Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field

Accepted Manuscript Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field Guoliang Jia, Satoru Nirasawa, Xiaohua Ji, Yongkang Luo, Haijie Liu PII: DOI: Reference:

S0308-8146(17)31292-X http://dx.doi.org/10.1016/j.foodchem.2017.07.138 FOCH 21522

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

19 February 2017 16 July 2017 25 July 2017

Please cite this article as: Jia, G., Nirasawa, S., Ji, X., Luo, Y., Liu, H., Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.07.138

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Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field Guoliang Jia a, b, Satoru Nirasawa c, Xiaohua Jia, b, Yongkang Luoa, b, Haijie Liu a, b, *

a

College of Food Science and Nutritional Engineering, China Agricultural University,

Beijing 100083, China b

Beijing Advanced Innovation Center for Food Nutrition and Human Health, China

Agricultural University, Beijing 100083, China c

Japan International Research Center for Agricultural Sciences, Tsukuba 305-8686,

Japan

*Corresponding author. Tel: +86-10-6273-7331; fax: +86-10-6273-7331; e-mail: [email protected]

E-mail addresses: Guoliang Jia: [email protected] Satoru Nirasawa: [email protected] Xiaohua Ji: [email protected] Yongkang Luo: [email protected]

1

Abstract: The application of a high-voltage electrostatic field (HVEF) is a novel method for thawing. To determine the effects of HVEF thawing (voltage range: -25 kV – 0 kV) on myofibrillar protein oxidation and/or denaturation and to provide a theoretical understanding of this process, pork tenderloin was thawed by traditional and HVEF methods. Based on the total sulfhydryl and carbonyl contents, further protein oxidation did not occur during HVEF thawing. It was proposed that the free radical-mediated oxidation of myofibrillar proteins was hindered by HVEF. The results of dynamic rheological analysis, protein aggregation and gel texture studies showed that HVEF thawing, especially -10 kV HVEF thawing, led to better indicators than those achieved under air thawing. A higher abundance of proteins extracted from -10 kV HVEF-thawed samples compared with air-thawed samples was found. Finally, this study showed that thawing under -10 kV conditions did not affect the structure of myofibrillar proteins.

Keywords: pork tenderloin, HVEF, myofibrillar proteins, oxidation, denaturation

Chemical compounds studied in this article: MgCl2 (PubChem CID: 5360315); potassium chloride (PubChem CID: 4873); EGTA (PubChem CID: 6207); PMSF (PubChem CID: 4784); EDTA (PubChem CID: 6049); DTNB (PubChem CID: 6254); urea (PubChem CID: 1176); DNPH (PubChem CID: 1176); trichloroethanoic acid (PubChem CID: 6421); formic acid (PubChem CID: 2

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1. Introduction Freezing meat is one of the most important methods for prolonging its shelf life and preserving it during exportation (Leygonie, Britz, & Hoffman, 2012). Accordingly, many novel thawing methods, including high-pressure, microwave and high-voltage electrostatic field (HVEF) thawing (Li & Sun, 2002; He, Liu, Nirasawa, Zheng, & Liu, 2013), have been recently developed. However, high-pressure thawing is expensive, and microwave thawing results in localized overheating (Li & Sun, 2002). In contrast, HVEF thawing, which has been applied to frozen pork tenderloin, tuna fish and rabbit meat (He, Liu, Nirasawa, Zheng, & Liu, 2013; Mousakhani-Ganjeh, Hamdami, & Soltanizadeh, 2015; Jia, Liu, Nirasawa, & Liu, 2017), is faster and results in higher quality products compared with other thawing methods (Dalvi-Isfahan, Hamdami, Le-Bail, & Xanthakis, 2016). The highest thawing rate for frozen pork tenderloin, tuna fish and rabbit meat thawed by HVEF was 1.60, 1.78 and 2.63 times greater than that obtained for the air-thawed samples (He, Liu, Nirasawa, Zheng, & Liu, 2013; Mousakhani-Ganjeh, Hamdami, & Soltanizadeh, 2015; Jia, Liu, Nirasawa, & Liu, 2017). The total viable count reduction of pork tenderloin and rabbit meat thawed by HVEF was more than 0.5 log CFU/g compared with that for air-thawed samples (He, Liu, Nirasawa, Zheng, & Liu, 2013; Mousakhani-Ganjeh, Hamdami, & Soltanizadeh, 2015; Jia, Liu, Nirasawa, & Liu, 2017). For the protein solubility, although Mousakhani-Ganjeh et al. (2015) found that protein solubility of thawed tuna fish 3

decreased with increasing voltage, higher sarcoplasmic protein solubility was observed for the -20 kV HVEF-thawed rabbit meat than air-thawed meat (Jia, Liu, Nirasawa, & Liu, 2017). Jia et al. (2017) reported that HVEF thawing under certain voltages did not lead to lipid oxidation of rabbit meat. However, few studies have focused on the physicochemical changes in myofibrillar protein isolate (MPI) extracted from meat thawed using HVEF methods. Myofibrillar proteins, which account for 55-65 % of muscle proteins, are vital for their water-holding capacity (WHC) and gelation ability (Leygonie, Britz, & Hoffman, 2012; Liu & Xiong, 1996). It was reported that MPI oxidation occurred in porcine longissimus dorsi during freeze-thaw cycles (Xia, Kong, Liu, & Liu, 2009). The remarkable and measurable changes that occurred during muscle protein oxidation included protein carbonyl formation, sulfhydryl reduction and protein cross-link formation (Lund, Heinonen, Baron, & Estevez, 2011). One study reported that an increase in MPI oxidation led to decreases in the WHC, tenderness and nutritional value (Lund, Heinonen, Baron, & Estevez, 2011). Therefore, it is essential to determine the effects of HVEF thawing on the degree of MPI oxidation. Traditional thawing methods used in households and industries worldwide include thawing in air and under running water (RW); therefore, these methods were chosen as controls in this study. Moreover, the HVEF voltages and electric field strengths tested in this work were much higher than those used in previous thawing experiments (He, Liu, Nirasawa, Zheng, & Liu, 2013; Mousakhani-Ganjeh, Hamdami, & Soltanizadeh, 2015). The negative corona discharge (EHD-) was adopted based on the previous 4

study (Jia, Liu, Nirasawa, & Liu, 2017). For the EHD-, the voltage at which the corona changes to a spark discharge is higher than that for the positive corona discharge (EHD+) (Dalvi-Isfahan, Hamdami, Le-Bail, & Xanthakis, 2016). The aims of this study were to compare the effects of HVEF thawing on MPI oxidation and denaturation to those of conventional thawing methods and to provide a theoretical foundation for the industrial application of this innovative food thawing method.

2. Materials and methods 2.1 Materials and chemicals The pork tenderloin used in this study was the same as that used in our previous study (Jia, He, Nirasawa, Tatsumi, Liu, & Liu, 2017). The pork tenderloin pieces (50 × 50 × 10 mm3, 45.3 ± 2.5 g) were randomly assigned to different experimental groups and stored at -20 °C until thawing. Trypsin (T1426-50MG) was obtained from Sigma (St. Louis, MO, USA). The other chemicals were at least analytical grade. 2.2 HVEF thawing experiments and temperature monitoring system As shown in Fig. S1, the experimental setup and temperature monitoring system were described in a previous work (Jia, He, Nirasawa, Tatsumi, Liu, & Liu, 2017); however, a miniature refrigerator was not required in this study. The HVEF was composed of a DC voltage generator, a treatment chamber and a multiple point-to-plate electrode. Four pieces of frozen pork tenderloin were placed on 5

aluminium foil above the bottom plate of the HVEF system. The voltages used in this study were -25 (breakdown voltage), -20, -15, -10, -5 and 0 kV. The inter-electrode distance was 50 mm. 2.3 MPI preparation The MPI was prepared according to the procedure of Liu and Xiong (1996) with some modifications. Minced muscle was homogenized in 4 volumes (v/w) of a 0.01 M phosphate buffer (pH 7.0) using an Ultra Turrax homogenizer (IKA T10 standard, German) at 4 °C for 2 × 30 s. The buffer consisted of 0.1 M KCl, 2 mM MgCl2, 1 mM ethylene glycol tetraacetic acid (EGTA) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Then, the homogenate was centrifuged at 2000 g for 15 min, and the supernatant was discarded. The pellet was washed two more times with 4 volumes of the same buffer under the same centrifugation conditions indicated above. The resulting myofibril pellet was then washed once with 4 volumes of 0.1 M NaCl. After centrifugation (rate: 2000 g; time: 15 min), the MPI was dissolved in a 0.6 M NaCl solution (pH 7.0), and the MPI concentration was measured by the biuret method. The MPI concentration was then adjusted to a suitable level for measuring protein oxidation and denaturation. 2.4 MPI oxidation 2.4.1 Total sulfhydryl (SH) content The SH content was measured using the method of Tadpitchayangkoon et al. (2010). MPI (2 M, 0.5 mL) was homogenized in 4.5 mL solubilizing buffer, which consisted of 0.2 M Tris-HCl, 8 M urea, 1 % SDS and 3 mM EDTA. Ellman’s reagent (0.5 mL, 2 6

mM DTNB, 0.2 M Tris-HCl, pH 8.0) was subsequently added to 4 mL aliquots of the supernatant, and the mixtures were incubated at 40 °C for 25 min. The SH content was calculated from the absorbance measured at 412 nm using a molar extinction coefficient of 13,600 M-1·cm-1. 2.4.2 Carbonyl content The MPI carbonyl content was measured as described by Xia et al. (2009) with some modifications. Briefly, 1 mL aliquots of MPI (2 M) were mixed with 1 mL 2,4-dinitrophenyl hydrazine (DNPH) (10 mM) dissolved in 2 M HCl. Then, the mixture was incubated in the dark for 1 h. The fractions were subsequently precipitated with 20 % (w/v) trichloroacetic acid (TCA) and centrifuged at 14,000 g for 5 min. The pellets were washed four times with 1 mL of a 1:1 (v/v) ethyl acetate:ethanol mixture, and the supernatant was discarded. The obtained pellets were dissolved in 3 mL of 6 M guanidine and 2 M HCl and then incubated at 37 °C for 15 min. After the sample was centrifuged at 14,000 g for 3 min, the absorbance of the supernatant was measured at 370 nm. The carbonyl content was determined using an absorption coefficient of 22,000 M-1·cm-1 and expressed as nmol fixed DNPH/mg MPI. 2.5 Differential scanning calorimetry (DSC) The degree of MPI denaturation in the thawed pork tenderloin was analysed using a differential scanning calorimeter (DSC-60, Shimadzu, Japan) according to the method reported by Jia et al. (2017) with modifications. The DSC sample consisted of 3–5 mg undiluted MPI. The heating rate and temperature scanning range were 10 °C/min and 7

25-90 °C, respectively. The main myosin and actin denaturation peaks were recorded, and the corresponding denaturation temperatures (Tpeak, °C) and enthalpies (∆H, J/g) were determined. 2.6 Protein aggregation The protein aggregation was examined using a light-scattering instrument (S3500, Microtrac, USA) according to the method of Sun et al. (2011). The MPI solution was centrifuged at 3000 g for 10 min. The resulting supernatant (4 mg/mL) was used for measurement. In particular, D4,3, D3,2, Dv,0.1 and Dv,0.5 were recorded. The D4,3 and D3,2 variables are the mean volume and surface diameters, respectively, and Dv,0.1 and Dv,0.5 denote the sizes at which 10 % and 50 % of the sample particles, respectively, have smaller diameters (Sun, Zhou, Zhao, Yang, & Cui, 2011). 2.7 Textural and dynamic thermo-mechanical analysis of the MPI gels formed by heat treatment Thermal gels were prepared as described by Liu et al. (2011) with some modifications. The MPI concentration used in the heat-induced gelation was 40 mg/mL, which was consistent with the work of Zhang et al. (Zhang, Yang, Tang, Chen, & You, 2015). The MPI was heated at 40 °C for 30 min and then at 90 °C for 30 min. After heating, the gels were immediately cooled by running tap water and then stored at 4 °C overnight before the texture profile analysis (TPA). The TPA was conducted using a TMS-Pro texture analyser (Food Technology Corporation, USA) with a 5-mm-thick aluminium platen probe (TMS 50 mm Diameter Platen) at a test speed of 1 mm/s. The gels (Height: 15 ± 1 mm, Diameter: 18 ± 1 mm) were axially compressed 8

to 75 % of their original height. The textural properties of the gels were described in terms of the hardness, springiness, cohesiveness, chewiness and adhesiveness. Dynamic rheological analysis of MPI during thermal gelation was performed using a Discovery Hybrid Rheometer (HR-2, TA Instruments, USA). The MPI samples were heated from 25 °C to 80 °C. Continuous oscillation at a frequency of 1 Hz and a strain of 0.01 were applied to monitor the dynamic moduli. The dynamic rheological properties of the heat-induced gels were described in terms of the shear storage modulus (G’, the elastic response of the MPI gels). 2.8 Comparative proteomic analysis of MPI 2.8.1 Protein digestion The digestion was based on the method reported by Shi et al. (2016). The MPI (100 µg proteins) was reduced with DTT (50 mM) for 60 min at 60 °C and alkylated with iodoacetamide (50 mM) in the dark for 45 min at 20 °C. After dilution with 200 µL of ammonium bicarbonate (50 mM), the proteins were digested with 20 µL of 0.1 mg/mL trypsin for 16 h at 37 °C. The digestion was stopped by the addition of 0.5 % formic acid. The resulting mixture was desalted using C18 ZipTip pipette tips and resuspended in 0.1 % formic acid (FA). 2.8.2 LC-MS/MS and database searches The resulting tryptic digests were eventually analysed by Easy nLC-LTQ-Orbitrap XL (Thermo, San Jose, CA) at the Institute of Biophysics, Chinese Academy of Sciences. Peptide samples were automatically loaded into the nano liquid chromatograph with a lab-made C18 trap column (150 µm×3 cm, 5 µm). The peptides 9

were separated on the lab-made C18 analytical column (75 µm×15 cm, 3 µm) using a gradient from 5% to 100% of buffer B (0.1% FA/80% acetonitrile (ACN)) at 300 nL/min flow over 90 min. The eluting peptides were analysed using the LTQ Orbitrap. Protein identification and label-free quantification were performed using Peaks 8 software

(Bioinformatics

Solutions

Inc.,

CA)

with the

Uniprot database

(http://www.uniprot.org/proteomes/UP000008227). The search parameters were set as follows: dynamic modification: oxidation (met), acetylation (protein N-term); static modification: carbamidomethylation (cys); enzyme: Trypsin; maximum missed cleavages per peptide: 2; precursor mass tolerance: 15 ppm; fragment mass tolerance: 0.5 Da; peptide false discovery rate (FDR): 0.01. 2.9 MPI structural analysis 2.9.1 Circular dichroism (CD) The CD spectra were measured over the range of 200-260 nm using a spectropolarimeter (Chirascan plus, Applied Photophysics, UK) according to the method reported by Jia et al. (2017) with modifications. The MPI (0.2 mg/mL) was placed in a 1 mm quartz cell and scanned at a rate of 120 nm/min. Changes in the secondary structure were revealed by the far-UV CD spectra. 2.9.2 Fluorescence spectroscopy The

fluorescence

measurements

were

performed

using

a

fluorescence

spectrophotometer (F-7000, Hitachi, Japan) based on the method reported by Liu et al. (Liu, Wang, Sun, McClements, & Gao, 2016) with modifications. The MPI concentration was 0.2 mg/mL, the excitation wavelength was 295 nm, and the 10

emission wavelength range was 310-400 nm. 2.9.3 Ca2+-ATPase activity The Ca2+-ATPase activity of the MPI was measured by the method described by Xia et al. (2009). Briefly, the MPI was diluted to a concentration of approximately 4.0 mg/mL. The reaction solution consisted of a 0.5 M Tris-maleic buffer (pH 7.0), 0.1 M CaCl2, 20 mM ATP (pH 7.0) and MPI. After the solution was allowed to react at 25 °C for 3 min, 15 % TCA was added to terminate the reaction. Then, the mixture was centrifuged at 10000 g for 2 min, and 4 mL of the supernatant was mixed with 3 mL of a Tris-MgCl2 buffer (pH 7.0) and 3 mL of 1 % ammonium molybdate in 1 mM H2SO4. The mixture was subsequently allowed to react at 45 °C for 30 min. Finally, the absorbance of the mixture was measured at 640 nm, and the results are expressed as µmol phosphate/mg MPI.

2.10 Statistical analysis All measurements were performed at least in triplicate, and the data are presented as the mean ± the standard deviation (SD). The least significant difference procedure was used to compare the mean values using a significance level of P < 0.05.

3. Results and discussion 3.1 MPI oxidation It is assumed that the post-mortem processing (e.g., chilling/freezing, high-pressure processing and irradiation) of meats accelerates protein oxidation (Soladoye, Juarez, 11

Aalhus, Shand, & Estévez, 2015; Fuentes, Ventanas, Morcuende, Estévez, & Ventanas, 2010). Proteins are susceptible to attack by reactive oxygen species (ROS) during these processes. Protein oxidation is considered to be a free radical chain reaction. The reactions of free radicals with proteins result in changes in both the protein backbone and amino acid side chains (Lund, Heinonen, Baron, & Estevez, 2011). Protein oxidation could reduce the meat quality, e.g., the tenderness and juiciness (Lund, Lametsch, Hviid, Jensen, & Skibsted, 2007). Fuentes et al. (2010) found that protein oxidation in dry-cured ham increased when a hydrostatic pressure of 600 MPa was applied. Therefore, whether HVEF thawing led to protein oxidation in our study was interesting to determine. As shown in Fig. 1, the total sulfhydryl and carbonyl contents were determined and used as MPI oxidation markers. Fig. 1A shows that the total sulfhydryl contents of the MPIs extracted from the pork tenderloins thawed at -10 kV and -25 kV were the highest, whereas the lowest total sulfhydryl content was observed in the MPI extracted from the pork tenderloin thawed in air. The total sulfhydryl content of the MPI extracted from the fresh pork tenderloin was 77.21 ± 8.48 µmol/g MPI. No significant differences were observed in the total sulfhydryl contents of the MPIs from the fresh pork tenderloin and the pork tenderloins thawed at -10 kV and -25 kV DC. Thiol oxidation leads to the formation of oxidized products such as disulfide cross-links (RSSR) and sulfenic acid. Bao et al. (2015) reported that pork stored in an oxygen (O2)-free environment had the highest thiol content. In this study, O2 was continuously consumed due to ozone (O3) formation under the HVEF (Fig. S2), 12

limiting radical-mediated MPI oxidation (Fig. S2A). In addition, it was previously reported that solid-state proteins were completely resistant to the action of ozone (Cataldo, 2003). The HVEF corona wind dried the pork tenderloin surface, especially at voltages higher than -10 kV (Fig. S3). Based on the two reasons above, HVEF could impede radical-mediated oxidation of MPI (Fig. S2), including lipid peroxyl radical formation via the chain reaction between lipid alkyl radicals and molecular oxygen (Fig. S2B). Thus, the free radical-mediated MPI oxidation would be partially inhibited under HVEF. It was previously reported that HVEF thawing at -10 kV reduced the thawing time of pork tenderloin by 37.5% relative to the thawing time in air (He, Liu, Nirasawa, Zheng, & Liu, 2013). In addition, the energy of -10 kV should be smaller than that of a higher voltage. Therefore, HVEF thawing at a voltage of -10 kV resulted in less protein oxidation than that when using the other tested thawing methods. The carbonyl contents of the different MPI samples are shown in Fig. 1B. The highest mean carbonyl content was observed for the MPI obtained from the pork tenderloin thawed in air. All the carbonyl contents were lower than 2 nmol/mg MPI. These results are consistent with those of a previous study of beef samples chilled for 6 days (Lund, Hviid, & Skibsted, 2007). Some differences in the mean carbonyl contents of the samples thawed by different methods were observed. It was previously proposed that carbonyl formation at specific amino acid side chains changes the myofibrillar protein conformation, leading to denaturation and loss of functionality (Estévez, 2011). However, Leygonie et al. (2011) noted that molecular oxygen did not 13

have a significant effect on ostrich protein carbonylation. Moreover, Lund et al. (2007) reported that protein cross-linking occurred, and the tenderness and juiciness of the studied samples were reduced, even when the protein carbonyl content was low.

3.2 Differential scanning calorimetry (DSC) Ishiwatari et al. (2013) used DSC to evaluate the denaturation degrees of beef proteins, including myosin and actin, which are the major components of myofibrillar proteins. The authors found that actin denaturation affected the weight loss more significantly than did myosin denaturation. However, myosin is closely related to the heat-induced gelation capacity. Therefore, the denaturation temperatures and enthalpies of both myosin and actin were evaluated in this study. To ensure that the obtained data were consistent with other data, MPI was also extracted from the macro pork tenderloin system, although the thermal denaturation enthalpies of muscle or meat proteins can be directly measured by DSC. In one study, it was assumed that the peak temperature and enthalpy decreased significantly due to a decrease in the protein thermal stability and due to changes in the protein structure (Huang, Liu, Xia, Kong, & Xiong, 2015). In this study, the denaturation temperatures of the different MPI samples varied significantly, whereas the denaturation enthalpies were similar (Table 1). No significant differences between the myosin and actin denaturation temperatures of the fresh pork tenderloin and the pork tenderloin thawed at -10 kV were observed. In addition, the differences in the actin denaturation temperature of the samples were significant; however, the overall differences were small, and the actin stability did not 14

decrease considerably. To be contrast, Mousakhani-Ganjeh et al. (2015) reported that HVEF thawing decreased protein solubility, compared with air thawing. Thawing rate is one of the most important considerations during the thawing process and controls the amount of time the proteins are oxidized in the presence of oxygen. Based on the results of thawing rate in our preliminary experiment and Mousakhani-Ganjeh et al. (Mousakhani-Ganjeh, Hamdami, & Soltanizadeh, 2016) respectively, it was found that the thawing rate for the pork tenderloin was improved much more than that for the tuna fish, which should be caused by different experimental set-up. Dalvi-Isfahan et al. (2016) reported that the generated corona wind could meet each other on the food surface without any energy losses for an optimal distance between adjacent wires.

3.4 Protein aggregation and texture [TPA and rheology] of the MPI gels The MPI average particle sizes, including the mean volume and surface diameters (D4,3 and D3,2, respectively), Dv,0.1 and Dv,0.5, for the fresh pork tenderloin and the pork tenderloins thawed in air, under running water, and under -10 kV and -20 kV DC HVEFs are given in Table 2. Except for -10 kV thawed meat, the mean diameter (D4,3) of MPI for the thawed meat samples increased significantly relative to those for the fresh sample. It was previously suggested that the carbonyls could react with free amino groups to form intermolecular disulfide and dityrosine cross-links between the protein molecules (Promeyrat, Gatellier, Lebret, Kajak-Siemaszko, Aubry, & 15

Santé-Lhoutellier, 2010). In addition to these oxidative reactions, using a voltage of -20 kV or higher in HVEF thawing might damage the structure of myoglobin (Jia, Liu, Nirasawa, & Liu, 2017). As the myofibrillar fraction contained some sarcoplasmic proteins including myoglobin, oxidation and denaturation of proteins could cause them to aggregate. The average MPI particle sizes in the samples thawed in air and under RW were much larger than those in the fresh sample, indicating that the degree of MPI (including some sarcoplasmic proteins) oxidation or denaturation in these two groups was higher than that in the fresh pork tenderloin group. In addition, Jia et al. (2017) reported that -20 kV HVEF led to less denaturation of the myofibrillar proteins than that under still air thawing. Table 2 also lists the textural parameters (hardness, springiness, cohesiveness, chewiness, adhesiveness) of the MPI gels obtained by heat treatment. Hardness is a very important protein gel functional property and might be related to protein denaturation and/or aggregation. Based on the results shown in Table 2, it was concluded that the MPI oxidation or denaturation that occurred in the pork tenderloin during the thawing process affected the protein gel texture. Although the mean hardness of the groups thawed by air, under RW and at -20 kV DC did not show significant differences, some measured data of TPA for the groups treated by RW or -20 kV DC were higher than those treated by air. Fig. 2 shows the storage modulus (G’) of MPI during thermal gelation. The G’ of MPI from fresh pork tenderloin remained stable from 25 °C to 48 °C and then increased from 48 °C to 52 °C, which was similar to the result reported by Zhao et al. 16

(2014). Subsequently, G’ declined from 52 °C to 57 °C due to the increased mobility of myosin molecules with heating. As the temperature increased (from 57 °C to 78 °C), the second increase in G’ resulted from the stronger interactions between denatured myosin molecules (Liu, Zhao, Xiong, Xie, & Qin, 2008). For the G’ of MPI from the thawed pork tenderloin, although the whole trend of G’ was similar to that of MPI from fresh pork tenderloin, the value of G’ was much smaller than the fresh value. Therefore, it was indicated that freezing and thawing processes led to an MPI gel with poor viscoelasticity. Note that the functionality of air-thawed MPI should be the least favourable compared with the other MPIs.

3.5 MPI quantification It was shown that the label-free quantitative proteomic method was a reliable quantification method for evaluating protein abundance in meat samples (Gallego, Mora, Aristoy, & Toldrá, 2015). To clearly compare the protein abundance differences in thawed pork tenderloin, the protein was extracted from samples thawed by air and at -10 kV DC. A total of 260 proteins were identified in our study, of which 103 proteins were differentially abundant between air-thawed pork tenderloin and -10 kV-thawed pork tenderloin (P < 0.05). Note that only 13 myofibrillar proteins were identified. Table 3 shows 7 differentially expressed myofibrillar proteins and 6 differentially expressed sarcoplasmic proteins. Among them, creatine kinase M-type and Heat shock 70 kDa protein 1B are potential biomarkers for WHC (Wu, Fu, Therkildsen, Li, & Dai, 2015; Di, Mullen, Elia, Davey, & Hamill, 2011), while 17

myosin-1 is a biomarker for meat tenderness (Wu, Fu, Therkildsen, Li, & Dai, 2015). Compared with air-thawed pork tenderloin, 12 proteins had a higher abundance in -10 kV-thawed pork tenderloin (Table 3). Therefore, -10 kV-treated pork tenderloin had better quality indicators, including WHC and tenderness.

3.6 MPI conformational changes 3.6.1 CD spectral analysis Changes in the far-UV CD signals can be used to monitor structural changes during unfolding, and thermal unfolding can be monitored by DSC (Kelly & Price, 1997). The secondary structures of the MPIs obtained from the fresh and thawed pork tenderloins were analysed by CD spectroscopy (Fig. 3A). The MPI extracted from the fresh sample exhibited two negative peaks at approximately 208 and 222 nm in the far-UV CD spectrum. After thawing the meat in air, the intensities of these peaks decreased, indicating the loss of the MPI α-helical conformation. Although Zhong et al. (2005) reported that the application of a pulsed electric field (PEF) could inactivate horseradish peroxidase (HRP) by changing the α-helical conformation, it should be noted that the voltage used in their study was much higher than that used in the present study.

3.6.2 Fluorescence spectral analysis Fluorescence spectroscopy is a common technique for studying the local tertiary structure of proteins. It is believed that conformational changes, such as the exposure 18

of non-polar groups in the hydrophobic interior of globular proteins to the surrounding aqueous phase, lead to a decrease in the fluorescence intensity (Cao & Xiong, 2015). Fig. 3B shows the fluorescence emission spectra of the MPIs obtained from the fresh and thawed pork tenderloins. The MPI fluorescence intensities of the thawed pork samples, especially the sample thawed in air, were lower than those of the fresh sample, suggesting that the MPI local structure changed during thawing.

3.6.3 Ca2+-ATPase activity Ca2+-ATPase is an indicator of myosin integrity, which is closely related to gelation (An, Peters, & Seymour, 1996). Furthermore, a decrease in the Ca2+ sensitivity might result from the denaturation or degradation of troponin, especially troponin C (Xia, Kong, Liu, & Liu, 2009). As shown in Fig. 3C, significant differences in the Ca2+-ATPase activity of the MPIs obtained from the samples thawed at -10 kV and under RW were observed. This result is consistent with the total sulfhydryl content results. Therefore, HVEF thawing at -10 kV should result in less myosin denaturation than that resulting from thawing under RW.

4. Conclusions In this study, frozen pork tenderloin was thawed under various applied voltages. Based on the MPI oxidation and thermal denaturation results, HVEF thawing did not lead to protein oxidation and/or denaturation compared with running water thawing or air thawing. On the contrary, air thawing led to the lowest total sulfhydryl content and 19

highest carbonyl content. The dynamic rheological analysis results and MPI aggregation and gel texture results were nearly consistent. A label-free strategy was applied to investigate the differences of protein abundance for -10 kV-thawed pork tenderloin and air-thawed pork tenderloin. The biomarkers in this study showed that -10 kV-thawed pork tenderloin had better WHC and meat tenderness. In addition, HVEF thawing at -10 kV had a smaller effect on the MPI conformation than did thawing in air. These results suggested that HVEF thawing at -10 kV should result in less MPI denaturation than that resulting from traditional thawing methods, thus making this a promising method for thawing pork tenderloin.

Acknowledgements This work was supported by the National Science Foundation of China (NSFC) (“Study on the characteristics and mechanism of freezing and thawing of meat under high-voltage electrostatic field”, grant number 31571908).

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Total sulfhydryl content (µmol/g MPI)

100

a a 80

b

b

RW

-5 kV

b

b

60

c 40

20

0 Air

-10 kV -15 kV -20 kV -25 kV

Carbonyl content (nmol/mg MPI)

Thawing method (A)

2

a ab

ab

b

ab

b

ab

1

0 Air

RW

-5 kV

-10 kV -15 kV -20 kV -25 kV

Thawing method (B)

Fig. 1. Effect of different thawing methods on the oxidation of myofibrillar protein isolate (MPI) from pork tenderloin.

25

3000

Storage modulus (Pa)

Fresh Air RW -10 kV DC -20 kV DC 2000

1000

0 20

30

40

50

60

70

80

Temperature (oC)

Fig. 2. Changes in the storage modulus (G') of pork tenderloin myofibrillar protein isolate (MPI) during thermal gelation.

26

Fresh

Air

Running water

8000

-10 KV DC

0

Fresh

Air

Running water

-10 kV DC

6000 Intensity

Circular Dichroism (mdeg)

5

-5

4000

2000

-10

-15 200

210

220

230

240

250

0 300

260

Wavelength (nm) (A)

320

340

360

380

400

Wavelength (nm) (B)

0.4 a

Ca-ATPase activity (µmol/mg MPI)

a b

b

0.3

0.2

0.1

0.0 Fresh

Air

Running water -10 kV DC

Thawing method (D)

Fig. 3. Effect of different thawing methods on the structure of myofibrillar protein isolate (MPI) from pork tenderloin.

27

Table 1 Effects of different thawing methods on myofibrillar protein denaturation in pork tenderloin. Myosin

Fresh Air RW -5 kV DC -10 kV DC -15 kV DC -20 kV DC -25 kV DC

Actin

Denaturation temperature (°C)

Denaturation enthalpy (J/g)

Denaturation temperature (°C)

Denaturation enthalpy (J/g)

61.68 ± 1.30a 60.75 ± 1.09b 59.80 ± 2.18b

-0.18 ± 0.054 -0.14 ± 0.015 -0.15 ± 0.017

74.99 ± 2.21a 72.43 ± 0.66a 71.40 ± 2.09b

-0.05 ± 0.01 -0.04 ± 0.00 -0.03 ± 0.01

58.76 ± 1.92bc

-0.18 ± 0.040

71.46 ± 2.53b

-0.04 ± 0.02

62.19 ± 0.00a

-0.19 ± 0.042

73.34 ± 1.54ab

-0.04 ± 0.01

59.95 ± 0.27b

-0.23 ± 0.015

71.83 ± 0.88b

-0.04 ± 0.01

59.51 ± 0.42b

-0.17 ± 0.021

72.47 ± 0.96ab

-0.04 ± 0.01

57.60 ± 1.84c

-0.19 ± 0.046

74.44 ± 1.07a

-0.04 ± 0.00

s of different thawing methods on the average particle size and gel textural properties of myofibrillar proteins. Average particle size

Textural property

D4,3 (µm)

D3,2 (µ m)

Dv,0.1 (µm)

Dv,0.5 (µ m)

Hardness (N)

Springiness (mm)

Cohesiveness

Chewiness (mJ)

A

14.51 ± 0.88c 32.83 ± 5.41ab 35.65 ± 2.14a 15.44 ± 2.34c 27.33 ± 2.58b

1.94 ± 0.15c 3.41 ± 1.27ab 4.56 ± 0.21a 2.45 ± 0.82b 1.77 ± 0.14c

0.64 ± 0.013b 0.78 ± 0.16b 0.95 ± 0.01a 0.78 ± 0.10b 0.63 ± 0.03b

8.41 ± 0.20b 18.82 ± 6.72a 23.58 ± 0.56a 3.18 ± 0.57b 6.76 ± 1.10b

0.84 ± 0.16a 0.44 ± 0.04b 0.51 ± 0.02b 0.73 ± 0.10a 0.55 ± 0.09b

1.02 ± 0.31b 0.46 ± 0.15c 0.53 ± 0.03c 1.39 ± 0.25a 0.69 ± 0.18bc

0.43 ± 0.01 0.41 ± 0.02 0.46 ± 0.02 0.44 ± 0.04 0.45 ± 0.02

0.45 ± 0.15a 0.08 ± 0.04b 0.12 ± 0.02b 0.47 ± 0.17a 0.17 ± 0.06b

0.0 0.0 0.0 0.0 0.0

28

Table 3 Differentially expressed proteins contained in the myofibrillar fraction between air-thawed pork tenderloin and -10 kV DC-thawed pork tenderloin by label-free mass spectrometry. Accession Myofibrillar proteins Sp | Q9TV62 | MYH4_PIG F1SS62 | F1SS62_PIG Q5XLD2 | Q5XLD2_PIG B5APU3 | B5APU3_PIG Sp | P68137 | ACTS_PIG Sp | A1XQV4 | TPM3_PIG Sp | P10668 | COF1_PIG Sarcoplasmic proteins Sp | Q5XLD3 | KCRM_PIG F1SHA2 | F1SHA2_PIG Sp | Q6S4N2 |

Description

Unique peptides

Sequence coverage (%)

Mol. Weight (kDa)

Air/-10 kV DC

P value

Myosin-4

39

71

223.234

0.97

<0.001

Myosin-1

13

75

170.268

1.92

<0.001

24

90

18.978

0.75

<0.001

1

2

44.761

0.19

<0.05

13

91

42.051

0.76

<0.001

13

89

33.058

0.39

<0.05

Cofilin-1

1

14

18.519

0.84

<0.001

Creatine kinase M-type

61

95

43.059

0.86

<0.001

5

25

24.934

0.98

<0.001

3

10

70.098

0.82

<0.01

Myosin regulatory light chain 2 Actin-related protein 2-like protein Actin alpha skeletal muscle Tropomyosin alpha-3 chain

Glycerol-3-phosphate dehydrogenase [NAD(+)] Heat shock 70 kDa

29

HS71B_PIG Sp | P11708 | MDHC_PIG F1SQ46 | F1SQ46_PIG Sp | P02189 | MYG_PIG

protein 1B Malate dehydrogenase cytoplasmic Triosephosphate isomerase Myoglobin

1

6

36.454

0.54

<0.001

8

37

26.668

0.88

<0.001

17

79

17.085

0.61

<0.05

30


HVEF thawing did not result in severe MPI oxidation and/or denaturation.
-10 kV thawing led to better textural properties than those of traditional methods.
A label-free strategy was applied to compare the protein abundance.
-10 kV thawing had less of effects on MPI conformation than that from air-thawing.

31