Effect of Combination of Time and Temperature on Quality Characteristics of Sous Vide Chicken Breast

The use of minimal thermal processing techniques such as sous vide technology to improve the quality of meat-based foods has gained a special focus in recent years. A proper combination of temperature and time parameters in sous vide processing plays an important role in the water-holding capacity, texture properties, and juiciness of the meat. The present study aimed to assess the impact of the one-step and two-step sous vide processing on different quality properties of chicken breast with special emphasis on the cooking loss, color, texture properties, protein solubility, and lipid oxidation. According to the results, chicken breast treated with a two-step temperature (50 and 60 °C) showed improved texture parameters (shear force, hardness, chewiness, and gumminess), lower cooking loss, acceptable redness values, and decreased lipid oxidation levels than the chicken breast treated with the one-step temperature of 60 °C. Moreover, the two-step sous vide technique revealed significantly higher total protein solubility of the chicken breast than the one-step sous vide. Based on pasteurization values, the two-step sous vide technique was equally safe as the one-step sous vide technique for vegetative cells’ inactivation for the main pathogens of interest (C. perfringens and L. monocytogenes).


Introduction
The global consumption of poultry meat is gradually increasing because of its beneficial effects on human health and the low price compared to the rest of the meats. The high level of protein, complete amino acid composition, essential micronutrients, and low levels of fat in poultry meat provide a healthy diet for different groups of people [1]. These features reduce the risk of obesity and prevent malnutrition and cardiovascular diseases in humans [2]. However, poultry meat consumption may be limited among the elderly because of the mastication and swallowing challenges due to different oral impairments [3,4]. Thus, in the case of chicken meat, breast muscles tend to show higher toughness and crumbliness as compared to chicken legs' muscles [5,6]. In this context, the food industry needs to develop novel poultry-based ready-to-eat products with an improved texture and high nutritional value to meet these demands.
Sous vide cooking is a minimal processing technology that has gained popularity in the food industry, catering sector, and restaurants in recent years [7,8]. Sous vide in French means "cooking under vacuum" and refers to mild heat treatment of food in a water bath under strictly monitored parameters (temperature and time) using thermostable vacuum pouches [9]. This technique provides better control over the texture of the product, inhibits lipid oxidation, improves sensorial characteristics, and minimizes protein denaturation depending on treatment conditions [10].
Identification of optimal temperature and time duration is often challenging in sous vide processing to achieve desired attributes of cooked meat such as tenderness or juiciness. Most researchers have studied only the influence of different combinations of a single temperature and time in sous vide processing on the quality attributes of beef [11][12][13], pork [14], turkey [15], and chicken [16,17]. On the other hand, several authors reported using additional pretreatments such as freezing technology [12] or thawing [18] before sous vide to achieve meat tenderization. Botinestean et al. [12] found that short-term freezing of beef steaks at −20 • C resulted in comparable shear force, hardness, and chewiness values with sous vide cooking alone. Ji et al. [18] concluded that freeze-thawing and freezing applied prior to sous vide significantly decreased the shear force of pork meat. However, from an economic perspective, these attempts do not seem feasible because they impose extra processing costs. Furthermore, including pretreatments such as thawing in sous vide processing may increase the weight loss of cooked meat [18]. Therefore, plantderived exogenous proteases such as papain, actinidin, zingibain, and bromelain were also tested [19][20][21][22]. These alternatives have significant proteolytic activity and proved effective to increase the tenderness of the treated meat [23][24][25]. However, if fresh plant extract is applied in meat, it could produce non-desirable off-flavors [26,27].
On the other hand, meat has its native endogenous proteolytic enzymes, the highest activity of which has been reported to be at temperatures between 40-50 • C [28]. Previous studies reported that the high activity of proteolytic enzymes at these temperatures is responsible for higher desmin degradation, which indicates an extension of meat tenderization [14,29]. The proteolytic activity of the calpain-1, calpain-2, and cathepsin B enzymes responsible for desmin degradation is more intensive between 40 and 55 • C and negligible at 60 • C and above [29]. Therefore, the inclusion of a low temperature as a first-step temperature in the sous vide technique may improve meat tenderness and other quality characteristics. There are no studies in the literature that examine the effect of sous vide with a two-step temperature on chicken breast muscles. Thus, this technique may provide an alternative cooking method to develop novel poultry-based ready-to-eat foods with an improved texture. Furthermore, it may provide valuable data to the food industry to use as a reference. Therefore, the objective of the present study was to investigate the effect of sous vide using the one-step temperature of 60 • C and the two-step temperatures (50 • C + 60 • C) applied in different time ratios of the same total treatment times, on physicochemical characteristics, texture attributes, lipid oxidation, and protein solubility of chicken breast muscles (pectoralis major). In addition, the pasteurization values of the chosen sous vide treatments were also calculated.

Experimental Design
Pectoralis major muscles (24 h post-mortem, pH 24 = 5.83 ± 0.12) were obtained from a slaughterhouse in Budapest, Hungary, and were kept overnight at a refrigerated temperature of 4 • C until sample preparation. The chicken breasts were trimmed of external fat and connective tissues and cut into uniform pieces of 2 ± 0.2 cm thick, weighing about 130 ± 2.4 g each. The samples were vacuum packaged in 90-µm PA/PE pouches (200 mm × 250 mm) with a vacuum machine (Multivac C100, MULTIVAC Sepp Haggenmüller SE & Co. KG, Wolfertschwenden, Germany) and were randomly assigned in one of the six groups of different temperature and time processing combinations (Table 1).
In the one-step sous vide treatments (T1 and T4), chicken breasts were cooked at 60 • C for 120 and 180 min, while in the two-step sous vide, temperatures of 50 • C and 60 • C were combined at different time ratios of the same total treatment times (Table 1). Three replicates were conducted for each processing treatment using a two-way (completely randomized) design. The two factors were total treatment time (two levels: 120 and 180 min) and treatment time ratio (three levels: 0:1, 1:2, 1:1). Sous vide processing was performed in two thermostatic water baths (Labor Müszeripari Müvek LP507/01). A T-type thermocouple inserted at the geometric center of the muscle was used to monitor the core temperature of the samples during thermal processing. The data logger recorded the temperature readings during processing at intervals of 2 s (scanning time) with a ±0.1 • C accuracy. After that, the cooked chicken breasts were put in ice-cold water at 1 • C for 60 min to cool down and then were maintained overnight at refrigerated conditions (2 • C) to reach a temperature of less than 4 • C for a period of 6 h, according to the recommended guidelines [30]. Physicochemical properties (moisture content, cooking loss, color attributes, texture properties), lipid oxidation, protein solubility, and pasteurization values were determined on the following days after thermal processing.

Pasteurization Values
The calculation of pasteurization values of each group was performed by integration of the time-temperature profile curves derived from the recorded time-temperature readings during treatments, according to the equation below: In Equation (1), t indicates the heating time (min), T refers to the measured core temperature ( • C) of the muscle during treatment, T ref indicates the reference temperature, and z value refers to the number of degrees of temperature required to increase the thermal death rate of the microorganism by a factor of 10. Three reference temperatures T ref (60,70, and 80 • C) for three target pathogens, with specific z-values of 6.74 • C (C. perfringens) [31], 10 • C (L. monocytogenes) [32], and 13 • C (C. botulinum spores) [33], respectively, were used to calculate the pasteurization values of the treatments included in the study. Calculations were done based on five independent batches for each group treatment.

Moisture Content and Cooking Loss
The moisture content of chicken breasts was measured in triplicate using the standard AOAC International 950.46 method [34]. Briefly, around 4 g of cooked chicken breast were weighed and subjected to drying in an air-forced oven (Labor Müszeripari Müvek, Budapest, Hungary) at 105 • C for 16 h. Cooking loss was calculated from the weight difference between the raw and cooked chicken breasts.

Color Measurement
The color attributes of chicken breasts were determined using the CIELAB [35] scoring system. A CR-410-type colorimeter (Konika Minolta Sensing Inc., Osaka, Japan) was used to measure the lightness, redness, and yellowness values of the samples after white calibration of the instrument. Five parallel readings were performed for each sample (n = 3).
The method described by Knispel [36] was used to calculate the total color difference (∆E) between groups using the obtained values of lightness L*, redness a*, and yellowness b*, based on the equation below: The texture of cooked samples was analyzed through texture profile analysis (TPA) following the method applied by Bourne [37]. Twelve chicken breast cores per treatment in duplicates (diameter = 12 mm, height = 12 mm), previously tempered at 4 • C, were compressed to 70% of their original height in two cycles with a P/35 cylinder probe (35 mm diameter) using a TA.XT Plus texture analyzer (Stable Micro System, Surrey, United Kingdom). A load cell (500 N) was applied at a speed of 2 mm s −1 from which maximum force-time deformation curves were obtained. Texture Expert software for Windows (Stable Micro System) was used for processing data to assess the TPA attributes: hardness, springiness, gumminess, cohesiveness, and chewiness. For shear force analysis, samples were cut on a slab shape in the size of 15 mm × 15 mm × 50 mm (width, thickness, length) and cut perpendicular to the orientation of fibers with a Warner-Bratzler knife blade with a flat end at 2 mm/s speed. The obtained peak force (N) was registered as a shear force value. Six parallel measurements were performed for each sample (n = 3).

Lipid Oxidation
To determine the lipid oxidation of chicken breasts, the thiobarbituric Acid Reactive Substances (TBARS) values were analyzed using the method from Dias et al. [38] with few modifications. The sample (5 g) was homogenized (Digital Ultra-Turrax, Staufen, Germany) for 2 min with 20 mL of 5% trichloroacetic acid (TCA) and 0.5 mL of the artificial antioxidant 0.15% butylated hydroxytoluene (BHT, Sigma-Aldrich, Munich, Germany). After that, centrifugation of the homogenized samples was performed at 5000× g for 10 min. The obtained supernatants were filtered using Whatman No. 1 filter paper into 25-mL volumetric flasks and adjusted to 25 mL with 5% TCA. Two milliliters of the filtrate were mixed with two milliliters of 0.08% thiobarbituric acid (TBA) in glass tubes and were heated in a water bath at 95 • C for half an hour. Solutions in glass tubes were cooled down to room temperature, vortexed, and then absorbances were measured against a blank (mixing 2 mL of 5% TCA and 2 mL of 0.08% TBA) at 532 nm by U 2900 UV-visible spectrophotometer (Hitachi Ltd., Tokyo, Japan). TBARS values represented the mean of triplicate measurements for each sample (n = 3) and were reported as mg malondialdehyde/kg of meat sample [39,40].

Protein Solubility
Protein solubility parameters were assessed using the method applied by Warner et al. [41]. One gram of sample was homogenized in triplicates with 10 mL of ice-cold 25 mM potassium phosphate buffer (pH = 7.2) by a Digital Ultra-Turrax homogenizer (Staufen, Germany) for the extraction of sarcoplasmic proteins. Homogenates were left for 20 h in refrigerated conditions (4 • C) and then were centrifuged (1500× g for 20 min) at 4 • C. Protein concentrations of the obtained supernatants were assessed following the Bradford method [42] using the BSE (bovine serum albumin) as a standard. Similarly, total protein solubility was determined by homogenizing 1 g of sample with 10 mL of 0.55 M potassium iodide and 0.05 M potassium phosphate buffer (pH = 7.2). Myofibrillar protein solubility was calculated as the difference between total protein solubility and sarcoplasmic protein solubility.

Statistical Analysis
The experimental data analysis was performed using IBM SPSS (Version 27.0, Armonk, NY, USA, 2020). Two-way multivariate MANOVA models were performed for moisture content and cooking loss, for color parameters (L*, a*, b*, and ∆E), for protein solubility parameters (sarcoplasmic, myofibrillar, and total protein solubility), and for TPA parameters (hardness, cohesiveness, springiness, gumminess, chewiness), and two-way ANOVA models for TBARS and shear force. The two factors analyzed were total treatment time (two levels: 120 and 180 min) and the time ratio (three levels: 0:1, 1:2, and 1:1). Normality of the residuals was tested by the Kolmogorov-Smirnov test (p > 0.05). Homogeneity of error variances was accepted by Levene's test (p > 0.05) except in the case of gumminess (p < 0.05). Multivariate overall test was evaluated based on the unexplained variance ratio (Wilk's lambda) and in the case of significant overall result, follow-up univariate ANOVA was performed using Bonferroni's correction to avoid familywise Type I error inflation. At the end, Tukey's post hoc tests were run if homogeneity of variances was satisfied and Games-Howell's method was used when this assumption was violated.

Investigation of Pasteurization Values of the Sous Vide Treatments
The pasteurization process has an important role in the sensorial attributes, nutritional value, and microbial stability of sous vide meat products [15,16]. Pasteurization values (p-values) are defined as the duration of the pasteurization process to achieve a particular reduction of a target pathogen at a specified temperature. To calculate pasteurization values of sous vide treatments, three target pathogens associated with sous vide meat products were considered: C. perfringens (P 6.74 60 ), L. monocytogenes P 10 70 , and C. botulinum spores (P 13 80 ). Pasteurization values (P 6.74 60 , P 10 70 , P 13 80 ) of the studied sous vide treatments derived from the recorded time-temperature profiles are presented in Table 2. As expected, chicken breasts treated with the one-step temperature obtained higher pasteurization values than the chicken breasts treated with the two-step temperature within the same total treatment time.  For pasteurization of sous vide foods, the European Chilled Food Federation (ECFF) [33] recommends at least a heat treatment equivalent to 70 • C for 2 min to ensure a 6-log reduction of L. monocytogenes (z-value = 10 • C). According to the results presented in Table 2, all the studied sous vide treatments achieved sufficient inactivation of L. monocytogenes from chicken breast. Previous studies have also reported successful inactivation of L. monocytogenes and other vegetative microorganisms by sous vide treatments [43,44]. On the other hand, both one-step and two-step sous vide treatments were insufficient to pasteurize chicken breast for C. botulinum spores as the pasteurization values (P 13 80 ) were lower than 26 min [34]. This poses a potential safety risk; thus, it requires proper storage at temperatures lower than 3 • C or at freezing temperatures to prevent the spore multiplication of C. botulinum [45].
Clostridium perfringens is a mesophilic spore-forming bacteria usually used as an indicator of thermal efficiency in sous vide treatments. From the thermal inactivation data of C. perfringens (D 60 = 5.3 min and z-value = 6.74 • C) [32], food products need to be pasteurized with a heating process equivalent to 60 • C for 32 min to achieve a 6-log vegetative cell reduction. From our results, it can be seen that all the studied sous vide treatments achieved the minimum pasteurization value of P 6.74 60 = 32 min.

Moisture Content and Cooking Loss
A comparison of the physicochemical properties of chicken breast (moisture content, cooking loss, and color attributes) between the studied sous vide treatments are displayed in Table 3. In the cases of moisture content and cooking loss, the two-way MANOVA overall result was significant for both factors (total treatment time: Wilk's lambda = 0.07, p < 0.001; time ratio: Wilk's lambda = 014, p < 0.001) with insignificant interaction (p = 0.22). The follow-up univariate ANOVA using Bonferroni's correction revealed significant results for both variables and both factors (all with p < 0.001).  Higher moisture content was observed when the application time of the low temperature (50 • C) was increased from 0 to 90 min in the 180-min sous vide treatments, with a significant increase only between T4 and T6 (p < 0.05). On the other hand, no significant increase in moisture content was observed between treatment time ratios within the 120-min sous vide treatments (T1, T2, T3) (p > 0.05). Our results are in agreement with those reported by other authors [15,46]. Bıyıklı et al. [15] found that moisture content was significantly higher in turkey cutlets cooked at lower sous vide cooking temperatures. The moisture content of meat depends on the degree of thermal protein denaturation, which causes shrinkage of muscle fibers [47], leading to a volume reduction and, consequently, water loss at high cooking temperatures.
Cooking losses during heat treatment contribute to a considerable share of food wastes in households and catering sector [48]. Sous vide presents a valuable method to reduce food waste by the combination of optimal temperature and time duration of cooking in heat-stable pouches. In our study, the cooking loss of chicken breasts cooked with different sous vide treatments ranged from 11.61% to 16.35% (Table 3). Chicken breasts cooked with the two-step sous vide treatment T3 had significantly lower cooking loss than the chicken breasts cooked with the one-step sous vide treatment T1 (p < 0.05). Low cooking loss correlates with higher juiciness, which has a positive impact on consumer preference for chicken meat [49]. A similar result was observed also between the 180-min one-step (T4) and two-step sous vide treatments (T5 and T6). Apparently, the application of the lower temperature (50 • C) in the two-step sous vide had a positive effect on lowering cooking loss and thus improving the juiciness of the chicken breast. In agreement with this result, Hwang et al. [46] reported that cooking loss of sous vide pork loins was lower when cooked at 50 • C compared to 55 and 60 • C. Zielbauer et al. [50] reported lower values of cooking loss compared with the trend during a long time of cooking pork at the temperature of 51 • C. This could be explained by collagen solubilization under denaturation, which results in gel formation and higher water content in meat [51].

Color Measurement
In Table 3 are presented the instrumental color properties (L*, a*, b*, and ∆E) of the chicken breast cooked in different combinations of temperature and time. Two-way MANOVA overall result was significant for both factors (total treatment time: Wilk's lambda = 0.23, p < 0.01; time ratio: Wilk's lambda = 0.003, p < 0.001) with significant interaction (p < 0.001). The follow-up univariate ANOVA using Bonferroni's correction revealed significant results for lightness (p < 0.05) and ∆E (p < 0.05) in the case of total treatment time and an insignificant result for a* (p = 0.91) and b* (p = 0.51). Meanwhile, univariate ANOVA resulted in a significant time ratio effect for all the four parameters (p < 0.001). The interaction was significant only for b* (p < 0.05).
Color is one of the main indicators in consumer perception of meat quality and safety [52]. Changes in the color properties of meat products treated with heat can be attributed to myoglobin denaturation and oxidation, Maillard reactions, packaging, or pH [51]. Compared to conventional cooking, sous vide-cooked meat obtains higher redness and lightness, which is due to myoglobin changes during denaturation, with deoxymyoglobin being the most resistant to protein denaturation from the three forms of myoglobin [53]. The present study showed that the two-step sous vide-treated samples had significantly lower lightness (L* value) than the one-step sous vide-treated samples within the same total treatment time (p < 0.05). This can be explained by the fact that higher temperatures can increase the protein denaturation process, which results in a higher brightness of meat [14]. Cooked chicken breast can be classified into three categories based on the degree of lightness: pale (L* < 53), normal (L* = 46-53), and dark (L* < 46) [54]. Based on the obtained lightness values, all the studied sous vide treatments belonged to the pale appearance classification group (L* > 53).
Redness values were between 1.34 and 2.46. The present study found no significant difference in redness when the total treatment time was increased from 120 min (T1) to 180 min (T4) at 60 • C (p > 0.05). In agreement with this result, Haghighi et al. [55] reported that redness values of sous vide chicken breasts were not affected when the cooking time was increased from 60 min to 150 min at the temperature of 60 • C. In contrast, Park et al. [56] reported that chicken breasts cooked at 60 • C for 180 min had lower redness compared to those cooked for 120 min. Our study revealed that chicken breast cooked with the 180-min sous vide had significantly higher redness values with a longer application time of the low temperature of 50 • C in the sous vide treatment. This might be attributed to myoglobin pigment concentration, which is presumed to be higher in the two-step sous vide-treated samples because its denaturation starts between 55 and 65 • C [53]. The pink color in poultry meat has been shown to be the first indicator that consumers check for a properly cooked product [57]. The degree of the pink color on chicken breast can be evaluated using a subjective pink threshold of a* = 3.8 [58]. In the present study, all the sous vide-treated samples obtained redness values under this threshold level.
The yellowness values of the chicken breasts cooked with the two-step temperature were significantly higher than those processed with the one-step temperature within the same total treatment time (p < 0.05). This might be due to a lower exposure of meat to the temperature of 60 • C during cooking in the two-step sous vide treatments (40-90 min) in comparison with the one-step sous vide treatments (120 and 180 min) ( Table 3).
Results of the total color difference (∆E) between raw and cooked chicken breasts are presented in Table 3. The highest total color difference (∆E) was obtained for treatment T4 (30.06) and the lowest, for treatment T2 (28.64). According to Mokrzycki and Tatol [59], an observer notices two different colors when ∆E > 5. In this context, the authors believe that for a better understanding of changes in the color attributes of the samples it is more common to evaluate the total color difference (∆E) between the studied sous vide treatments. Total color difference (∆E) results between sous vide treatments were classified based on five categories as described by Mokrzycki and Tatol [59] and are presented below in Table 4. According to the study of Tomasevic et al. [60], a clear color difference between meat products is noticeable if ∆E > 3.5. From the pairwise comparison between the 120-min sous vide treatments, a clear color difference can be noticed between the chicken breast cooked with the one-step temperature treatment (T1) and the two-step temperature treatment (T3) where the total color difference (∆E) was 3.94 (Table 4). Similar results were obtained for total color difference (∆E) between the 180-min one-step (T4) and two-step sous videtreated chicken breasts (T5, T6) (∆E > 3.5). On the other hand, the total color difference between chicken breast cooked at one-step temperature treatments for 120 and 180 min can be perceived only by an experienced observer (∆E > 2). It can be inferred that the first-step temperature of 50 • C and the time duration in which it was used in the two-step sous vide treatments had a major impact on the color attributes between the studied sous vide treatments.

Texture Properties
Shear force is the most common instrumental method to evaluate the tenderness of cooked meat. Shear force and texture profile analysis (TPA) parameters of the studied sous vide treatments on the chicken breast are displayed in Table 5. Two-way ANOVA revealed significant total treatment time (p < 0.01) and treatment time ratio effects (p < 0.001) on shear force with insignificant interaction effect (p > 0.05). Chicken breasts cooked with the two-step temperature had significantly lower shear force values than chicken breasts cooked with the one-step temperature in the case of 180-min treatments (p < 0.05). These results showed the effect of the first-step temperature (50 • C) applied in the 180-min twostep temperature treatments on increasing the tenderness of sous vide chicken breast. In agreement with this result, Hwang et al. [46] reported that shear force values were lower when pork loins were cooked at 50 • C compared to those cooked at 55 or 60 • C. This tenderization effect might be attributed to the specific proteolytic enzymes for desmin degradation, which are largely active up to 50 • C [29]. Chicken breasts cooked with the one-step sous vide obtained higher shear values because at 60 • C the main proteolytic enzymes, calpains, are inactivated [61] while cathepsins operate with only 50% of their proteolytic activity [62].  The results of TPA parameters are shown in Table 5. The two-way MANOVA overall result was significant for both factors (total treatment time: Wilk's lambda = 0.04, p < 0.001; time ratio: Wilk's lambda = 0.03, p < 0.001) with significant interaction (p < 0.001). The follow-up univariate ANOVA using Bonferroni's correction revealed significant results for cohesiveness, gumminess, and chewiness in the case of total treatment time (p < 0.05) and an insignificant result for hardness and springiness (p > 0.38). Univariate ANOVA resulted in a significant time ratio effect for hardness, gumminess, and chewiness (p < 0.05) and it was insignificant for cohesiveness and springiness (p > 0.23). The interaction was significant for cohesiveness, gumminess, and chewiness (p < 0.05).
The highest TPA-hardness mean value (49.78 N) was obtained in chicken breasts cooked with the 120-min one-step sous vide (T1), which was significantly higher compared to chicken breast cooked with the 120-min two-step sous vide (T2 and T3) (p < 0.05). However, hardness was not significantly different in the 180-min two-step sous vide treatments (T5 and T6) compared to the 180-min one-step sous vide treatment (T4). On the other hand, no significant difference was observed in hardness between chicken breast cooked with the one-step sous vide at 60 • C for 120 min (T1) and 180 min (T4). Similarly, Park et al. [56] found that the hardness of chicken breast was not affected by cooking time when cooked at the temperature of 60 • C for different time durations (60, 120, and 180 min). No significant differences were observed between one-step and two-step sous vide on cohesiveness and springiness within the same total treatment time (p > 0.05) ( Table 5). Previous studies have also reported no difference in springiness and cohesiveness of the sous vide chicken breast [56] and beef [11] cooked at different combinations of temperature and time durations. In addition to hardness, two other important texture parameters especially for elderly consumers are chewiness and gumminess. According to the results presented in Table 5, gumminess was significantly reduced in the two-step sous vide-treated samples in comparison with the one-step sous vide-treated ones within the same total treatment time, except for T5. The trends for gumminess values were just slightly different from chewiness values in sous vide chicken breast treated at the same total treatment time. The chewiness was also reduced in the case of the 120-min two-step sous vide-cooked chicken breast with T2 treatment having a significant decrease. The reduced shear force and hardness (initial bite tenderness) and the reduced gumminess and chewiness obtained in two-step sous vide-treated chicken breast present important texture attributes for an increase in meat consumption by elderly consumers [63].

Lipid Oxidation
To evaluate the rate of oxidation in sous vide-treated chicken breast, the level of the secondary lipid oxidation compounds such as aldehydes (like malonaldehyde) was measured using a TBARS test. This method is used to indirectly detect the level of oxidative rancidity in meat products. The results of TBARS values of sous vide-treated chicken breast with different combinations of temperatures and time durations are shown in Table 6. Two-way ANOVA revealed significant total treatment time and treatment time ratio effects on TBARS (p < 0.001) with insignificant interaction effect (p = 0.83). Increasing the total treatment time from 120 min to 180 min within the same treatment time ratio caused an increase in TBARS values of the cooked chicken breast. In agreement with our result, Karpinska-Tymoszczyk et al. [17] reported that increasing the cooking time from 90 to 150 min at 61 • C increased the TBARS values of sous vide chicken breast fillets. Chicken breasts treated with the one-step temperature treatments obtained significantly higher TBA values than those treated with the two-step temperature treatments within the same total treatment time (p < 0.05) ( Table 6). This might be due to the longer application time of the end step temperature in the two-step sous vide treatments, which caused higher protein denaturation, thus higher release of iron ions, which have a strong pro-oxidant effect [64]. The highest mean TBA value (0.520 mg MDA/kg sample) was observed in chicken breasts cooked at 60 • C for 180 min (T4 treatment). According to Akoglu et al. [65], consumers are not able to detect the oxidative rancidity in sous vide-treated chicken breast with TBARS values below 1 mg/kg. None of the tested two-step sous vide treatments exceeded this threshold level (Table 6).

Protein Solubility
The results for sarcoplasmic, myofibrillar, and total protein solubility of chicken breasts cooked at different combinations of temperatures and time durations are shown in Table 6. The two-way MANOVA overall result was significant for both studied factors (total treatment time: Wilk's lambda = 0.13, p < 0.001; time ratio: Wilk's lambda = 0.04, p < 0.001) with significant interaction (p < 0.001). The follow-up univariate ANOVA using Bonferroni's correction revealed significant results for myofibrillar and total protein solubility in the case of total treatment time (p < 0.001) and an insignificant result for sarcoplasmic protein solubility (p = 0.64). Univariate ANOVA resulted in a significant time ratio effect for all the three protein solubility variables (p < 0.05). The interaction was significant for sarcoplasmic and myofibrillar protein solubility (p < 0.05).
The present study showed a significant decrease in total protein solubility of chicken breast cooked with the one-step sous vide in comparison with the ones cooked with the twostep sous vide within the same total treatment time. Lower total protein solubility indicates the heat denaturation of sarcoplasmic and myofibrillar proteins. Protein denaturation increased with longer cooking time durations of chicken breast at the high temperature of 60 • C, which is in agreement with the results of Cropotova et al. [66]. Sarcoplasmic protein solubility of chicken breasts cooked with the two-step sous vide was increased at a higher rate than myofibrillar protein solubility in comparison to chicken breasts cooked with the one-step sous vide in the 120-min total treatment time but not in the 180-min (Table 6). There is a relation between protein solubility, moisture content, and cooking loss of meat [67,68]. Murphy and Marks reported a strong correlation between protein solubility and cooking loss in heat-treated chicken breast patties [67]. The results of our study are in accordance with this observation (data not shown). Moisture content was also significantly correlated with both myofibrillar and total protein solubility (R = 0.889 and R = 0.785, both with p < 0.001).

Conclusions
The two-step sous vide technique can provide a valuable cooking alternative for elderly consumers as it reduced the main texture parameters (shear force, hardness, chewiness, and gumminess), but at the same time preserved the water content, redness, and oxidative stability in chicken breasts. Furthermore, significantly lower cooking loss and increased protein solubility values were observed in chicken breast cooked with the two-step sous vide compared to the ones cooked with the traditional one-step sous vide. The present study also found that the treatment time ratio between the first-and second-step temperatures (50 and 60 • C) had a significant effect on cooking loss, lightness, total color difference, gumminess, TBARS, sarcoplasmic, and total protein solubility of sous vide-treated chicken breasts within the same total treatment time. Meanwhile, the total treatment time was significant only for moisture content, cooking loss, and TBARS values of cooked chicken breasts within the same treatment time ratios. Regarding microbiological safety, all the two-step sous vide treatments were able to successfully inactivate the vegetative cells of two main pathogens of interest (C. perfringens and L. monocytogenes) in chicken breast. As expected, none of the studied sous vide heat treatments were enough to inactivate the C. botulinum spores; thus, proper refrigeration storage of these products is required. Future investigations need to be done to evaluate the sensory characteristics and the shelf life of chicken breast cooked with the two-step sous vide to determine consumer acceptance, with special emphasis among elderly consumers.