Effects of high pressure modification on conformation and gelation properties of myofibrillar protein

Food Chemistry 217 (2017) 678–686

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Effects of high pressure modification on conformation and gelation properties of myofibrillar protein Ziye Zhang a,b,c,⇑, Yuling Yang a,⇑, Peng Zhou b,c, Xing Zhang a, Jingyu Wang a a College of Food Science and Engineering/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing University of Finance and Economics, Nanjing 210023, PR China b State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province 214122, PR China c Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Wuxi, Jiangsu Province 214122, PR China

a r t i c l e

i n f o

Article history: Received 21 June 2016 Received in revised form 5 September 2016 Accepted 6 September 2016 Available online 7 September 2016 Keywords: Myofibrillar protein High pressure Conformation Gelation property SEM DSC

a b s t r a c t The effects of high pressure (HP) treatment (100–500 MPa) on conformation and gelation properties of myofibrillar protein (MP) were investigated. As pressure increased (0.1–500 MPa), a-helix and b-sheet changed into random coil and b-turn, proteins unfolded to expose interior hydrophobic and sulfhydryl groups, therefore surface hydrophobicity and formation of disulfide bonds were strengthened. At 200 MPa, protein solubility and gel hardness reached their maximum value, particle size had minimum value, and gel microstructure was dense and uniform. DSC data showed that actin and myosin completely denatured at 300 MPa and 400 MPa, respectively. Rheological modulus (G0 and G00 ) of HP-treated MP decreased as pressure increased during thermal gelation. Moderate HP treatment (5200 MPa) strengthened gelation properties of MP, while stronger HP treatment (P300 MPa) weakened the gelation properties. 200 MPa was the optimum pressure level for modifying MP conformation to improve its gelation properties. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction It is known that the gelation properties of myofibrillar protein (MP) are very important for muscle products. The high pressure (HP) has been demonstrated to be able to cause changes of protein conformation, structure, and hence improving the gelation properties of muscle proteins (Hsu, Hwang, Yu, & Jao, 2007). HP treatment could cause variable alterations on protein conformational structures depended on the pressure level used. Under pressurization, the quaternary structure dissociates at moderate pressures (100–200 MPa), the tertiary structure is significantly affected at pressure level above 200 MPa and secondary structure changes take place at stronger high pressures (300–700 MPa) (Ahmed, Ramaswamy, Kasapis, & Boye, 2010). Acero-Lopez, Ullah, Offengenden, Jung, and Wu (2012) studied the effect of HP treatment on ovotransferrin, and found the secondary structure changed from helices, sheets, turns, and aggregated strand to mostly intermolecular b-sheets or aggregated strands at 200 MPa, but switched back to original structure at higher pressures. Maria, Ferrari, and Maresca (2016) pointed out that HP treatment ⇑ Corresponding authors at: College of Food Science and Engineering, Nanjing University of Finance and Economics, PR China (Y. Yang). E-mail addresses: [email protected] (Z. Zhang), [email protected] (Y. Yang). http://dx.doi.org/10.1016/j.foodchem.2016.09.040 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

on bovine serum albumin (BSA) would induce a-helix unfolding into disordered structure and turns. Cao, Xia, Zhou, and Xu (2012) reported that the microstructure of myosin gels below 200 MPa were filament structure with many small cavities, while gels upon 300 MPa were globular aggregates with big cavities. These changes in protein conformation and gel microstructure have profound effects on a protein’s gelation properties and its possible food applications. Gelation properties of proteins have a direct relationship with its conformational structure (Ramaswamy, Singh, & Sharma, 2015). HP treatment can cause modification of the quaternary and tertiary structure of a protein that can lead to denaturation, aggregation and gelation, and improvement of the functional properties by enhancing moisture-protein and protein-protein interactions (Singh, Sharma, & Ramaswamy, 2015). HP modification can lead to the enhancement or weakness of protein gelation properties depended on the pressure level used. Moderate HP treatment (5200 MPa) would induce the increase of protein solubility, while stronger HP treatment (P300 MPa) would cause reduction of solubility (Bravo, Felipe, López-Fandiño, & Molina, 2015; Marcos & Mullen, 2014; Van der Plancken, VanLoey, & Hendrickx, 2005). HP-induced protein unfolding could lead to the reversible or irreversible gelation of the proteins, with repercussions on the viscoelastic characteristics of the protein solution (Ahmed, Ramaswamy, Alli, & Ngadi, 2003). It has been reported that HP

Z. Zhang et al. / Food Chemistry 217 (2017) 678–686

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treatment prior to heating enhanced thermal gelation of muscle proteins. Pressurization (150 MPa, 10 min) enhanced heatinduced (70 °C, 10 min) gelation of myosin from ovine muscle at low ionic-strength (Suzuki & Macfarlane, 1984). The hardness of heat-induced peanut protein isolate gel increased by 50% after HP treatment at 0.1–100 MPa, while gradually decreased with further increased pressures (He et al., 2014). Previous literature pointed out the mechanism of HP-induced myosin gelation was that under pressurization, myosin unfolded, following the exposure of interior hydrophobic and sulfhydryl groups, protein denatured and associated to form gels (Cao et al., 2012). The purpose of this work was to assess changes in the conformation structure and gelation properties of myofibrillar proteins for use in new food systems with good textures. A deeper understanding of the mechanisms of governing the conformation and gelation properties by HP treatment would be very useful to design novel processes addressed to the improving functionality of MP in meat products.

as the pressure-transfer medium. Prior to pressurization, MP samples were sealed in polyethylene bags without trapping air bubbles, and treated by pressures at 100, 200, 300, 400, 500 MPa (±10 MPa). HP increased at a speed of 3.5 MPa/s to designed pressures and held for 10 min, then released within 5 s. The control was MP sample without pressurization (0.1 MPa). After HP treatment, MP were diluted to 40 mg/mL for Raman spectrum testing, 30 mg/mL for preparing MP gel, 5 mg/mL for solubility measurement and 1 mg/mL for sulfhydryl group content, surface hydrophobicity and particle size measurement. MP gel was prepared as follows: 2 mL of MP solutions (30 mg/mL) were placed in 7 mL capped plastic centrifuge tubes. The tubes were heated in a water bath at a rate of 1 °C/min from 20 to 65 °C and kept at 65 °C for 20 min. Then, the tubes were cooled to room temperature (25 °C) and then kept overnight at 4 °C for SEM and textural tests.

2. Materials and methods

The determination of amino acid content was according to Park and Xiong (2007) with some modifications. HP-treated MP samples were mixed with 6 M HCl in 10 mL capped glass tubes, hydrolyzed at 110 °C for 24 h; after cooled to room temperature (25 °C), opened the tubes and concentrated the hydrolyzate to dryness with a nitrogen gas purge equipment, then dissolved the dried substance with 0.02 M HCl and moved into a 50 mL volumetric flask, set to 50 mL with deionized water (Milli-Q, Millipore, Boston, MA, USA); part of the solution was transferred to a centrifuge tube, centrifuged at 10,000g for 5 min; filtered the supernatant with 0.22 lm membrane, then transferred into a 2 mL vial, using an automatic amino acid analyzer (L-8900, Hitach Corporation, Tokyo, Japan) for amino acid analysis. The calibration standards were Lamino acids (Sigma-Aldrich Inc., St. Louis, MO, USA) in nano pure water.

2.1. Materials Six-week-old commercial broilers (Arbor Acres Plus, 2.6 ± 0.1 kg in weight, female) were selected and slaughtered according to the National Standard of the People’s Republic of China for Operating Procedure of Chicken Slaughtering (GB/T 19478-2004, 2004) on a chicken farm in Nanjing, China. The breast meats (36–40 h postmortem) were purchased, then frozen at 18 °C and brought to the laboratory. Meats were used within one month. ANS (8-ani lino-1-naphthalenesulphonic acid, CAS: 82-76-8), DTNB (5,5dithiobis [2-nitrobenzoic acid], CAS: 69-78-3), EDTA (ethylenediaminetetraacetic acid, CAS: 60-00-4), EGTA (ethylene-bis (oxyethylenenitrilo) tetraacetic acid, CAS: 67-42-5) and urea (CAS: 57-13-6) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). BSA (bovine serum albumin, CAS: 9048-46-8) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All other chemicals were analytical grade or better. 2.2. Extraction of myofibrillar protein (MP) MP was extracted from chicken breast meat carried out as described by Zhang, Yang, Tang, Chen, and You (2016) with some modifications. Before extraction, meat was thawed at 4 °C for 12 h in a refrigerator. Trimmed muscle (40 g) was cut into small pieces (about 0.5  0.5  0.5 cm3) and homogenized in 8 volumes of a buffer (0.1 M NaCl, 2 mM MgCl2, 1 mM EGTA, 0.5 mM dithiothreitol, and 10 mM Na2HPO4, pH 7.0) in a homogenizer (DS-1, Shanghai Specimen Model Factory, Shanghai, China). The homogenates were centrifuged (4 °C) at 2000g for 20 min (Avanti J-26XP centrifuge, Beckman Coulter, Brea, CA, USA). After decanting the supernatant, pellets were re-suspended using homogenizer and centrifuged using the same conditions twice more. After that, the pellet was re-suspended in 8 volumes of another buffer (0.1 M NaCl, 1 mM NaN3, pH 6.0), filtered with clean and dry gauzes, and centrifuged (2000g for 20 min). After decanting the supernatant, MP pellets were re-suspended, filtered and centrifuged twice more. The protein content of MP was determined by the Biuret method using BSA as standard and used in 48 h. 2.3. HP treatment and preparation of MP gel HP treatment was carried out in a high pressure unit (UHPF750 MPa, Baotou Kefa, China). An oil (bis (2-ethylhexyl) sebacate, Li-Dong precision machinery company, Shenzhen, China) was used

2.4. Amino acid analysis

2.5. Secondary structure The secondary structure proportions of MP samples were measured by Raman spectroscopy using a Jobin Yvon Labram HR800 spectrometer (Horiba/Jobin. Yvon, Longjumeau, France). MP samples were spread on a glass slide during measurement. Raman spectra were recorded under the following conditions: laser power, 100 mW; laser spot diameter reaching the sample, 1 lm; spectral resolution, 2.0 cm1; number of sample scans, 3. The time required for the acquisition of 1 spectrum was about 1 min. Spectra were smoothed, baseline corrected, normalized against the phenylalanine band at 1003 cm1, as it was insensitive to the microenvironment, and Amide I was analyzed using Labspec version 3.01c (Horiba/Jobin. Yvon, Longjumeau, France). Protein secondary structures were determined as percentages of a-helix, b-sheet, b-turn, and random coil using Alix’s method (Alix, Pedanou, & Berjot, 1988).

2.6. Surface hydrophobicity Surface hydrophobicity (S0-ANS) was determined using ANS as a fluorescence probe. 25 lL of ANS solution (8 mM ANS in 0.1 M NaH2PO4 buffer, pH 6.0) solution was added to 5 mL of MP solution (1 mg/mL) and mixed well. Samples were kept under dark conditions at 25 °C for 20 min. Fluorescence intensity was measured by a fluorescence spectrophotometer (F-7000, Hitachi Corp., Japan) at an excitation wavelength of 374 nm and an emission wavelength of 485 nm. The initial slope of the plot of fluorescence intensity versus protein concentration was referred to as S0-ANS.

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2.7. Sulfhydryl group content To determine the total SH group content, 4.5 mL of a solution containing 8 M urea and 10 mM EDTA (pH 6.0), and 100 lL Ellman’s reagent (10 mM DTNB in 0.1 M NaH2PO4 buffer) were added to 0.5 mL MP solutions (1 mg/mL), the mixture was set in dark at room temperature (25 °C) for 25 min. The absorbance was measured at 412 nm by a UV–vis spectrophotometer (U-3900, Hitachi Corp., Japan) to calculate the total SH group content using a molar extinction coefficient of 13,600 M1 cm1. Reactive SH group contents were measured using the same system in the absence of urea. 2.8. Protein solubility The solubility of MP was determined as follows: MP solutions (5 mg/mL) were centrifuged at 10,000g for 20 min. The supernatant was collected and protein content was measured by the Biuret method, using BSA as a standard. Protein solubility was expressed as the percentage in comparison with that obtained before high pressure treatment. 2.9. Particle size One milliliter MP solution (1 mg/mL) was injected in a clear zeta cell for particle size measurement. The particle size was measured by a Zetasizer (Nano-ZS90, Malvern Instruments Ltd., Malvern, Nottinghamshire, UK). Particle size data were presented as mean diameter (nm). Three replicates were performed for each sample. 2.10. Scanning electron microscopy (SEM) MP Gel samples were fixed in 2.5% glutaraldehyde in 0.1 M NaH2PO4 buffer solution at pH 7.2 for 2 h. Ethanol dehydration was then performed using a series of solutions (ethanol-water ratios of 50, 75, 90, 95 and 100%), then samples were transferred into tertiary butyl alcohol. Samples were freeze-dried by a freeze dryer (FreeZone 4.5 Plus, Labconco, Kansas City, MO, USA) and sputter-coated with 10 nm of gold. Samples were observed at an accelerating voltage of 15 kV and a magnification of 2000 using a scanning electron microscope (TM 3000, Hitachi Corp., Japan). 2.11. Gel strength Gel hardness was determined using a texture analyzer (TA.XT. Plus, Stable Micro Systems, Surrey, UK) equipped with a stainless steel probe of P/6. The parameters were set as follows: pre-test speed, 5.0 mm/s; test speed, 1.0 mm/s; post-test speed, 5.0 mm/s; trigger type, auto-5 g, and distance, 5 mm. Tests were triplicated. 2.12. Differential scanning calorimetry (DSC) DSC data were obtained by a Perkin-Elmer DSC (DSC 8000, Perkin-Elmer instruments, Waltham, MA, USA). 10 mg of HPtreated MP samples were accurately weighed and hermetically sealed for analysis. Samples were heated from 20 to 100 °C at a rate of 10 °C/min. A sealed empty pan was used as a reference. Denaturation enthalpies (DHd) and peak temperatures (Td in °C) were calculated from the thermograms by Pyris-12 software (Perkin-Elmer Instruments, USA). 2.13. Rheological characteristics The gelation property of MP solutions (30 mg/mL) was monitored by a rheometer (MCR302, Anton Paar, Graz, Austria), equipped with a parallel plate (PP50, 50 mm in diameter).

Measurements were at a strain of 0.02, a constant frequency of 0.1 Hz and a 0.5 mm gap. Temperature was increased at a rate of 1 °C/min from 20 to 80 °C. Storage modulus (G0 ) and loss modulus (G00 ) values were recorded continuously. 2.14. Statistical analysis Statistical analysis of results was performed using SPSS software (SPSS Inc., Ver.19, IL, USA), all data were presented as means ± standard deviation (SD). A one-way analysis of variance (ANOVA) was employed to determine the statistical difference. Significant differences between means were identified using Duncan’s multiple range test (p < 0.05). 3. Results and discussion 3.1. Conformational structure 3.1.1. Primary structure The primary structure of a protein refers to the amino acid sequence. Changes in amino acid content of a protein can be used to reflect the alteration on its primary structure. Table 1 showed the effect of HP treatment on amino acid content of MP samples. Compared with the control (0.1 MPa), the content of all amino acids of HP-treated MP had no significant changes (p > 0.05). As the energy could be provided by 10,000 MPa was 8.37 kJ/mol (Morild, 1981), while the disruption energy of covalent bonds such as disulfide bond required energy of 213.1 kJ/mol (Pauling, 1960) was far in excess of 8.37 kJ/mol, so the covalent bonds (such as peptide bonds) could not be destroyed by HP treatment. From the perspective of both amino acid contents and covalent bonds, there were no loss of amino acids and rupture of covalent bonds, thus HP treatment had no disruption on protein primary structure. Hayakawa, Linko, and Linko (1996) investigated the effects of high pressure on ovalbumin, bovine serum albumin (BSA) and b-lactoglobulin, found HP treatment had no effect on covalent bonds and the primary structure was not affected. Previous studies found that after HP treatment, electrophoresis showed that there were no new bands of lower molecular weight appeared (Cao et al., 2012; Zhang, Yang, Tang, Chen, & You, 2015), which might also suggest that there were no cleavage of protein covalent bonds and primary structure. 3.1.2. Secondary structure The amide bond of proteins has several distinct vibrational modes, of which the amide I (1600–1700 cm1) is very useful for investigating protein secondary structure (Acero-Lopez et al., 2012; Cando, Herranz, Borderías, & Moreno, 2015; Savadkoohi, Bannikova, Mantri, & Kasapis, 2016). In this study, the amide I band was centered at 1640–1645 cm1 and 1680–1690 cm1 (b-turn), 1645–1660 cm1 (a-helix), 1660–1670 cm1 (random coil) and 1670–1680 cm1 (b-sheet) respectively (Carey, 1982). The secondary structure proportions of MP samples were calculated by analyzing the amide I Raman spectrum region and shown in Table 2. As to the control (0.1 MPa), a-helix is the major secondary structure (40.49%). Compared with the control (0.1 MPa), the ordered secondary structure proportions of pressurized MP decreased: a-helix decreased gradually from 40.49 (0.1 MPa) to 30.68% (500 MPa) and b-sheet reduced from 22.11 (0.1 MPa) to 9.79% (500 MPa); the proportions of disordered secondary structure increased: b-turn increased from 21.75 (0.1 MPa) to 31.16% (500 MPa) and random coil increased from 15.97 (0.1 MPa) to 27.87% (500 MPa). This phenomenon might be due to that a-helix was stabilized by intra hydrogen bonds of peptide chains,

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Z. Zhang et al. / Food Chemistry 217 (2017) 678–686 Table 1 Amino acid analysis of non-pressurized and pressurized MP. Amino acid

Ala Arg Asp Cys Glu Gly His Ile Leu Lys Met Phe Ser Thr Tyr Val

High pressure (MPa) 0.1

100

200

300

400

500

10.00 ± 0.04a 27.17 ± 0.03a 4.62 ± 0.03a 11.83 ± 0.06a 6.49 ± 0.07a 9.35 ± 0.05a 23.26 ± 0.08a 15.73 ± 0.03a 16.85 ± 0.07a 21.11 ± 0.02a 13.47 ± 0.03a 18.82 ± 0.08a 5.84 ± 0.04a 5.28 ± 0.03a 17.79 ± 0.02a 12.35 ± 0.07a

9.99 ± 0.05a 27.19 ± 0.04a 4.62 ± 0.05a 11.86 ± 0.05a 6.49 ± 0.01a 9.35 ± 0.07a 23.24 ± 0.05a 15.76 ± 0.05a 16.88 ± 0.08a 21.13 ± 0.05a 13.50 ± 0.08a 18.84 ± 0.04a 5.84 ± 0.02a 5.28 ± 0.08a 17.82 ± 0.03a 12.37 ± 0.06a

10.00 ± 0.04a 27.17 ± 0.04a 4.63 ± 0.03a 11.86 ± 0.01a 6.49 ± 0.02a 9.35 ± 0.03a 23.25 ± 0.08a 15.78 ± 0.04a 16.89 ± 0.07a 21.11 ± 0.02a 13.50 ± 0.03a 18.83 ± 0.06a 5.85 ± 0.04a 5.29 ± 0.06a 17.83 ± 0.01a 12.37 ± 0.02a

10.02 ± 0.06a 27.17 ± 0.05a 4.65 ± 0.02a 11.85 ± 0.07a 6.51 ± 0.03a 9.37 ± 0.05a 23.26 ± 0.07a 15.76 ± 0.05a 16.89 ± 0.02a 21.11 ± 0.08a 13.50 ± 0.06a 18.83 ± 0.04a 5.87 ± 0.06a 5.3 ± 0.07a 17.81 ± 0.07a 12.37 ± 0.06a

9.99 ± 0.04a 27.17 ± 0.08a 4.61 ± 0.04a 11.85 ± 0.03a 6.48 ± 0.04a 9.35 ± 0.05a 23.25 ± 0.03a 15.75 ± 0.04a 16.87 ± 0.07a 21.11 ± 0.02a 13.49 ± 0.05a 18.82 ± 0.03a 5.83 ± 0.05a 5.27 ± 0.05a 17.81 ± 0.07a 12.35 ± 0.04a

9.99 ± 0.03a 27.19 ± 0.04a 4.61 ± 0.02a 11.87 ± 0.05a 6.48 ± 0.02a 9.35 ± 0.03a 23.26 ± 0.06a 15.76 ± 0.02a 16.89 ± 0.03a 21.11 ± 0.05a 13.50 ± 0.09a 18.83 ± 0.05a 5.83 ± 0.04a 5.27 ± 0.08a 17.81 ± 0.04a 12.37 ± 0.06a

Different superscripted letter in the same line means significant differences (p < 0.05).

Table 2 Secondary structure, surface hydrophobicity and sulfhydryl group content of non-pressurized and pressurized MP. High pressure (MPa)

Secondary structure proportion (%)

a-helix 0.1 100 200 300 400 500 a–f

b-sheet a

40.49 ± 0.93 38.57 ± 0.76ab 36.89 ± 0.73b 35.57 ± 0.52bc 33.89 ± 0.57c 30.68 ± 0.39d

b-turn

Random Coil a

22.11 ± 0.45 19.73 ± 0.31b 17.65 ± 0.33c 15.32 ± 0.32d 13.81 ± 0.25e 9.79 ± 0.22f

SH content (mol/104 g protein)

S0-ANS

a

Total SH a

15.97 ± 0.37 17.91 ± 0.31b 19.22 ± 0.29c 21.69 ± 0.38d 23.71 ± 0.35e 27.87 ± 0.33f

21.75 ± 0.42 23.69 ± 0.47b 25.94 ± 0.41c 26.82 ± 0.53cd 28.09 ± 0.47d 31.16 ± 0.36e

a

442.8 ± 5.6 460.3 ± 6.7b 605.1 ± 10.3c 664.7 ± 8.9d 714.5 ± 7.5e 751.8 ± 10.6f

Reactive SH a

12.95 ± 0.26 12.72 ± 0.23a 12.22 ± 0.21ab 11.91 ± 0.15b 11.34 ± 0.17bc 10.88 ± 0.13c

7.16 ± 0.21a 7.89 ± 0.13ab 7.97 ± 0.19b 8.58 ± 0.17bc 9.10 ± 0.24c 9.49 ± 0.22c

Different superscripted letter in the same column means significant differences (p < 0.05).

b-sheet relied on inter hydrogen bonds between peptide chains, as pressure increased, protein unfolded, hydrogen bonding was weakened, resulted in the disruption of a-helix and b-sheet and the formation of random coil and b-turn. Puppo et al. (2004) used circular dichroism spectra to investigate the impact of HP treatment on soybean protein isolates (SPI), and pointed out that a-helix content decreased, and random coil content increased with the increase of pressure level. Zhang, Li, Tatsumi, and Kotwal (2003) pointed out that some ordered structures of a-helix and b-structure of glycinin were destroyed and converted to random coil after processed at 500 MPa for 10 min. Cao (2012) used FT-IR investigated the effects of high pressure on rabbit myosin, and found that the ordered structures of a-helix and b-sheet increased, the disordered structures of b-turn and random coil decreased. Savadkoohi et al. (2016) found that pressurization of soy glycinin at 600 MPa for 15 min resulted in the reduction of a-helix and b-sheet proportions, and the increase of random coil proportion. 3.1.3. Tertiary and quaternary structure HP treatment could alter the tertiary and quaternary structure of protein molecules, the changes of surface hydrophobicity (S0-ANS) and sulfhydryl (SH) group content could be used to reflect tertiary and quaternary structure changes of protein molecules after pressurization (Tian & Du, 2007). Table 2 showed the changes of surface hydrophobicity, total and reactive SH content of MP after HP treatment. Compared with the control (0.1 MPa), the S0-ANS of pressurized MP increased significantly (p < 0.05) after pressurization (100–500 MPa) (Table 2), which might be due to that after HP treatment, protein molecules stretched and unfolded, conformation structure became loose and destabilized, under the unstable tertiary and quaternary structure, the hydrophobic groups which

were previously buried in interior regions of protein and surrounded by non-polar environment, got exposed in aqueous environment. More ANS had access to bind with the previously masked non-polar parts of protein molecules, thus surface hydrophobicity increased. The positive relationship between pressure level and S0-ANS indicated an increased unfolding of protein and greater exposure of hydrophobic groups with the increase of pressure. Alvarez, Ramaswamy, and Ismail (2008) found that as pressure increased, protein structure becoming more open, thereby allowing ANS molecules to enter to the hydrophobic core of the protein subunits. It was also possible that pressure treatment allowed entry of water into the interior of the protein, and thus causing protein conformation from folding to unfolding. In a similar study, it was found that HP treatments caused an increase in the surface hydrophobicity of egg white protein (Van der Plancken et al., 2005). SH groups belong to weak secondary bonds and maintain the tertiary structure of proteins (Tian & Du, 2007), changes in SH content can reflect the alteration degree of tertiary and quaternary structure. As shown in Table 2, the reactive SH contents increased with the increase of pressure. Several authors attributed the increase of reactive/free SH content to the disruption of SAS bonds (Condés, Speroni, Mauri, & Añón, 2012), however, as we mentioned in Section 3.1.1, the covalent bonds (such as SAS) could not be destroyed by HP treatment. We speculated the increase of reactive SH content was due to the stretching and unfolding of protein molecules, interior SH groups exposed under pressurization. Most of the reactive/free SH groups in native proteins were masked to the attack by Ellman’s reagents, due to their location in poorly accessible regions of the polypeptide chain. The application of an external treatment, such as pressure, modified the protein conformational structure with the consequent unmasking and activation

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pressures (0.1–200 MPa) improved protein solubility, while stronger HP treatment (P300 MPa) induced the formation of large protein aggregates. Fig. 1B showed the effect of HP treatment on particle size of MP in dispersions. Pressurization of MP samples induced a reduction of particle size from 2061.1 (0.1 MPa) to 1975 nm (100 MPa) and a sharp decrease to 652.8 nm (200 MPa), which seemed to be explained by denaturation and non-covalent bond rupture of protein molecules, because HP treatment could trigger the dissociation of muscle proteins, depolymerize actin and actomyosin (Cheftel & Culioli, 1997; Zhu, Lanier, & Farkas, 2015). As pressure further increased, the particle size increased to 849.4 nm (300 MPa), 853.8 nm (400 MPa) and 856.6 nm (500 MPa), which suggested that stronger HP treatment (P300 MPa) improved the formation of protein aggregates, while there were no significant changes between particle size of stronger HP-treated (300– 500 MPa) MP samples (p > 0.05). The increase of protein particle size was most probably due to formation of aggregates which generated through intermolecular disulfide bridges and hydrophobic interactions. Bravo et al. (2015) investigated the transmission electron micrographs of pressurized milk protein and casein micelle size, and found that the mean micellar diameters were reduced by HP, reaching a minimum at 450 MPa, after pressurized at pressures P450 MPa, the mean diameter increased. Through Fig. 1A and B, we speculated that protein solubility was inversely associated with particle size. This might be due to that protein solubility was determined as the protein content in the supernatant after centrifugation at 10,000g for 20 min, smaller mean diameters of protein particles would be expected to result in less protein in the precipitate after centrifugation and therefore stronger solubility. Moreover, smaller particle size means greater surface area and increased protein-water interactions, which contributed to better protein functionality.

of SH groups, which could be detected according to the protocol of the Ellman’s reaction. The total SH content decreased significantly as pressure increased (p < 0.05) (Table 2). The decrease in total SH groups might be due to the formation of disulfide bonds (Cando et al., 2015). After pressurization, more buried SH groups got exposed, and the strengthened hydrophobic interactions (Table 2) shortened the distances between intermolecular sulfhydryl groups, when oxygen was present, more reactive SH groups and shorter distance contributed to disulfide bonds formation. Cando et al. (2015) found HP treatment (150–300 MPa) decreased SH group contents to form disulfide bonds in Alaska Pollock surimi gels. Hsu et al. (2007) found that the total SH contents of actomyosin from tilapia decreased substantially when pressure was above 200 MPa. Van der Plancken et al. (2005) also had similar observation of decreasing total SH content on pressurized egg white protein. Comprehensively, HP treatment changed the tertiary and quaternary structure of protein molecules, inducing exposure of interior hydrophobic groups and sulfhydryl groups, strengthened the surface hydrophobicity and formation of disulfide bonds, which contributed to protein-protein interactions.

3.2. Solubility and particle size In order to follow the protein molecular interactions involved in the gelation properties development of MP induced by HP treatment, solubility of MP was necessary analyzed. The solubility changes of non-pressurized and pressurized MP samples were shown in Fig. 1A. The solubility increased significantly from 18.50 (0.1 MPa) to 47.86% (200 MPa), this might be due to that the quaternary structure dissociated at moderate pressures (100– 200 MPa) (Ahmed et al., 2010), and HP depolymerized actin and actomyosin to promote solubilization of myofibrillar proteins (Cheftel & Culioli, 1997). As pressure increased (300–500 MPa), solubility decreased gradually to 43.9% (500 MPa). The reduction of protein solubility was due to the formation of HP-induced insoluble protein aggregates (Marcos & Mullen, 2014). After HP treatment, interior hydrophobic resides and sulfhydryl groups got exposed (Table 2), which favored the non-disulfide and disulfide bond cross-linking interactions, inducing the formation of protein aggregates and reduction of protein solubility (Van der Plancken et al., 2005; Zhang et al., 2015). Marcos and Mullen (2014) found that the solubility of myofibrillar protein from beef muscle increased at pressure of 0.1–200 MPa and deceased in 200– 400 MPa. Bravo et al. (2015) reported that milk protein solubility of ultracentrifugation supernatants increased from 10.51 (0.1 MPa) to 12.92 mg/mL (250 MPa), then decreased gradually to 10.20 mg/mL (900 MPa). These data suggested that moderate

A

The physical property of gel texture was highly dependent on its microstructure. The microstructural characteristics of nonpressurized and pressurized MP gel were showed in Fig. 2A–F. Gels with irregular cavities were present in non-pressurized MP samples (0.1 MPa). At 100 MPa, MP gels contained many filaments and irregular cavities as protein began to unfold. Compared with 100 MPa, MP gel pressurized at 200 MPa had denser and homogeneous network. This might be due to that MP had the smallest particle size at 200 MPa (Fig. 1B), which contributed to more regular space arrangement of proteins during microstructure formation. Besides, more hydrophobic residues and sulfhydryl groups exposed, protein-protein interactions enhanced, which contributed

B c

cd

40 30 20

a

b

2000 d Particle Size (nm)

c

50 Solubility (%)

3.3. Gel microstructure and hardness

b a

1500 d

1000

d

d

c 500

10

0

0 0.1

100 200 300 400 High Pressure (MPa)

500

0.1

100 200 300 400 High Pressure (MPa)

500

Fig. 1. Changes in solubility (A) and particle size (B) of non-pressurized and pressurized MP (mean ± SD, n = 3). Different letters (a–d) on top of a column indicate significant difference (p < 0.05) among samples treated under different pressures.

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B (100 MPa)

A (0.1 MPa)

C (200 MPa)

E (400 MPa)

D (300 MPa)

G

60 b

50 Hardness (g)

F (500 MPa)

c d

40

e

30 a

a

20 10 0 0.1

100 200 300 400 High Pressure (MPa)

500

Fig. 2. SEM micrographs at 2000 (A–F) and hardness (G) of non-pressurized and pressurized MP gel. Different letters (a–e) on top of a column indicate significant difference (p < 0.05) among samples treated under different pressures.

to the formation of uniform network. After stronger HP treatment (P300 MPa), MP gel cavities became larger and heterogeneous, a possible explanation might be that the formation of gel microstructure were mostly depended on the relative speed of protein unfolding and aggregation: when aggregation speed was faster than unfolding speed, it would form dense and uniform gel microstructure, if the aggregation speed was slower than unfolding speed, it would form coarse and heterogeneous gel microstructure (Lefèvre, Fauconneau, Quail, & Culioli, 1998). After stronger HP treatment (P300 MPa), MP denatured excessively, interior hydrophobic and sulfhydryl groups exposed. At this time, when MP solutions were heated for gelation, considering the HPinduced unfolded proteins, the unfolding speed provided by the native protein molecules would be much slower, aggregation speed would be much higher than unfolding, protein segments aggregated to form globular aggregates and irregular network. Cao et al. (2012) also had similar microstructure observations of heat-induced rabbit myosin gel after HP treatment (100–400 MPa). Fig. 2G showed the hardness of non-pressurized (0.1 MPa) and pressurized (100–500 MPa) MP gel. Gel hardness increased from 20.25 (0.1 MPa) to 46.6 g (200 MPa), then decreased gradually to 33.3 g (500 MPa). This might be due to that, after mild pressurization (100–200 MPa), MP denatured and stretched moderately, which made the protein structure destabilized, more

hydrophobic groups and sulfhydryl groups got exposed. When heated for gelation, exposed hydrophobic residues cross-linked to aggregate, sulfhydryl groups reacted to form disulfide bonds, therefore stronger protein-protein interactions contributed to homogeneous gel microstructure (Fig. 2A–F) and stronger gel hardness (Fig. 2G). Suzuki and Macfarlane (1984) also reported that mild pressure treatment (150 MPa) prior to heating enhanced thermal gelation of myosin. While stronger HP treatment (P300 MPa) resulted in the degradation or depolymerization of myofibrillar proteins (Cheftel & Culioli, 1997; Zhu et al., 2015), proteins aggregated irregularly, formed a heterogeneous gel microstructure and induced the reduction of hardness. Cando et al. (2015) found moderate HP (150 MPa) treated surimi gel had higher breaking force, stronger HP (300 MPa) pressurized gel had lower breaking force. Ma et al. (2013) found that compared with the non-pressurized pork gel, 100 MPa significantly increased the hardness of SSMP-CK (salt-soluble meat protein, CaCl2 and j-carrageenan) gels, while 300–400 MPa decreased the textural parameters (hardness, elasticity, cohesiveness and chewiness) of protein gels (p < 0.05). Comprehensively considering the gel microstructure and hardness data (Fig. 2), MP gels with higher hardness had smaller, denser and homogeneous gel microstructure, while gels with lower harness had larger cavities and coarse microstructure.

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3.4. DSC and gelation characteristics 3.4.1. Differential scanning calorimetry (DSC) The denaturation of proteins is the very important in thermal gelation, which initiates various protein-protein interactions that affects gelation properties of MP. DSC could be used to study individual proteins denatured in different temperature ranges, yielding distinguishable peaks. We adopted DSC to investigate and pinpoint the temperature range of denaturation transitions of specific proteins after HP treatment. Comprehensively considering the previous references (Speroni, Szerman, & Vaudagna, 2014; Stabursvik & Martens, 1980), we attributed our observed protein transitions of myofibrillar proteins as follows: HP-induced structure at 48–51 °C (peak 0), myosin at 57–60 °C (peak 1) and actin at 69–72 °C (peak 2) (Fig. 3). Changes of enthalpy in Fig. 3 were utilized in order to estimate the amount of denaturation in pressurized samples, which was calculated as [100  (DHpressurized/DH0.1MPa)]. Fig. 3 showed the thermograms of non-pressurized (0.1 MPa) and pressurized (100–500 MPa) MP samples. Pressurization caused protein denaturation that led to smaller peaks than that of the control sample. As pressure increased, the DH of endothermal transitions had a decreasing tendency, indicating the continual

denaturation of proteins. In other reports, it was found that HP treatments caused a decrease in the enthalpy of denaturation (Singh et al., 2015; Van der Plancken et al., 2005). At 0.1 MPa, the non-pressurized MP samples showed a typical DSC thermogram with two main endothermal transitions (peak 1 and peak 2) ranging at 57–60 °C and 68–71 °C, generally pertaining to denaturation of myosin (57.66 ± 0.23 °C) and actin (68.42 ± 0.17 °C) (Stabursvik & Martens, 1980). Compared with the control (0.1 MPa), at 100 MPa, the DH of peak 1 decreased 14.60% (compared with 0.1 MPa, same as follows), peak 2 decreased 44.37%, indicating a moderate denaturation of myosin and great denaturation of actin. At 200 MPa, there was a sharp DH reduction of peak 1 (60.40%) and DH of peak 2 decreased 50.70%, more than half of myosin and actin severely denatured, indicating HP treatment could trigger the degradation of muscle proteins and depolymerize actin (Cheftel & Culioli, 1997; Zhu et al., 2015). At 300 MPa, protein denaturation was clearly visible and was confirmed by the reduction in the enthalpy of denaturation (DH) of peak 1 (61.82%). The reduction in DH was considered as signifying the breakdown of the structure leading to denaturation (Ahmed, Ramaswamy, Ayad, All, & Alvarez, 2007), which led to the disappearance of peak 2 and appearance of a new peak (peak 0). The appearance of peak 0 might be resulted from the formation of HP-induced structure (aggregates) (Singh

Fig. 3. DSC thermograms of non-pressurized (A) and pressurized (B–F) MP.

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A

350

30

B

300

25

250 20 0.1MPa 100MPa

150

200MPa 300MPa

100

0.1MPa

G''/Pa

G'/Pa

200

15

100MPa 200MPa 300MPa

10

400MPa

400MPa

500MPa

500MPa

5

50 0 20

40

60

80

0 20

Temperature ( )

40

60

80

Temperature ( )

Fig. 4. Changes in G0 (A) and G00 (B) of non-pressurized and pressurized MP during gelation.

et al., 2015; Speroni et al., 2014). At 400 MPa, the DH of peak 0 increased 70.19% (compared with 300 MPa, same as follows), and peak 1 disappeared, suggesting the further formation of HPinduced aggregates and the complete denaturation of myosin. At 500 MPa, only peak 0 existed and the DH increased 103.37%, indicating the further increase of HP-induced aggregates. DSC dada showed that actin and myosin completely denatured at 300 MPa and 400 MPa, respectively, myosin had higher pressure resistance, and actin performed greater sensitivity to HP treatment. 3.4.2. Rheological characteristics Rheological behavior is an important property used to describe protein functionality. The storage modulus (G0 ) measures the stored energy representing the elastic portion, and the loss modulus (G00 ) measures the energy dissipated as heat representing the viscous portion (Meyers & Chawla, 1999). The G0 and G00 of nonpressured and pressure-treated MP during gelation were showed in Fig. 4. Compared with non-pressurized MP samples, the changes of G0 and G00 of pressurized samples were similar, both decreased with the increased pressures. Cando et al. (2015) found similar changes on G0 of HP-pretreated (150–300 MPa) surimi during gelation. Compared with the control (0.1 MPa), at 100 MPa, G0 and G00 of MP samples had no evident changes, while 44.37% actin denatured (Fig. 3), which suggested actin had not participate in strengthening of MP gelation. At 200 MPa, the G0 and G00 of MP samples had a significant reduction (p < 0.05), and the denaturation peak occurred at 48 °C disappeared, which was mostly due to the severe denaturation of myosin (Fig. 3), this might explain the important role of myosin in gelation. At 300 MPa, G0 and G00 further decreased (p < 0.05), which was resulted from the continual denaturation of myosin and actin. While between 400 MPa and 500 MPa, there were no significant changes in G0 and G00 (p > 0.05), indicating the denaturation of protein molecules were almost complete. Thus, the HP-induced protein denaturation was directly related to the reduction of G0 and G00 values.

HP treatment (=300 MPa) heavily denatured MP, most proteins became destabilized, interior hydrophobic and sulfhydryl groups exposed in aqueous environment, promoted the formation of disulfide bonds and strength of hydrophobic interactions. Stronger protein-protein interactions and cross-linking promoted the generation of insoluble large aggregates, resulted in the reduction of MP solubility. Textural properties of protein gel greatly depended on its microstructure. The formation of gel microstructure were mostly depended on the relative speed of unfolding and aggregation: faster aggregation speed than unfolding contributed to dense and uniform gel microstructure, slower aggregation speed than unfolding led to coarse and heterogeneous gel microstructure (Lefèvre et al., 1998). At moderate pressures (5200 MPa), protein mildly unfolded, most molecules were native, when heated for gelation, the unfolding speed was faster than aggregation, which contributed to uniform gel microstructure and stronger hardness. At stronger HP treatment (=300 MPa), protein heavily unfolded, few native molecules remained, when heated for gelation, the unfolding speed provided by the native protein was slower than aggregation, which resulted in heterogeneous gel microstructure and weaker harness. HP induced protein denaturation affected rheological properties of MP samples (Fig. 4). Generally, during thermal gelation, the protein denaturation and unfolding contributed to the increase of G0 and G00 . As HP became stronger, more protein denatured (Fig. 3), when heat for gelation, there would be fewer native protein molecules to strengthen gelation, therefore the G0 and G00 decreased. Thus HP-induced protein denaturation was directly related to the reduction of G0 and G00 values. Comprehensively, moderate HP (5200 MPa) moderately denatured protein conformation, promoted gelation properties of MP, while stronger HP (=300 MPa) excessively denatured proteins, heavier protein-protein interactions induced unbeneficial gelation properties of MP. Data presented in this work suggested that 200 MPa was the optimum pressure level for modifying MP conformation to improve gelation properties.

3.5. Relationship between conformation and gelation properties of myofibrillar protein

4. Conclusions

HP would produce a variable degree of conformation structure changes depends on the pressure level used, which in turn led to changes in the gelation properties of MP. Moderate HP treatment (5200 MPa) mildly denatured MP, dissociated protein quaternary structure (Ahmed et al., 2010), depolymerized actin and actomyosin (Cheftel & Culioli, 1997), which promoted solubilization of myofibrillar proteins. While stronger

This work evaluated the conformational change of myofibrillar protein and its effect on the gelation properties under high pressure. HP treatment changed a-helix and b-sheet into random coil and b-turn, disrupted tertiary and quaternary structure, and led to the unfolding proteins, exposure of interior hydrophobic and sulfhydryl groups. Moderate HP treatment (5200 MPa) increased the solubility of MP and the microstructure and hardness

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of MP gel, while stronger HP treatment (P300 MPa) weakened these functionality. 200 MPa was the optimum pressure level for modifying conformation to improve gelation properties of MP.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31371798).

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