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The effect of pressure-assisted heating on the water holding capacity of chicken batters
Innovative Food Science and Emerging Technologies 45 (2018) 280–286
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The effect of pressure-assisted heating on the water holding capacity of chicken batters
T
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Hai-bo Zhenga,b, Min-yi Hanc, Hui-juan Yangd, Xing-lian Xue, , Guang-hong Zhoue a
Key Laboratory of Meat Processing, Ministry of Agriculture, Nanjing 210095, PR China Food and Drug College, Anhui Science and Technology University, Fengyang 233100, PR China c Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, PR China d Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing 210095, PR China e College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Pressure assisted heating Water holding capacity Protein denaturation Water distribution and mobility Microstructure
The ability of gel-type meat products to hold water is an important quality attribute, which is affected by processing. The aim of this research was to investigate the effects of pressure-assisted heating, which can disrupt myofibrils and hinder heat-induced protein denaturation, on the water holding capacity of chicken meat batters. High pressure-assisted heating (100–400 MPa, 65 °C, and 30 min) and heating-only (0.1 MPa, 65 °C, and 30 min) was applied to chicken meat batters, the centrifugal loss, water distribution and mobility, microstructure, and residual denaturation enthalpy were determined. A threshold pressure of between 300 and 400 MPa was found, below which the WHC was improved, but impaired at greater pressures. Distributed exponential analysis of the T2 relaxation revealed three states of water binding (T2b, T21 and T22), each of which was significantly correlated with WHC. Pressure-treated batters had a higher amount of bound water than the heat-only batters, and showed a decrease in immobilized water and an increase in free water with increasing pressure. Myofibril structures were degraded by high pressure. High pressure resulted in a porous microstructure which held more water. However, pressures greater than the threshold caused loose gel-networks and decreased water holding capacities. The heat-denaturation of meat proteins was affected by high pressure. Actin was denatured by high pressure instead of heating, while collagen and some myosin derivatives were preserved from being denatured by heating. The changes in protein denaturation and batter microstructure were correlated with water distribution properties. The results contributed to a better understanding of the effects of high-pressure with heat on the water holding capacity of chicken batters. Industrial relevance: A beneficial threshold pressure of between 300 and 400 MPa was found, below which the water holding capacity was improved, and above which water holding capacity was reduced. As the effect of high pressure on physical properties and sterilization were not always consistent, this finding reminds the meat industry need to adopt a suitable pressure to achieve a balance between physical properties and sterilization. The low filed nuclear magnetic resonance could be adopted in a routine examination of product quality.
Chemical compounds studied in this article: Sodium chloride (PubChem CID: 5234) Sodium tripolyphosphate (PubChem CID: 24,455)
1. Introduction The water holding capacity of meat products is an important quality attribute, not just for the sensory aspects, but also for economic benefits. Meat has a water content of approximately 75% which is distributed in intra- and extra-myofibrillar spaces (Pace & Rathbun, 1945). Further, additional water is usually added to gel-type meat products during processing, making it difficult for the meat products to retain water. When the meat product is cooked, the heat-induced shrinkage of myofibrils results in the migration of water from intra-myofibrillar to the extra-myofibrillar spaces, potentially resulting in water loss ⁎
(Tornberg, 2005; Van der Sman, 2007). Understanding the water distribution and its migration is essential for developing meat product formulations and processing technology. The application of high pressure processing (HPP) has increased rapidly during the last 30 years. HPP has being used in the food industry for various purposes including its effectiveness for reducing numbers of viable microorganisms, meat tenderization, protein gelation, starch gelatinization and food frozen/unfrozen (Balasubramaniam & Farkas, 2008; Suzuki, 2002). HPP has long been recognized as a “cold processing” technology, mostly because of its “cold pasteurization” effect (Heinz & Buckow, 2010). However, a combination of high pressure
Corresponding author. E-mail address: [email protected] (X.-l. Xu).
https://doi.org/10.1016/j.ifset.2017.11.011 Received 12 October 2017; Received in revised form 10 November 2017; Accepted 17 November 2017 Available online 21 November 2017 1466-8564/ © 2017 Elsevier Ltd. All rights reserved.
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chamber was heated to 65 °C and kept constant by a water bath (ILBWCS, STIK Shanghai Co., Ltd.). The temperature in the sample chamber was monitored during processing by a thermocouple located inside the top of the chamber. A heating time of 30 min as suggested by Fernandez Martin et al. (1997), was used for all samples. Compression led to a temperature increase of 3 °C/100 MPa and decompression led to a temperature decrease of 4 °C/100 MPa. The initial temperature of the compression fluid was reduced before each run to counteract the temperature increase caused by adiabatic heating. The pressurization and depressurization rates were 5 MPa/s and 20 MPa/s, respectively. All samples were then cooled in a running tap water immediately for 1 h after being heated, and then stored in a 0–4 °C room.
and high temperature, namely heating under pressure (HUP), that is, applying two physical effects simultaneously to a foodstuff, can improve the food product. This has not been thoroughly explored. In meat products, one of the beneficial effects is reducing water loss. This has been reported by several studies on chicken, pork and beef (Fernandez Martin, Fernandez, Carballo, & Colmenero, 1997; Jiménez-Colmenero, Fernández, & Carballo, 1998; Sikes & Tume, 2014; Zheng et al., 2015; Zheng et al., 2017). Several changes related to the WHC of HUP-treated meat products have been suggested, such as the breakdown of myosin molecules, a denaturation-preserving effect, and disruption of myofibrils (JimenezColmenero, Cofrades, Carballo, Fernandez, & Fernandez-Martin, 1998; Zheng et al., 2015; Zheng et al., 2017). However, these findings were based on indirect determinations or subjective observations and were unable to explain the actual pattern of water distribution and movement within the cellular environment. Several studies have shown that low-field nuclear magnetic resonance (LF-NMR) proton relaxometry can provide information about water mobility and distribution based on the measurement of transverse water proton relaxation (Berendsen, 1992; Bertram, Aaslyng, & Andersen, 2005). Also, it is a rapid, noninvasive, and nondestructive tool for the characterization of water properties (Han, Wang, Xu, & Zhou, 2014). The aim of this study was to investigate the effect of high pressure assisted heating on the water holding capacity of chicken meat batters using low-field NMR proton T2 relaxometry, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) in combination with traditional meat quality measurements. This work can contribute to the application of high pressure processing in the meat industry.
2.4. Determination of water distribution and mobility Water distribution and mobility were determined according to the method of Han et al. (2014) by using low-field nuclear magnetic resonance (LF-NMR), with minor modifications. A sub-sample (approximately 2 g) was cut from the sausage and placed in a cylindrical glass tube (15 mm in diameter). The relaxation measurements were performed on a bench top pulsed NMR analyser (PQ001, Niumag Electric Corporation, China) with a corresponding resonance frequency for protons of 22.6 MHz. Transverse relaxation, T2, was measured using the Carr-Purcell-Meiboom-Gill (CPMG) sequence (Carr & Purcell, 1954; Meiboom & Gill, 1958). The T2 measurements were performed with a τ value (time between 90° pulse and 180° pulse) of 200 μs. The repetition time between two succeeding scans was 5 s. Data were acquired from 3000 echoes as an average of 8 repetitions. Each measurement was performed in quintuplicate. The transverse low-field NMR relaxation data were fitted to a multi-exponential curve with the MultiExp Inv Analysis software (Niumag Electric Corporation, China). This analysis yielded a plot of relaxation amplitude for individual relaxation processes versus relaxation time. The relaxation peak time (T2) and peak area (P2) were calculated from the curve.
2. Material and methods 2.1. Materials and chemicals Chicken breast meat (M. pectoralis major) was transported from a meat company (Jiangsu Tyson Foods Co, LTD., China) at low temperature (0–4 °C) within 24 h after slaughtering. All chemicals used were of analytical grade, except sodium chloride and sodium polyphosphates, which were food grade.
2.5. Determination of water holding capacity Centrifugal loss was determined by the centrifugation method as described by Zheng et al. (2017). Samples were cut into 1 cm long cylinders, weighed (Wstart) and wrapped with a filter paper. Then, the sample was centrifuged at 10,000 ×g for 10 min at 10 °C. After removing the filter paper, the sample was reweighed (Wend). The centrifugal loss was expressed as the percentage of water loss to total weight.
2.2. Preparation of batters A total of 10 kg chicken meat was trimmed to remove visible fat and connective tissue, cut into strips, and then minced through a meatgrinding machine (MOD TC/12E, Sirman, Italy) fitted with a plate made up of 5-mm-holes. Then, the minced meat was mixed and divided into 5 equal parts before being chopped separately. The minced chicken (80 g/100 g) was chopped with ice (17.7 g/100 g), sodium chloride (2 g/100 g), and sodium tripolyphosphate (0.3 g/100 g) for 5 min in a chopper (K15E, Talsabell S. A., Spain) at 1800 rpm. The temperature of chicken batters during the cutting was maintained below 13 °C. The meat batter was subjected to vacuum to remove the air bubbles before stuffing into plastic casings with a diameter of 26 mm and linked every 20 cm. The sausages (120 g each) were vacuum packaged in vacuum bags separately and then stored at 0–4 °C prior to further treatment on the following day.
Centrifugal loss (%) =
Wstart − Wend × 100 Wstart
2.6. Structural observation Subsamples were taken from the central part of sausages and cut into 0.1 mm × 1 mm × 2 mm blocks and fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer solution (pH 7.0). The ethanol dehydration, freeze-drying and sputter-coating were performed according to the procedure of Cao, Xia, Zhou, and Xu (2012). Samples were observed and photographed by using a scanning electron microscope (S-3000N, Hitachi, Japan) at a voltage of 10 kV.
2.3. Pressure/thermal treatments 2.7. Determination of thermal properties Vacuum packaged chicken meat batters were subjected to 0.1, 100, 200, 300, or 400 MPa and heat at 65 °C for 30 min. Heat-only (0.1 MPa) treatment was performed in a water bath (TW20, JULABO Technology Co. Ltd., Germany). The pressure assisted heating (100–400 MPa) was performed in a 0.3 L high-pressure unit (S-FL-850-9-W/FPG5620YHL, Stansted Fluid Power Ltd., UK) by using a mixture of water and propylene glycol (7:3, w/v) as the compression fluid. The high-pressure
Samples (15–20 mg) were encapsulated in aluminum pans and hermetically sealed. The samples were equilibrated at 25 °C for 2 min before scanning from 25 to 90 °C at 5 °C/min in a differential scanning calorimeter (DSC1, Mettler-Toledo International Inc., Switzerland) with an empty aluminum pan as a reference. Dry matter content was determined for thermal data normalization to dry matter content by 281
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800
desiccation at 105 °C for 16 h. The endothermal curve of each sample was recorded.
T2 (ms)
Experiments were carried out with at least in 5 independent replicates and the results were expressed as mean ± standard deviation (SD). Analysis of variance was carried out with the software SAS 9.0 (SAS Institute Inc., USA), and differences among means were evaluated by using Duncan's new multiple range test (p < 0.05).
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3.1. Water distribution and mobility
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Changes in water distribution and mobility in meat products can be determined using low-field nuclear magnetic resonance (LF-NMR). NMR transverse relaxation of water (T2) can be separated into the relaxation decay of three exponential populations, representing three water compartments in meat products: bound water (T2b), immobilized water (T21), and free water (T22) (Pearce, Rosenvold, Andersen, & Hopkins, 2011). Fig. 1 shows the distributions of T2 relaxation times of three peaks centered at approximately 0.5–2.5 ms (T2b), 30–110 ms (T21), and 300–800 ms (T22). As shown in Fig. 2, pressure-treated batters, generally, had lower T2b (1.15–1.23 ms) but higher T21 (57.65–60.22 ms) and T22 (513.34–624.18 ms) than heat-only samples, except the 400 MPatreated batter had a lower T22 (513.34 ms). Since the relaxation time T2 reflects the probability of a water molecule interacting with surrounding surfaces, the changes in T2 implies alterations in the space which acts as relaxation sinks (Pearce et al., 2011). Pressure-treated batters had shorter T2b but longer T21 than heat-only batters, indicating a smaller space for bound water but a larger space for immobilized water. The decreasing T22 from 624.18 ms of the 100 MPa-treated samples to 513.34 ms of 400 MPa-treated sample also indicated that the space for free water had decreased with increased pressure. An increase in P2 population indicates an increase in water population (Pearce et al., 2011). High pressure significantly (p < 0.05) increased the proportion of P2b from 1.24% for heat-only samples to 2.65–2.78% for pressure-treated samples, but there was no significant (p > 0.05) difference among pressure-treated samples. As the proportion of P2b represents the amount of bound water, the higher proportion of P2b in the pressure-treated samples indicated that more water was bound with pressure-treated proteins. The samples heated at 100–300 MPa had almost the same P21 (96.47–96.68%) and greater than that of the heat-only (95.74%) and
Fig. 2. Low-field NMR relaxometry T2 relaxation times of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min. a–d, means (n = 5) without a common letter within the same T2 relaxation time indicate significant difference (p < 0.05).
P2b a
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Pressure (MPa) Fig. 3. Low-field NMR relaxometry proportions of peak areas of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min. a–c, means (n = 5) without a common letter within the same P2 population indicate significant difference (p < 0.05).
30 a
Centrifugal loss (%)
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8 0.1 MPa 100 MPa 200 MPa 300 MPa 400 MPa
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2.8. Statistical analysis
Amplitude (a.u.)
T2b a
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Pressure (MPa) Fig. 4. Centrifugal loss of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, and 400 MPa for 30 min. a–c, means (n = 5) with different letters indicate significant difference (p < 0.05).
0 1
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T2 (ms) Fig. 1. Distribution of T2 relaxation times for chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min.
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Fig. 5. SEM microstructure of pressure/thermal treated chicken batters treated by heating at 65 °C and at pressures of 0.1 (a), 100 (b), 200 (c), 300 (d), or 400 MPa (e) for 30 min.
400 MPa-treated (95.64%) samples, indicating more water was immobilized in 100–300 MPa-treated samples than the others. A decrease in P21 normally was accompanied by an increase in P22 (Bertram et al., 2001). The heat-only sample showed the highest P22 (3.02%), followed by the 400 MPa-treated sample (1.65%), while the samples treated at 100–300 MPa showed the lowest P22 (0.6, 0.67, and 0.8%, respectively), indicating that the heat-only sample had the most free water, followed by the 400 MPa-treated sample, while the samples treated at 100–300 MPa had the lowest free water.
reduction in centrifugal loss indicated that the WHC was improved by HUP treatments. The improvement in WHC had also been characterized by cook loss as reported by Jimenez-Colmenero et al. (1998), who showed that pressure-treated samples had a lower water loss which decreased with increasing pressure. However, a significant (p < 0.05) increase in centrifugal loss to 18.21% was observed as the pressure was increased to 400 MPa, indicating that WHC decreased when pressure was too high. The results of centrifugal loss were in agreement with those of LF-NMR.
3.2. Centrifugal loss
3.3. Microstructure
The effect of HUP treatment (0.1–400 MPa) on water holding capacity was characterized by centrifugal loss (Fig. 4). Heat-only sample had the highest centrifugal loss, up to 25.77%, followed by the 400 MPa-treated samples, which was 18.21%, whereas those heated at 100–300 MPa had the lowest centrifugal loss of 10.19% to 11.11%. The
The myofibril fragments in the heat-only samples were essentially intact and were be easily recognized, but they became disrupted and became less visible in pressure-treated samples, see Fig. 5. The structure of myofibrils in the 100 MPa-treated samples was still evident but was barely present in the 200 MPa-treated samples and completely 283
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samples, the proteins that had a denaturation temperature below 65 °C should be denatured. However, an endothermic peak around 64 °C was found in almost all pressure-treated samples, indicating that collagen was preserved from being heat denatured. This was in agreement with the reports that collagen does not undergo appreciable changes when subjected to HUP because its hydrogen-bonded structure was strengthened by pressure (Buckow, Sikes, & Tume, 2013; FernandezMartin, 2007). However, at 100 MPa, the pressure appeared to be too low to protect collagen from being heat denatured, as the collagen peak was not found in the 100 MPa-treated samples. A weak endothermic peak at 60 °C was observed in the 100 MPa-treated samples, and it was attributed to myosin aggregates (Chapleau, Mangavel, Compoint, & Lamballerie-Anton, 2004), which have been found to remain native-like at conditions up to 400 MPa at a temperature up to 75 °C (FernandezMartin, 2007). However, this peak could not be distinguished in samples at higher pressures, probably because this peak was too small and too close to the large collagen peak. Actin had an endothermic peak near 71 °C and remained native in the heat-only samples. However, no actin peak was found in the pressure-treated samples, indicating that actin had been denatured by pressurization. This was consistent with previous reports where it was found that actin was a thermostable but a pressure labile protein (Fernandez-Martin, 2007). The less denatured collagen, the myosin aggregates, and the denatured actin were present in the pressure-treated samples, indicating that pressure treatment induced a different degree or type of denaturation as compared to heat-only sample, which may have affected its affinity for water. Pressure-treated samples showed a shorter T2b relaxation time (Fig.2) and a larger P2b proportion (Fig. 3) than heat-only sample, indicating that the proteins in the pressure-treated samples had greater affinity than those of heat-only sample.
0.02 W/g
Heat flow
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Temperature (oC) Fig. 6. Thermograms of raw chicken meat batter.
Heat flow
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4. Discussion
200 MPa
4.1. The effect of HUP treatment on WHC, LF-NMR, microstructure and protein denaturation
100 MPa 0.1 MPa 30
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The effect of pressure-assisted heating on WHC varied with pressure intensity. It was obvious that pressures between 100 MPa and 300 MPa had a positive influence on WHC, while a pressure at 400 MPa reduced WHC, indicating there was a threshold of between 300 MPa and 400 MPa for WHC at the specified temperature. It was also obvious that the negative effect of high pressure on WHC commenced at 300 MPa, as the P21 showed a drop and P22 showed an increase at 300 MPa. Further, the pore size of the gel network showed an obvious increase at 300 MPa. Although protein denaturation that was preserved by high pressure has been reported to be one of the important properties of HUP treatment (Kanaya, Hara, Nakamura, & Hiramatsu, 1996), there was no obvious correlation between the degree of denaturation of proteins and WHC. The water holding capacity between the 300 MPa-treated and 400 MPa-treated samples was significantly different (p < 0.05), but there was no obvious difference in thermograms. Therefore, it would be more reasonable to ascribe the changes in WHC to a different type rather than the different extent of denaturation. Villamonte, Simonin, Duranton, Cheret, and de Lamballerie (2013) proposed that it was the type of denaturation, not the total protein denaturation, which influenced the hardness of pressure-only treated meat batters. The microstructure changed progressively with increasing pressure (Fig. 5), showing two changing features: the disruption of myofibrils and the formation of porous networks. The disruption of myofibrils was caused by high pressure, however, the formation of stable porous network was probably promoted by both heating and high pressure. The pore size of the gel networks, changed significantly as the pressure increased from 100 MPa to 400 MPa, implying that the heat-induced interactions between meat proteins were also affected by high pressure. This was also consistent with our view that HUP treatment induced a
90
o
Temperature ( C) Fig. 7. Thermograms of chicken batters treated by pressure/thermal at 65 °C and at 0.1, 100, 200, 300, or 400 MPa for 30 min.
disrupted in 300 MPa and 400 MPa-treated samples. The disruption of myofibrils contributed to the formation of a porous microstructure both inside and outside the myofibrils, which was consistent with the report of Zheng et al. (2017). Heat-only samples had few protein networks outside the myofibrils, and no porous microstructure inside the myofibrils (Fig. 5a). In pressure-treated samples, although the higher the pressure the greater the disruption (Iwasaki, Noshiroya, Saitoh, Okano, & Yamamoto, 2006), this did not necessarily result in a better microstructure (Fig. 5e). The gel network of pressure-treated samples became more regular and porous as pressure increased from 100 MPa to 200 MPa. However, the microstructure became less porous and with larger cavities as pressure increased to 300 MPa, and at 400 MPa, aggregates and fractures appeared. 3.4. Differential scanning calorimetry (DSC) For raw batter, typical endothermic peaks were observed at 56 °C, 64 °C and 71 °C (Fig. 6), which were ascribed to the thermal denaturation of myosin, sarcoplasmic proteins and collagen, and actin, respectively (Speroni, Szerman, & Vaudagna, 2014). As a heating temperature at 65 °C was used for all the heated 284
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new type of denaturation.
Acknowledgments
4.2. Connections between WHC, LF-NMR, microstructure and protein denaturation
This research was supported by the China Agriculture Research System (CARS-41), the Natural Science Foundation of China (31471601), the Key Projects of Young and Middle-aged Talents in Colleges and Universities in 2016, China (gxfxZD2016184), and the Fundamental Research Funds for the Central Universities (KYZ201543). The assistance provided by Dr. Ron Tume (Honorary Guest Professor at NAU) in the preparation of this manuscript is gratefully acknowledged.
As the amount of water loosely bound correlates with water loss (Bertram, Donstrup, Karlsson, & Andersen, 2002), the higher proportion of free water the higher the tendency to lose water. The proportion of free water (P22) showed a highly positive correlation (r = 0.9892, p = 0.0013) with centrifugal loss, as reported by Bertram, Purslow, and Andersen (2002) and Zheng et al. (2015), where it was found that the higher P21 or the lower P22, the higher water holding capacity. As the variations in WHC resulting from pressure differences can be predicted by LF-NMR, this technique can be used to provide an accurate and convenient means to evaluate the WHC of HUP-treated meat products. Several reports have shown that the gel microstructure (gel capillarity) played an important role in determining the water holding capacity, as the major part of the water was immobilized by capillary force (Liu, Lanier, & Osborne, 2016; Stevenson, Dykstra, & Lanier, 2013). In our present work we showed that heat-only and 400 MPatreated samples had a less porous microstructure than the samples treated at 100–300 MPa, so were accompanied with higher centrifugal loss (Fig. 4). The microstructure studies also agreed with the LF-NMR data. The desired microstructure, resulting from the disruption of myofibrils, and the size of the porous gel network was optimal at a pressure of 200 MPa (Fig. 5c). It was also found that T21 and P21 reached their maximum values at 200 MPa. Similarly, the 400 MPatreated samples, which had an impaired microstructure, had lower T21 and P21 values. It was concluded that the variations in T21 and P21 were consistent with the changes in microstructure. As bound water refers to water that is closely associated with proteins (Shao et al., 2016), the variations in protein structure should affect the T2 relaxation time of bound water. Han et al. (2014) reported that an increase in heat-induced denaturation was accompanied by an increase in T2b. In this study, actin, which accounts for about 15% of total protein, was completely denatured at all high pressures. However, its denaturation was less, or not affected, in the heat-only samples as the temperature was lower than its denaturation temperature (Fig. 7). This may partially explain the difference in T2b and P2b between pressure-treated and heat-treated samples. Collagen was denatured at 100 MPa but was less denatured at greater pressures, however, there were no significant (p > 0.05) changes in T2b and P2b, indicating that it did not affect T2b and P2b. The myosin, accounting for about 30% of the total protein, was thought to be partly transformed to myosin aggregates which remained partially non-denatured at 100 MPa. However, this peak was not observed in other pressure-treated samples. As there were no clear connections between bound water and degree of protein denaturation degree, it appeared likely that it was the different structure induced by high pressure that led to the difference in bound water between pressure-treated and heat-only samples.
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Studies of the effect of hydrostatic pressure pretreatment on thermal gelation of chicken myofibrils and pork meat patty. Food Chemistry, 95(3), 474–483. Jimenez-Colmenero, F., Cofrades, S., Carballo, J., Fernandez, P., & Fernandez-Martin, F. (1998). Heating of chicken and pork meat batters under pressure conditions: Protein interactions. Journal of Agricultural and Food Chemistry, 46(11), 4706–4711. Jiménez-Colmenero, F., Fernández, P., & Carballo, J. (1998). High-pressure-cooked lowfat pork and chicken batters as affected by salt levels and cooking temperature. Journal of Food Science, 63(4), 656–659. Kanaya, H., Hara, K., Nakamura, A., & Hiramatsu, N. (1996). Time-resolved turbidimetric measurements during gelation process of egg white under high pressure. In R. Hayashi, & C. Balny (Vol. Eds.), Progress in biotechnology. Vol. 13. Progress in biotechnology (pp. 343–346). Elsevier. Liu, W., Lanier, T. C., & Osborne, J. A. (2016). Capillarity proposed as the predominant mechanism of water and fat stabilization in cooked comminuted meat batters. Meat Science, 111, 67–77. Meiboom, S., & Gill, D. (1958). Modified spin-echo method for measuring nuclear relaxation times. Review of Scientific Instruments, 29(8), 688–691. Pace, N., & Rathbun, E. N. (1945). Studies on body composition. 3. The body water and chemically combined nitrogen content in relation to fat content. Journal of Biological Chemistry, 158, 685–691. Pearce, K. L., Rosenvold, K., Andersen, H. J., & Hopkins, D. L. (2011). Water distribution and mobility in meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality attributes - A review. Meat Science, 89(2), 111–124. Shao, J.-H., Deng, Y.-M., Jia, N., Li, R.-R., Cao, J.-X., Liu, D.-Y., & Li, J.-R. (2016). Lowfield NMR determination of water distribution in meat batters with NaCl and polyphosphate addition. Food Chemistry, 200, 308–314. Sikes, A. L., & Tume, R. K. (2014). Effect of processing temperature on tenderness, colour and yield of beef steaks subjected to high-hydrostatic pressure. Meat Science, 97(2), 244–248. Speroni, F., Szerman, N., & Vaudagna, S. R. (2014). High hydrostatic pressure processing of beef patties: Effects of pressure level and sodium tripolyphosphate and sodium
5. Conclusions Below a threshold of between 300 and 400 MPa, pressure-assisted heating contributed to the formation of a gel having a high water holding capacity. The increase in bound water found in pressure-treated batters likely resulted from the different protein structure induced by high pressure. The increased immobilized water and the decreased free water in pressure-treated batters were ascribed to the disruption of myofibrils and the formation of a porous microstructure. Heating under a pressure higher than the threshold pressure impaired the water holding capacity. This was probably caused by disturbance in the interaction of meat proteins during denaturation imposed by high pressure. A different type of denaturation, instead of denaturation degree, plus the disruption of myofibrils, contributed to the distinct different water distribution and mobility in pressure-treated batters. 285
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Villamonte, G., Simonin, H., Duranton, F., Cheret, R., & de Lamballerie, M. (2013). Functionality of pork meat proteins: Impact of sodium chloride and phosphates under high-pressure processing. Innovative Food Science & Emerging Technologies, 18, 15–23. Zheng, H., Han, M., Yang, H., Tang, C., Xu, X., & Zhou, G. (2017). Application of high pressure to chicken meat batters during heating modifies physicochemical properties, enabling salt reduction for high-quality products. LWT - Food Science and Technology, 84, 693–700. Zheng, H., Xiong, G., Han, M., Deng, S., Xu, X., & Zhou, G. (2015). High pressure/thermal combinations on texture and water holding capacity of chicken batters. Innovative Food Science & Emerging Technologies, 30, 8–14.
chloride concentrations on thermal and aggregative properties of proteins. Innovative Food Science & Emerging Technologies, 23, 10–17. Stevenson, C. D., Dykstra, M. J., & Lanier, T. C. (2013). Capillary pressure as related to water holding in polyacrylamide and chicken protein gels. Journal of Food Science, 78(2), C145–C151. Suzuki, A. (2002). High pressure-processed foods in Japan and the world. Progress in Biotechnology, 19, 365–374. Tornberg, E. (2005). Effects of heat on meat proteins–Implications on structure and quality of meat products. Meat Science, 70(3), 493–508. Van der Sman, R. G. M. (2007). Moisture transport during cooking of meat: An analysis based on Flory-Rehner theory. Meat Science, 76(4), 730–738.
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Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset
The effect of pressure-assisted heating on the water holding capacity of chicken batters
T
⁎
Hai-bo Zhenga,b, Min-yi Hanc, Hui-juan Yangd, Xing-lian Xue, , Guang-hong Zhoue a
Key Laboratory of Meat Processing, Ministry of Agriculture, Nanjing 210095, PR China Food and Drug College, Anhui Science and Technology University, Fengyang 233100, PR China c Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, PR China d Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing 210095, PR China e College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Pressure assisted heating Water holding capacity Protein denaturation Water distribution and mobility Microstructure
The ability of gel-type meat products to hold water is an important quality attribute, which is affected by processing. The aim of this research was to investigate the effects of pressure-assisted heating, which can disrupt myofibrils and hinder heat-induced protein denaturation, on the water holding capacity of chicken meat batters. High pressure-assisted heating (100–400 MPa, 65 °C, and 30 min) and heating-only (0.1 MPa, 65 °C, and 30 min) was applied to chicken meat batters, the centrifugal loss, water distribution and mobility, microstructure, and residual denaturation enthalpy were determined. A threshold pressure of between 300 and 400 MPa was found, below which the WHC was improved, but impaired at greater pressures. Distributed exponential analysis of the T2 relaxation revealed three states of water binding (T2b, T21 and T22), each of which was significantly correlated with WHC. Pressure-treated batters had a higher amount of bound water than the heat-only batters, and showed a decrease in immobilized water and an increase in free water with increasing pressure. Myofibril structures were degraded by high pressure. High pressure resulted in a porous microstructure which held more water. However, pressures greater than the threshold caused loose gel-networks and decreased water holding capacities. The heat-denaturation of meat proteins was affected by high pressure. Actin was denatured by high pressure instead of heating, while collagen and some myosin derivatives were preserved from being denatured by heating. The changes in protein denaturation and batter microstructure were correlated with water distribution properties. The results contributed to a better understanding of the effects of high-pressure with heat on the water holding capacity of chicken batters. Industrial relevance: A beneficial threshold pressure of between 300 and 400 MPa was found, below which the water holding capacity was improved, and above which water holding capacity was reduced. As the effect of high pressure on physical properties and sterilization were not always consistent, this finding reminds the meat industry need to adopt a suitable pressure to achieve a balance between physical properties and sterilization. The low filed nuclear magnetic resonance could be adopted in a routine examination of product quality.
Chemical compounds studied in this article: Sodium chloride (PubChem CID: 5234) Sodium tripolyphosphate (PubChem CID: 24,455)
1. Introduction The water holding capacity of meat products is an important quality attribute, not just for the sensory aspects, but also for economic benefits. Meat has a water content of approximately 75% which is distributed in intra- and extra-myofibrillar spaces (Pace & Rathbun, 1945). Further, additional water is usually added to gel-type meat products during processing, making it difficult for the meat products to retain water. When the meat product is cooked, the heat-induced shrinkage of myofibrils results in the migration of water from intra-myofibrillar to the extra-myofibrillar spaces, potentially resulting in water loss ⁎
(Tornberg, 2005; Van der Sman, 2007). Understanding the water distribution and its migration is essential for developing meat product formulations and processing technology. The application of high pressure processing (HPP) has increased rapidly during the last 30 years. HPP has being used in the food industry for various purposes including its effectiveness for reducing numbers of viable microorganisms, meat tenderization, protein gelation, starch gelatinization and food frozen/unfrozen (Balasubramaniam & Farkas, 2008; Suzuki, 2002). HPP has long been recognized as a “cold processing” technology, mostly because of its “cold pasteurization” effect (Heinz & Buckow, 2010). However, a combination of high pressure
Corresponding author. E-mail address: [email protected] (X.-l. Xu).
https://doi.org/10.1016/j.ifset.2017.11.011 Received 12 October 2017; Received in revised form 10 November 2017; Accepted 17 November 2017 Available online 21 November 2017 1466-8564/ © 2017 Elsevier Ltd. All rights reserved.
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chamber was heated to 65 °C and kept constant by a water bath (ILBWCS, STIK Shanghai Co., Ltd.). The temperature in the sample chamber was monitored during processing by a thermocouple located inside the top of the chamber. A heating time of 30 min as suggested by Fernandez Martin et al. (1997), was used for all samples. Compression led to a temperature increase of 3 °C/100 MPa and decompression led to a temperature decrease of 4 °C/100 MPa. The initial temperature of the compression fluid was reduced before each run to counteract the temperature increase caused by adiabatic heating. The pressurization and depressurization rates were 5 MPa/s and 20 MPa/s, respectively. All samples were then cooled in a running tap water immediately for 1 h after being heated, and then stored in a 0–4 °C room.
and high temperature, namely heating under pressure (HUP), that is, applying two physical effects simultaneously to a foodstuff, can improve the food product. This has not been thoroughly explored. In meat products, one of the beneficial effects is reducing water loss. This has been reported by several studies on chicken, pork and beef (Fernandez Martin, Fernandez, Carballo, & Colmenero, 1997; Jiménez-Colmenero, Fernández, & Carballo, 1998; Sikes & Tume, 2014; Zheng et al., 2015; Zheng et al., 2017). Several changes related to the WHC of HUP-treated meat products have been suggested, such as the breakdown of myosin molecules, a denaturation-preserving effect, and disruption of myofibrils (JimenezColmenero, Cofrades, Carballo, Fernandez, & Fernandez-Martin, 1998; Zheng et al., 2015; Zheng et al., 2017). However, these findings were based on indirect determinations or subjective observations and were unable to explain the actual pattern of water distribution and movement within the cellular environment. Several studies have shown that low-field nuclear magnetic resonance (LF-NMR) proton relaxometry can provide information about water mobility and distribution based on the measurement of transverse water proton relaxation (Berendsen, 1992; Bertram, Aaslyng, & Andersen, 2005). Also, it is a rapid, noninvasive, and nondestructive tool for the characterization of water properties (Han, Wang, Xu, & Zhou, 2014). The aim of this study was to investigate the effect of high pressure assisted heating on the water holding capacity of chicken meat batters using low-field NMR proton T2 relaxometry, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) in combination with traditional meat quality measurements. This work can contribute to the application of high pressure processing in the meat industry.
2.4. Determination of water distribution and mobility Water distribution and mobility were determined according to the method of Han et al. (2014) by using low-field nuclear magnetic resonance (LF-NMR), with minor modifications. A sub-sample (approximately 2 g) was cut from the sausage and placed in a cylindrical glass tube (15 mm in diameter). The relaxation measurements were performed on a bench top pulsed NMR analyser (PQ001, Niumag Electric Corporation, China) with a corresponding resonance frequency for protons of 22.6 MHz. Transverse relaxation, T2, was measured using the Carr-Purcell-Meiboom-Gill (CPMG) sequence (Carr & Purcell, 1954; Meiboom & Gill, 1958). The T2 measurements were performed with a τ value (time between 90° pulse and 180° pulse) of 200 μs. The repetition time between two succeeding scans was 5 s. Data were acquired from 3000 echoes as an average of 8 repetitions. Each measurement was performed in quintuplicate. The transverse low-field NMR relaxation data were fitted to a multi-exponential curve with the MultiExp Inv Analysis software (Niumag Electric Corporation, China). This analysis yielded a plot of relaxation amplitude for individual relaxation processes versus relaxation time. The relaxation peak time (T2) and peak area (P2) were calculated from the curve.
2. Material and methods 2.1. Materials and chemicals Chicken breast meat (M. pectoralis major) was transported from a meat company (Jiangsu Tyson Foods Co, LTD., China) at low temperature (0–4 °C) within 24 h after slaughtering. All chemicals used were of analytical grade, except sodium chloride and sodium polyphosphates, which were food grade.
2.5. Determination of water holding capacity Centrifugal loss was determined by the centrifugation method as described by Zheng et al. (2017). Samples were cut into 1 cm long cylinders, weighed (Wstart) and wrapped with a filter paper. Then, the sample was centrifuged at 10,000 ×g for 10 min at 10 °C. After removing the filter paper, the sample was reweighed (Wend). The centrifugal loss was expressed as the percentage of water loss to total weight.
2.2. Preparation of batters A total of 10 kg chicken meat was trimmed to remove visible fat and connective tissue, cut into strips, and then minced through a meatgrinding machine (MOD TC/12E, Sirman, Italy) fitted with a plate made up of 5-mm-holes. Then, the minced meat was mixed and divided into 5 equal parts before being chopped separately. The minced chicken (80 g/100 g) was chopped with ice (17.7 g/100 g), sodium chloride (2 g/100 g), and sodium tripolyphosphate (0.3 g/100 g) for 5 min in a chopper (K15E, Talsabell S. A., Spain) at 1800 rpm. The temperature of chicken batters during the cutting was maintained below 13 °C. The meat batter was subjected to vacuum to remove the air bubbles before stuffing into plastic casings with a diameter of 26 mm and linked every 20 cm. The sausages (120 g each) were vacuum packaged in vacuum bags separately and then stored at 0–4 °C prior to further treatment on the following day.
Centrifugal loss (%) =
Wstart − Wend × 100 Wstart
2.6. Structural observation Subsamples were taken from the central part of sausages and cut into 0.1 mm × 1 mm × 2 mm blocks and fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer solution (pH 7.0). The ethanol dehydration, freeze-drying and sputter-coating were performed according to the procedure of Cao, Xia, Zhou, and Xu (2012). Samples were observed and photographed by using a scanning electron microscope (S-3000N, Hitachi, Japan) at a voltage of 10 kV.
2.3. Pressure/thermal treatments 2.7. Determination of thermal properties Vacuum packaged chicken meat batters were subjected to 0.1, 100, 200, 300, or 400 MPa and heat at 65 °C for 30 min. Heat-only (0.1 MPa) treatment was performed in a water bath (TW20, JULABO Technology Co. Ltd., Germany). The pressure assisted heating (100–400 MPa) was performed in a 0.3 L high-pressure unit (S-FL-850-9-W/FPG5620YHL, Stansted Fluid Power Ltd., UK) by using a mixture of water and propylene glycol (7:3, w/v) as the compression fluid. The high-pressure
Samples (15–20 mg) were encapsulated in aluminum pans and hermetically sealed. The samples were equilibrated at 25 °C for 2 min before scanning from 25 to 90 °C at 5 °C/min in a differential scanning calorimeter (DSC1, Mettler-Toledo International Inc., Switzerland) with an empty aluminum pan as a reference. Dry matter content was determined for thermal data normalization to dry matter content by 281
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800
desiccation at 105 °C for 16 h. The endothermal curve of each sample was recorded.
T2 (ms)
Experiments were carried out with at least in 5 independent replicates and the results were expressed as mean ± standard deviation (SD). Analysis of variance was carried out with the software SAS 9.0 (SAS Institute Inc., USA), and differences among means were evaluated by using Duncan's new multiple range test (p < 0.05).
bc
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Changes in water distribution and mobility in meat products can be determined using low-field nuclear magnetic resonance (LF-NMR). NMR transverse relaxation of water (T2) can be separated into the relaxation decay of three exponential populations, representing three water compartments in meat products: bound water (T2b), immobilized water (T21), and free water (T22) (Pearce, Rosenvold, Andersen, & Hopkins, 2011). Fig. 1 shows the distributions of T2 relaxation times of three peaks centered at approximately 0.5–2.5 ms (T2b), 30–110 ms (T21), and 300–800 ms (T22). As shown in Fig. 2, pressure-treated batters, generally, had lower T2b (1.15–1.23 ms) but higher T21 (57.65–60.22 ms) and T22 (513.34–624.18 ms) than heat-only samples, except the 400 MPatreated batter had a lower T22 (513.34 ms). Since the relaxation time T2 reflects the probability of a water molecule interacting with surrounding surfaces, the changes in T2 implies alterations in the space which acts as relaxation sinks (Pearce et al., 2011). Pressure-treated batters had shorter T2b but longer T21 than heat-only batters, indicating a smaller space for bound water but a larger space for immobilized water. The decreasing T22 from 624.18 ms of the 100 MPa-treated samples to 513.34 ms of 400 MPa-treated sample also indicated that the space for free water had decreased with increased pressure. An increase in P2 population indicates an increase in water population (Pearce et al., 2011). High pressure significantly (p < 0.05) increased the proportion of P2b from 1.24% for heat-only samples to 2.65–2.78% for pressure-treated samples, but there was no significant (p > 0.05) difference among pressure-treated samples. As the proportion of P2b represents the amount of bound water, the higher proportion of P2b in the pressure-treated samples indicated that more water was bound with pressure-treated proteins. The samples heated at 100–300 MPa had almost the same P21 (96.47–96.68%) and greater than that of the heat-only (95.74%) and
Fig. 2. Low-field NMR relaxometry T2 relaxation times of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min. a–d, means (n = 5) without a common letter within the same T2 relaxation time indicate significant difference (p < 0.05).
P2b a
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Pressure (MPa) Fig. 3. Low-field NMR relaxometry proportions of peak areas of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min. a–c, means (n = 5) without a common letter within the same P2 population indicate significant difference (p < 0.05).
30 a
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Pressure (MPa) Fig. 4. Centrifugal loss of chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, and 400 MPa for 30 min. a–c, means (n = 5) with different letters indicate significant difference (p < 0.05).
0 1
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T2 (ms) Fig. 1. Distribution of T2 relaxation times for chicken batters treated by heating at 65 °C and at pressures of 0.1, 100, 200, 300, or 400 MPa for 30 min.
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Fig. 5. SEM microstructure of pressure/thermal treated chicken batters treated by heating at 65 °C and at pressures of 0.1 (a), 100 (b), 200 (c), 300 (d), or 400 MPa (e) for 30 min.
400 MPa-treated (95.64%) samples, indicating more water was immobilized in 100–300 MPa-treated samples than the others. A decrease in P21 normally was accompanied by an increase in P22 (Bertram et al., 2001). The heat-only sample showed the highest P22 (3.02%), followed by the 400 MPa-treated sample (1.65%), while the samples treated at 100–300 MPa showed the lowest P22 (0.6, 0.67, and 0.8%, respectively), indicating that the heat-only sample had the most free water, followed by the 400 MPa-treated sample, while the samples treated at 100–300 MPa had the lowest free water.
reduction in centrifugal loss indicated that the WHC was improved by HUP treatments. The improvement in WHC had also been characterized by cook loss as reported by Jimenez-Colmenero et al. (1998), who showed that pressure-treated samples had a lower water loss which decreased with increasing pressure. However, a significant (p < 0.05) increase in centrifugal loss to 18.21% was observed as the pressure was increased to 400 MPa, indicating that WHC decreased when pressure was too high. The results of centrifugal loss were in agreement with those of LF-NMR.
3.2. Centrifugal loss
3.3. Microstructure
The effect of HUP treatment (0.1–400 MPa) on water holding capacity was characterized by centrifugal loss (Fig. 4). Heat-only sample had the highest centrifugal loss, up to 25.77%, followed by the 400 MPa-treated samples, which was 18.21%, whereas those heated at 100–300 MPa had the lowest centrifugal loss of 10.19% to 11.11%. The
The myofibril fragments in the heat-only samples were essentially intact and were be easily recognized, but they became disrupted and became less visible in pressure-treated samples, see Fig. 5. The structure of myofibrils in the 100 MPa-treated samples was still evident but was barely present in the 200 MPa-treated samples and completely 283
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samples, the proteins that had a denaturation temperature below 65 °C should be denatured. However, an endothermic peak around 64 °C was found in almost all pressure-treated samples, indicating that collagen was preserved from being heat denatured. This was in agreement with the reports that collagen does not undergo appreciable changes when subjected to HUP because its hydrogen-bonded structure was strengthened by pressure (Buckow, Sikes, & Tume, 2013; FernandezMartin, 2007). However, at 100 MPa, the pressure appeared to be too low to protect collagen from being heat denatured, as the collagen peak was not found in the 100 MPa-treated samples. A weak endothermic peak at 60 °C was observed in the 100 MPa-treated samples, and it was attributed to myosin aggregates (Chapleau, Mangavel, Compoint, & Lamballerie-Anton, 2004), which have been found to remain native-like at conditions up to 400 MPa at a temperature up to 75 °C (FernandezMartin, 2007). However, this peak could not be distinguished in samples at higher pressures, probably because this peak was too small and too close to the large collagen peak. Actin had an endothermic peak near 71 °C and remained native in the heat-only samples. However, no actin peak was found in the pressure-treated samples, indicating that actin had been denatured by pressurization. This was consistent with previous reports where it was found that actin was a thermostable but a pressure labile protein (Fernandez-Martin, 2007). The less denatured collagen, the myosin aggregates, and the denatured actin were present in the pressure-treated samples, indicating that pressure treatment induced a different degree or type of denaturation as compared to heat-only sample, which may have affected its affinity for water. Pressure-treated samples showed a shorter T2b relaxation time (Fig.2) and a larger P2b proportion (Fig. 3) than heat-only sample, indicating that the proteins in the pressure-treated samples had greater affinity than those of heat-only sample.
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Temperature (oC) Fig. 6. Thermograms of raw chicken meat batter.
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4. Discussion
200 MPa
4.1. The effect of HUP treatment on WHC, LF-NMR, microstructure and protein denaturation
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The effect of pressure-assisted heating on WHC varied with pressure intensity. It was obvious that pressures between 100 MPa and 300 MPa had a positive influence on WHC, while a pressure at 400 MPa reduced WHC, indicating there was a threshold of between 300 MPa and 400 MPa for WHC at the specified temperature. It was also obvious that the negative effect of high pressure on WHC commenced at 300 MPa, as the P21 showed a drop and P22 showed an increase at 300 MPa. Further, the pore size of the gel network showed an obvious increase at 300 MPa. Although protein denaturation that was preserved by high pressure has been reported to be one of the important properties of HUP treatment (Kanaya, Hara, Nakamura, & Hiramatsu, 1996), there was no obvious correlation between the degree of denaturation of proteins and WHC. The water holding capacity between the 300 MPa-treated and 400 MPa-treated samples was significantly different (p < 0.05), but there was no obvious difference in thermograms. Therefore, it would be more reasonable to ascribe the changes in WHC to a different type rather than the different extent of denaturation. Villamonte, Simonin, Duranton, Cheret, and de Lamballerie (2013) proposed that it was the type of denaturation, not the total protein denaturation, which influenced the hardness of pressure-only treated meat batters. The microstructure changed progressively with increasing pressure (Fig. 5), showing two changing features: the disruption of myofibrils and the formation of porous networks. The disruption of myofibrils was caused by high pressure, however, the formation of stable porous network was probably promoted by both heating and high pressure. The pore size of the gel networks, changed significantly as the pressure increased from 100 MPa to 400 MPa, implying that the heat-induced interactions between meat proteins were also affected by high pressure. This was also consistent with our view that HUP treatment induced a
90
o
Temperature ( C) Fig. 7. Thermograms of chicken batters treated by pressure/thermal at 65 °C and at 0.1, 100, 200, 300, or 400 MPa for 30 min.
disrupted in 300 MPa and 400 MPa-treated samples. The disruption of myofibrils contributed to the formation of a porous microstructure both inside and outside the myofibrils, which was consistent with the report of Zheng et al. (2017). Heat-only samples had few protein networks outside the myofibrils, and no porous microstructure inside the myofibrils (Fig. 5a). In pressure-treated samples, although the higher the pressure the greater the disruption (Iwasaki, Noshiroya, Saitoh, Okano, & Yamamoto, 2006), this did not necessarily result in a better microstructure (Fig. 5e). The gel network of pressure-treated samples became more regular and porous as pressure increased from 100 MPa to 200 MPa. However, the microstructure became less porous and with larger cavities as pressure increased to 300 MPa, and at 400 MPa, aggregates and fractures appeared. 3.4. Differential scanning calorimetry (DSC) For raw batter, typical endothermic peaks were observed at 56 °C, 64 °C and 71 °C (Fig. 6), which were ascribed to the thermal denaturation of myosin, sarcoplasmic proteins and collagen, and actin, respectively (Speroni, Szerman, & Vaudagna, 2014). As a heating temperature at 65 °C was used for all the heated 284
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new type of denaturation.
Acknowledgments
4.2. Connections between WHC, LF-NMR, microstructure and protein denaturation
This research was supported by the China Agriculture Research System (CARS-41), the Natural Science Foundation of China (31471601), the Key Projects of Young and Middle-aged Talents in Colleges and Universities in 2016, China (gxfxZD2016184), and the Fundamental Research Funds for the Central Universities (KYZ201543). The assistance provided by Dr. Ron Tume (Honorary Guest Professor at NAU) in the preparation of this manuscript is gratefully acknowledged.
As the amount of water loosely bound correlates with water loss (Bertram, Donstrup, Karlsson, & Andersen, 2002), the higher proportion of free water the higher the tendency to lose water. The proportion of free water (P22) showed a highly positive correlation (r = 0.9892, p = 0.0013) with centrifugal loss, as reported by Bertram, Purslow, and Andersen (2002) and Zheng et al. (2015), where it was found that the higher P21 or the lower P22, the higher water holding capacity. As the variations in WHC resulting from pressure differences can be predicted by LF-NMR, this technique can be used to provide an accurate and convenient means to evaluate the WHC of HUP-treated meat products. Several reports have shown that the gel microstructure (gel capillarity) played an important role in determining the water holding capacity, as the major part of the water was immobilized by capillary force (Liu, Lanier, & Osborne, 2016; Stevenson, Dykstra, & Lanier, 2013). In our present work we showed that heat-only and 400 MPatreated samples had a less porous microstructure than the samples treated at 100–300 MPa, so were accompanied with higher centrifugal loss (Fig. 4). The microstructure studies also agreed with the LF-NMR data. The desired microstructure, resulting from the disruption of myofibrils, and the size of the porous gel network was optimal at a pressure of 200 MPa (Fig. 5c). It was also found that T21 and P21 reached their maximum values at 200 MPa. Similarly, the 400 MPatreated samples, which had an impaired microstructure, had lower T21 and P21 values. It was concluded that the variations in T21 and P21 were consistent with the changes in microstructure. As bound water refers to water that is closely associated with proteins (Shao et al., 2016), the variations in protein structure should affect the T2 relaxation time of bound water. Han et al. (2014) reported that an increase in heat-induced denaturation was accompanied by an increase in T2b. In this study, actin, which accounts for about 15% of total protein, was completely denatured at all high pressures. However, its denaturation was less, or not affected, in the heat-only samples as the temperature was lower than its denaturation temperature (Fig. 7). This may partially explain the difference in T2b and P2b between pressure-treated and heat-treated samples. Collagen was denatured at 100 MPa but was less denatured at greater pressures, however, there were no significant (p > 0.05) changes in T2b and P2b, indicating that it did not affect T2b and P2b. The myosin, accounting for about 30% of the total protein, was thought to be partly transformed to myosin aggregates which remained partially non-denatured at 100 MPa. However, this peak was not observed in other pressure-treated samples. As there were no clear connections between bound water and degree of protein denaturation degree, it appeared likely that it was the different structure induced by high pressure that led to the difference in bound water between pressure-treated and heat-only samples.
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