peak I: Tb =45 °C; peak II: Tb =45 and 51 °C, peak IV: Tb =45, 51 and 60 °C) the amount of native protein gets reduced with increasing time. However it is worth noting that for peak III (maximum at 68 °C), temperatures up to 51 °C do not have any denaturing effect even for longer times.

Fig. 5 Total specific enthalpy depending on cooking time for different cooking temperatures

Discussion Freezing of the samples was necessary due to logistic reasons. Although it is known that meat quality attributes are influenced by freezing and thawing [16], not all of the underlying mechanisms are understood yet. Concerning the parameters investigated here (cooking loss and protein denaturation), findings in the literature are not fully consistent. Kim et al. [17] found a decrease in cooking loss of pork due to freezethawing and attributed it to the fact that freeze-thawing lead to an increased amount of exudate before cooking, thus leaving less water available that can be easily removed during cooking. On the other hand it was found that cooking loss increased in beef after frozen storage periods longer than 30 days [18] and cooking loss was not affected by freezethawing of ostrich meat, which the authors explained by the fact that water lost during cooking originates mostly from chemically bound water as well as molten fats which would be unaffected by freeze-thawing [19]. Findings concerning the protein denaturation are somehow ambiguous. For bovine [20] as well as porcine [21] muscle, it was found that the denaturation enthalpy of mainly the first DSC peak decreased, while no significant differences in the DSC curves of fresh and frozen pork samples were observed [22]. The effect of freezing was not systematically studied here, but test DSC measurements with fresh and thawed samples indicated at most in general a slight reduction in total enthalpy from fresh to frozen samples, but no systematic influence of the freezing time. Only samples that were frozen have been compared in this study. The DSC profile for raw pork (Fig. 1a) corresponds well to those known from literature [21, 22]. This general shape is

Author's personal copy Food Biophysics Fig. 6 Percentages of remaining native protein as determined from the areas under the DSC curves for the individual peaks depending on time and temperature of the heat treatments

similar for meat from different species, while the precise position and relative depth of the peaks varies. Tentative assignments of the three main peaks for beef [9, 10, 23, 24], rabbit [25, 26] and chicken [27, 28] have been done by several authors via comparison of the DSC profiles of whole meat with those of meat fractions such as connective tissue, sarcoplasmic proteins or myofibrillar proteins as well as with those of isolated proteins. Less work has been done on pork [28], where the assignment was mainly made by comparison with profiles from other animal species [29]. This assignment is difficult because interactions between proteins such as myosin and actin change the denaturation temperature of the individual proteins [30] and the transition profile of e.g. the myosin subfragments is strongly dependent on environmental factors such as ionic strength and pH [26, 31, 32]. It therefore cannot necessarily be assumed that transition temperatures found for isolated proteins hold for the same protein when it is integrated into the complex muscle structure. However, the following assignments for the main peaks are widely used: peak I corresponds to the denaturation of myosin heads, in the region of peak II myosin tails, sarcoplasmic proteins as well as collagen denature and peak IV is assigned to actin, while also isolated titin from porc has been shown to denature in the respective temperature range [33]. The assignment of myosin rods to peak II seems debatable, because second harmonic generation (SHG) microscopy measurements suggest that myosin rods denature already between 40 and 53 °C [34] and also DSC studies of isolated myosin rods have shown that their major transitions are below 60 °C [32]. However, the third peak, although observed in a number of studies [21, 22, 25, 35], has not yet been assigned. Stabursvik et al. [36] discuss a shoulder of Peak II in beef and attribute it to sarcoplasmic

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proteins by comparison with the DSC curves of drip juice. Similarly, they attribute Peak II to collagen by comparison with connective tissue. A peak in sarcoplasmic proteins of pork with Tmax slightly higher than that observed for peak II in whole meat was also mentioned by Deng et al. [37]. To verify these findings for pork filet as used here, centrifugal loss, which is considered to be composed mainly of sarcoplasmic proteins [15], and separated collagen were compared to the DSC measurements of the whole meat from the same filet (Fig. 3). The small endothermic peak visible between 28 and 30 °C in all curves is resulting from small amounts of fat [38]. Our measurements of centrifugal loss show three endothermic peaks, of which the third one possesses a Tmax of 68.2 °C, which is almost identical to that of peak III in whole meat (Tmax =68.0 °C). Also, the second and deepest peak in centrifugal loss is located between peak II and peak III of the whole meat. Connective tissue shows a well pronounced peak at 64.8 °C, which is located between peaks II and III of centrifugal loss (Fig. 3). These results show, that the thermogram of whole meat especially in the region of peak II and III is a complex superposition of signals stemming from different proteins, thus making a clear assignment difficult. It is also interesting to mention that the enthalpy of peak III of the whole meat is independent of cooking time for cooking temperatures up to 51 °C (Fig. 4), which might indicate its connection to a protein requiring long times for denaturation, such as collagen. figure 4 illustrates the effect of different cooking temperatures and times on the denaturation state of the meat proteins. Figure 4a shows that cooking meat to the respective denaturation temperatures Td of certain proteins as given by the peak temperatures in the DSC thermogram, results in a complete


removal of the signal up to the cooking temperature after 10 min, meaning that the proteins responsible for this peak have been denatured. More interesting are the results shown in Fig. 4b–d, which make clear that denaturation, may also occur for cooking temperatures below Td, if the time for which the sample is held at that temperature is increased. In a very simplified view, thermal protein denaturation can be described as a reaction for which to occur a certain energy barrier with height Ed has to be overcome. Figure 7a illustrates this situation in terms of free energies. Cooking at a certain temperature Tb (temperature of the water bath) reduces the energy barrier for denaturation by kBTb (Fig. 7b), where kB is the Boltzmann constant. The crossing of the remaining energy barrier EdkBTb, i.e. the denaturation of the protein, is a statistical process with probability p. E d −k B T b ð1Þ p∝exp − kBT b The smaller the barrier becomes (i.e. the higher the bath temperature Tb), the larger becomes the probability of denaturation. There is a certain probability for denaturation even for kBTb 50 °C, incubation at 43 °C for a prolonged time also led to a slow denaturation and aggregation [40]. We do not aim here to derive any more specific conclusions concerning the denaturation kinetics of individual protein species on the basis of our data from whole meat, since this seems to be problematic because most likely several proteins contribute to each of the peaks visible in the DSC measurements. Also, the precise denaturation pathways of all of the proteins are not known and it has been shown that in the case of twostep irreversible denaturation processes (Lumry-Eyring models) DSC transitions can be distorted if the irreversible step is fast at temperatures in the range of or below that of reversible unfolding [41]. However, for meat and other biological materials, such models seem to be oversimplified. It is generally accepted that water loss during cooking is related to thermal denaturation of proteins and subsequent changes in protein conformation and the increase of energy. However, the exact mechanisms are complex and not yet fully understood. 1 H NMR T2 relaxation measurements of meat indicated the presence of several water fractions with different relaxation t i m e s t h a t w e r e a t t r i b u t e d t o i n t e r m y o f i b r i l l a r, intramyofibrillar and hydration water or water closely associated to the proteins, with decreasing relaxation times, respectively [42–44]. This shows that an examination of water loss during cooking has to take into account processes on different length scales. While water located between the myofibrils is squeezed out due to disruption of cell walls and shrinking of myofibrils, the water binding abilities of the proteins themselves on a molecular scale have also to be considered. Cooking meat leads to shrinkage of meat first transverse to the fiber axes at temperatures of about 45–60 °C and then parallel to the muscle fibers at 60–90 °C [45, 46]. These shrinkages are attributed to the denaturation and following coagulation of myofibrillar proteins as well as shrinkage of collagen [2], which occurs as a first step of collagen denaturation [34, 47], when hydrogen bonds stabilizing the compact (non-laterally cross-linked) triple helix are broken [25] and it is transformed into three single stranded polyampholite polymers. As a second step, the intrahelical hydrogen bonds of the individual alpha helices are disrupted [48] accompanied by


the breaking of crosslinks [47]. While an incomplete denaturation leading to shrinking of the fiber could contribute to an increase in cooking loss [49], this finally results in the solubilization of collagen and its conversion to gelatin that occurs only after long times [13, 48]. The charged amino acids are then exposed to water, which forms hydrate shells in the vicinity of the water soluble amino acids. These processes lead to improved water binding and counterbalance the cooking losses at longer times. It has been found by SHG microscopy that the shrinkage of collagen fibers in muscle fibers and endomysium of pork starts at 58 and 57 °C, respectively [34]. A major loss in juiciness from 63 to 73 °C was related to actin denaturation and shrinkage of myofibrils. [9] On the other hand, protein denaturation can lead to gelation of sarcoplasmic proteins and myosin [2, 50, 51]. Thus a direct correlation of cooking loss to protein denaturation appears to be difficult and is always related to the composition of a certain piece of meat. Some authors found a correlation between cooking loss at certain temperatures and denaturation of certain proteins [52, 53]. Water loss upon cooking has also been described by the Flory-Rehner theory, where the Flory-Huggins interaction parameter varied with temperature in order to describe protein denaturation and was used to derive a swelling pressure, the gradient of which is then used in Darcy’s law to predict the cooking loss.[54] The measurements performed here for cooking loss (Fig. 2a) and amount of total protein denaturation (Fig. 5) suggest a more complicated situation. Although in principle, it is expected that the water loss is also governed by arguments suggested via Eq. (1), no obvious dependence of the rate of water loss is observed here. Taking into account the above discussed statements about effects of protein denaturation in different temperature ranges, the course of the cooking loss shown in Fig. 2 can be discussed as follows: For the reference sample rested at room temperature in an evacuated bag no protein denaturation occurs and thus the water loss has to be attributed to the vacuum packaging, as already discussed. At very long times, reabsorption of water into the meat structure seems to occur. For a cooking temperature of 45 °C, the time course of the cooking loss looks similar to the one at room temperature, but the cooking loss is much higher. This can be explained by the transverse shrinking of myofibrils, leading to larger gaps between the muscle fiber bundles allowing the intermyofibrillar water to be squeezed out more easily. The cooking loss for long times at 51 °C drops even below that of 45 °C. This could be a sign of protein denaturation processes leading to gel formation and thus improved water binding at temperatures between 45 and 60 °C for times longer than 240 min. Also, increased cathepsin activity at those temperatures [8] results in shorter collagen fragments that have a potential for increased water binding (and faster denaturation). The

strongest increase in cooking loss occurs from 60 to 74 °C, a result that confirms earlier findings for beef [2, 52] and is most likely related to the denaturation and shrinking of actin fibers (Peak IV), while shrinking of collagen is also expected to play a role. A strong shrinking of the sample had been observed also visibly especially at 74 °C. This also leads to a changed situation concerning the effect of the plastic bag, this also being the reason why no attempt was made to subtract this background water loss. At 74 °C, while no signal of remaining native proteins could be detected in the DSC measurements even at the shortest times, cooking loss for the same temperature is increasing until a cooking time of 240 min. Thus, water loss appears to occur on longer time scales compared to protein denaturation, which seems plausible taking into account that this is a macroscopic process and water has to be transported out of the sample, while protein denaturation occurs on molecular scales. The slower dynamics of the water transport could also be related to the slow relaxation of the protein matrix [54] following to the mechanical stresses occurring in consequence of protein denaturation and shrinking of fibers.

Conclusion It has been shown that protein denaturation and water binding in pork filet are strongly dependent not only on temperature but also on time. If meat proteins are heated for prolonged times, proteins are denatured even if cooking temperatures are well below the actual respective denaturation temperatures. This can be understood by statistical arguments. It has important gastronomical implications, since the ongoing protein denaturation at long times might lead to products with undesired properties if they are for example held at a constant temperature until serving. Water loss is also increasing with increasing times and temperatures, with the strongest increase observed from 60 to 74 °C. However, kinetics of protein denaturation as measured by DSC and water loss are different, indicating the involvement of different processes such as the squeezing out of water due to protein denaturation and muscle fiber shrinkage on the one hand and improved water binding induced by gelation of myosin, sarcoplasmic proteins or collagen on the other hand. It also has to be taken into account that protein denaturation and water transport occur on different time scales.

Acknowledgments The authors thank Marta Ghebremedhin (Max Planck Institute for Polymer Research) for assistance with sample preparation as well as Hubertus Tzschirner (esskunst) and Peter Fischer (Komet) for helpful discussions about sous-vide cooking techniques. The work was supported by the German Ministry for Economic Affairs (BMWi) via the ZIM program.


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