Effect of low and high pulsed electric field on the quality and nutritional minerals in cold boned beef M. longissimus et lumborum

Innovative Food Science and Emerging Technologies 41 (2017) 135–143

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Effect of low and high pulsed electric field on the quality and nutritional minerals in cold boned beef M. longissimus et lumborum Ammar Ahmad Khan a,f, Muhammad Atif Randhawa a, Alan Carne b, Isam A. Mohamed Ahmed c, David Barr d, Malcolm Reid d, Alaa El-Din A. Bekhit e,⁎ a

National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand Department of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia d Department of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand e Department of Food Science, University of Otago, PO Box 56, Dunedin, New Zealand f University Institute of Diet and Nutritional Sciences, The University of Lahore, Pakistan b c

a r t i c l e

i n f o

Article history: Received 24 January 2017 Received in revised form 2 March 2017 Accepted 2 March 2017 Available online 06 March 2017 Keywords: Pulsed electric field Tenderness Beef Intensity Minerals Lipid oxidation

a b s t r a c t The present research investigated the effects of low PEF (LPEF, 2.5 kV, 200 Hz and 20 μs) and high PEF (HPEF, 10 kV, 200 Hz and 20 μs) on the quality of cold-boned beef loins at 1 and 14 days of post-treatment. HPEF increased (P b 0.001) the temperature of the beef M. longissimus et lumborum (LL) samples. HPEF samples had higher (P = 0.007) shear force than LPEF samples and control samples were not different from either. HPEF beef LL samples had higher L* values and lower a* values (P b 0.001) compared to LPEF and control samples. Higher lipid oxidation (P = 0.013) was found in HPEF samples compared to LPEF samples. Lower (P b 0.01) P, K and Fe concentrations were found in HPEF samples compared to LPEF samples. The results suggest that high intensity PEF treatment can negatively affect the quality of beef. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Pulsed electric field (PEF) technology has been shown to have several useful applications in food processing through the manipulation of cell structure that allows significant improvements in sensory, yield and safety attributes to be obtained in an extremely short processing time, normally in the order of seconds and generally without the need for heating (Barba et al., 2015). The potential for use of PEF in meat processing has attracted some attention and the technology has been reported to improve ham processing through enhanced microdiffusion of a brine solution (McDonnell, Allen, Chardonnereau, Arimi, & Lyng, 2014; Töpfl & Heinz, 2007). Research on fresh beef from our lab (Bekhit, van de Ven, Hopkins, Suwandy, & Fahri, 2014; Suwandy, Carne, van de Ven, Bekhit, & Hopkins, 2015a, 2015b, 2015c, 2015d) reported reduced shear force and increased proteolysis of key structural proteins under certain PEF processing conditions, which were dependent on muscle type and rigor state of the muscles. Also, other researchers have found PEF beef LL treated samples (1.4 kV/cm, 10 Hz, 20 μs, 300 and 600 pulses) had higher sensory rating as being more tender compared to non-treated controls (Arroyo et al., 2015) and ⁎ Corresponding author. E-mail address: [email protected] (A.E.-D.A. Bekhit).

http://dx.doi.org/10.1016/j.ifset.2017.03.002 1466-8564/© 2017 Elsevier Ltd. All rights reserved.

treatment of beef M. Semitendinosus muscle (1.1–2.8 kV/cm, 5–200 Hz, 12.7–226 kJ/kg) can lead to structural changes but no significant impact on shear force (O'Dowd, Arimi, Noci, Cronin, & Lyng, 2013). Collectively, these reports support a promising role for PEF in meat tenderization and meat quality enhancement. PEF technology enhances the permeability of cellular components under mild voltage and frequency processing conditions (Jaeger, Balasa, & Knorr, 2008) and therefore has the potential of accelerating tenderization rate through the release of Ca++ and activation of μ-calpain early post-mortem, resulting in an increase in meat tenderization. The key in commercializing PEF processing conditions is to identify the processing parameters that can consistently be applied to various meat cuts, which will vary depending on the anatomical location of the muscle types (Bekhit et al., 2014; Suwandy et al., 2015a, 2015b, 2015c, 2015d). To date most of the studies mentioned above have demonstrated the capability of PEF within a range of operating parameters that can deliver a positive outcome, i.e. increased tenderness, without the impact of higher treatment regimes. This can enable determination of the impact of meat quality attributes, such as tenderness, color stability, cooking and purge loss and lipid oxidation, and structural changes without examining maximum PEF limits for meat, i.e. high and low PEF treatment conditions. However, there is no information on the impact of PEF on the mineral content of treated meat. Red meat is marketed as an excellent source of minerals required

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for a healthy diet, such as iron (Fe), zinc (Zn) and phosphorus (P). With increased permeability of the cellular structure after PEF treatment, the potential loss of these important nutritional minerals may be high during storage or during cooking. A wide range of PEF conditions have been used to induce structural and biological modifications of a wide range of biomaterials (Puértolas & Barba, 2016; Puértolas, Koubaa, & Barba, 2016). However, the minimum and maximum limits of PEF intensity for beef are 2.5 and 10 kV, respectively, as reported (Bekhit et al., 2014; Suwandy et al., 2015a, 2015b, 2015c, 2015d). The aim of the present study was to investigate the effects of low PEF (LPEF, 2.5 kV, 200 Hz and 20 μs) and high PEF (HPEF, 10 kV, 200 Hz and 20 μs) on the quality attributes (pH, purge and cooking loss, lipid oxidation, color measurement and shear force) of cold-boned beef M. longissimus et lumborum (LL) at 1 and 14 days post-treatment. The impact of these treatments on the concentrations of P, Fe, Zn and potassium (K) were also investigated in raw and cooked samples. 2. Materials and methods 2.1. Samples and treatments Loins (M. longissimus et lumborum, LL) were obtained from 6 dairy cows (average age was 6.2 ± 0.4 years and cold carcase weight was 204.2 ± 21.8 kg) raised on pasture and slaughtered by the Alliance Group limited (Pukeuri plant, Oamaru, NZ). The carcasses were electrically stimulated [square mono wave, 80 V, 25 s, pulse width of 68 msec and at a pulse rate per sec of 15 pps]. The loins from the left and right sides were removed from the 6 carcases at 24 h post-mortem. All visible fat and connective tissue were trimmed and discarded. The trimmed meat samples were processed into blocks (average weight = 346.6 ± 26.5 g) to fit the PEF treatment chamber (dimensions 13 × 8 × 5 cm) and assigned to treatments [control 1 day post treatment (con 1); control 14 day post treatment (con 14), low PEF 1 day post treatment (2.5 kV, 200 Hz and 20 μs; total treatment time = 30 s; LPEF 1), low PEF 14 day post treatment (2.5 kV, 200 Hz and 20 μs; total treatment time = 30 s; LPEF 14), high PEF 1 day post treatment (10 kV, 200 Hz and 20 μs; total treatment time = 30 s; HPEF 1) and high PEF 14 day post treatment (10 kV, 200 Hz and 20 μs; total treatment time = 30 s; HPEF 14)]. The LPEF and HPEF conditions selected were based on preliminary trials and previous experiments (Bekhit, Suwandy, Carne, van de Ven, & Hopkins, 2016; Suwandy et al., 2015c). The treatment chamber had two electrodes consisting of two parallel stainless steel plates that were positioned apart at a distance of 8 cm by a Teflon insulating material. PEF treatment parameters (10 kV and 200 Hz) were chosen based on earlier observations that higher PEF treatment parameters can generate an undesirable slight cooking effect on the edges of the meat samples (Bekhit et al., 2016) and that the maximum frequency limit that can be used under the present experimental conditions was 200 Hz. The PEF system (Elcrack-HPV5, DIL, Quakenburck, Germany) was used in batch mode and the meat fiber direction was parallel to the electrodes. An oscilloscope (Model UT2025C, Uni-Trend Group Ltd., Hong Kong, China) was used to monitor the pulse shape used (square wave bipolar). The calculation of total specific energy was carried out according to the method as described by Arroyo et al. (2015) using the following equation: Q¼

V2 τ σ A N  d m

where V is the input voltage (V), τ is the pulse width (s), σ is the average conductivity of the samples (S/cm), A is the electrode contact area (cm2), d is the distance between the electrodes (cm), N is the number of pulses and m is the mass of the sample (kg). Total specific energy was approximately 12.4 KJ.kg−1 for LPEF and 149.8 KJ.kg−1 for HPEF. As the HPEF treatment generated high temperature in the beef samples, an additional 6 loins (average cold carcase weight was 220.4 ±

16.6 kg) were obtained from dairy cattle and processed as control and heated control (control heated). The samples were divided into blocks (average weight 362.3 ± 10.5 g), randomly assigned to non-treated control (control) and samples incubated in a water bath at 53 ± 1 °C (the highest temperature reached with 10 kV treatment) for 25 min (heated Control), then the samples were processed as described above. The weight, pH, temperature and conductivity of the samples were measured before and after treatment. A small sample was obtained from control and treated samples for visualization of the effect of PEF treatment on the ultra-structure of the beef (see below). The samples were then individually vacuum-packed and stored at 4 °C for either 1 or 14 days post-treatment times. On the day of the assigned post processing time, the pH, weight and conductivity of samples were measured. The blocks were then cut into 3 slices, avoiding any slightly cooked edges on HPEF samples, and one of them was randomly assigned for display at 4 °C to measure the color changes. The other 2 slices were vacuum packed and one was kept at −40 °C for further biochemical analysis (mineral content and lipid oxidation) and the other was frozen and stored at −20 °C for subsequent tenderness (shear force) measurement within 1 week of freezing. Color measurements, and consequently lipid oxidation, were not determined on the heated control samples. The maximum PEF operating condition (HPEF) in the present study was at an input voltage of 10 kV and frequency of 200 Hz. This generated a slight cooking effect around the edges in the HPEF treatments due to the high heat generated in that treatment (Table 1). The average temperature of the beef LL samples before treatment was 10.1 ± 1.38 °C. HPEF treated samples had higher (P b 0.001) temperature change during treatment (ΔT = 40.5 °C) compared with LPEF treated samples (ΔT = 1.9 °C). The increase in temperature in the present study was much higher than those found in earlier studies from our lab (ranged between 0.4 and 8.5 °C using 5–10 kV and 20–90 Hz and up to 16.2 °C with repeated PEF treatments at 10 kV and 90 Hz) using cold boned beef LL (Bekhit et al., 2014; Suwandy et al., 2015a, 2015b) due to the higher frequency used in the present study. 2.2. Measurements 2.2.1. pH The pH measurement of the meat samples was carried out using a Hanna pH meter and electrode (Model HI 98150) calibrated at ambient temperature. The pH of meat samples was determined before and after the pulsed electric field (PEF) treatment and at the time of opening the vacuum packed samples at the designated post treatment storage time (1 and 14 days of storage at 4 °C). 2.2.2. Purge loss percentage Purge loss of the meat samples was measured at day 1 and day 14 post-treatment aging in vacuum packaging at 4 °C. The samples were blotted by using paper towels on the day of allocated storage time and Table 1 Changes (post-treatment – pre-treatment) in average temperature, conductivity, weight and pH of beef LL muscle under low (2.5 kV, 200 Hz and 20 μs for 30 s) and high (10 kV, 200 Hz and 20 μs for 30 s) pulsed electric field treatments compared to non-treated control (Control) and samples incubated at 51 °C for 25 min (Heated control). Treatment⁎ Heat incubation PEF@

a,b

Control Heated control Control Low High SEM

ΔT (°C)

Δσ (mS·cm−1)

ΔpH

– 41.1(1.4)⁎ – 1.9b 40.5a 3.3

– 6.2(1.1) – 1.3b 9.0a 0.9

– −0.03(0.03) – −0.005 −0.035 0.022

Within each treatment in each column, means that have different superscripts are significantly different at P b 0.05. ⁎ Mean values. The values in brackets are standard error. @ The values are the predicted means.

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weighed. The purge loss percentage was calculated using the following formula; Purge loss ð%Þ ¼ ½ðweight of sample before storage−weight of sample after storageÞ= weight of sample before storage  100

2.2.3. Cooking loss and shear force The meat was weighed and cooked from frozen state in a water bath at 80 °C. Vacuum packed meat samples were immersed individually in the water bath until they attained an internal temperature of 75 °C (Chrystall & Devine, 1991). The internal temperature of the meat was measured individually by using a Fluke type K temperature probe attached to a Fluke 52 pH-meter. After cooking, the meat was kept on ice to cool and then re-weighted after blotting dry with paper towels. The cooking loss was measured by calculating the difference in weight before and after cooking. The shear force of cooked meat was determined by using a MIRINZ tenderometer as described by Chrystall and Devine (1991) based on 8 measurement replications for each slice. The cooked samples were cut into strips parallel to the muscle fiber direction with a cross section area of 10 mm × 10 mm using a doublebladed scalpel with the blades set 10 mm apart. Meat tenderness was measured by using a MIRINZ tenderometer (AgResearch MIRINZ, Hamilton, New Zealand). The tenderness values were obtained by measuring the force required to cut across the meat strip and mean force values (converted to Newtons, N) were used in the statistical analysis. 2.2.4. Temperature The temperature measurement was carried out in the center of each meat block using a combination puncture pH electrode immediately before and after PEF treatment. In addition the temperature was recorded in several locations (8 locations per block) using a hand held infrared thermometer (Tech imports, Auckland, NZ) and a slight temperature gradient was found in some of the treatment conditions. Means of the 8 measurements were used for further analysis. The post treatment results are reported as the change in sample temperature (the temperature after treatment – temperature before treatment). 2.2.5. Electrical conductivity σ The electrical conductivity of meat from each block was measured immediately before and after the PEF treatment and at the designated post treatment time of vacuum packaging, using a hand held electrical conductivity meter (LF-Star, Matthaus, Pottmes, Germany). Three readings from different locations were measured in each sample and the average values were used for the statistical analysis. 2.2.6. Lipid oxidation The extent of lipid oxidation was determined using the thiobarbituric acid reactive substances (TBARS) assay as described by Suwandy et al. (2015a). Subsamples were obtained from samples at 1 and 11 days of display following the color measurements (Section 2.2.8). The samples were frozen in liquid nitrogen, vacuum packed and stored at − 80 °C until analysis. The amount of malondialdehyde (MDA) (mg·kg−1) in the sample was determined by using the following equation; Mg MDA=kg meat ¼ A@531  E  72:063=100  dilution factor

where E is the molar extinction coefficient of 156.000 M−1·cm−1 Molar mass of malondialdehyde = 72.063 g·mol−1

MDA

=

137

2.2.7. Determination of ultrastructure changes in PEF treated beef muscles using light microscopy The ultra-structure changes of the beef muscles were determined for control and PEF treated samples. A small piece of each muscle sample was put in a labelled cassette and fixed in 10% neutral buffered formalin (NBF) for 24 h. Each cassette was then put into 70% (v/v) alcohol until processing on a Thermo Excelsior ES tissue processor (Thermo Scientific, Waltham, MA, USA). Each processed meat tissue sample was embedded in a wax block, then cut on a Lecia RM 2135 rotatory microtome. Sections (4 μm) were floated in a 40 °C water bath and picked up on glass slides. The slides were then heated for 1 h in a 60 °C oven before being stained. After heating, the slides were stained using a Verhoeff's van Geisen staining technique (Sheehan and Hrapchak, 1980). After staining, the slides were covered with coverslips and ultra-structure changes were examined with a light microscope (Model No. Olympus BX50 F-3) at × 20 and × 40 magnification. Images were taken using a camera (Olympus company, Ltd., Japan) attached to the microscope. 2.2.8. Color measurement of PEF treated beef muscles Color measurement of control and PEF treated beef muscles was conducted on steaks that had been cut from the blocks after 1 and 14 days of post-treatment. Color measurement was conducted as described by Bekhit, Farouk, Cassidy, and Gilbert (2007). The samples were placed in polystyrene trays and covered with oxygen permeable polyvinylchloride film (O2 permeability N2000 mL·m−2·atm−1·24 h−1 at 25 °C, AEP FilmPac (Ltd), Auckland, New Zealand). Samples were exposed to fluorescent cool light (1076 lx) and color measurements were performed at 24, 72, 120, 168, 216 and 264 h of display at 4 °C.·The color measurement was made using a MiniScan XE (Model 45/0-L, Hunter Associates Laboratory Inc., Reston, VA). The equipment was calibrated using a black standard plate and a white standard (C2–36852). Measurements were CIE L*, a* and b* values and spectral reflectance (400– 700 nm) using illuminant C and a 10° observer with an aperture size of 3.5 cm. The chroma (C = [a⁎2 + b⁎2][1/2]), and hue angle (HA = tan−1 b⁎ / a⁎) were calculated (AMSA, 1991). 2.2.9. Minerals measurements An Agilent 7500ce quadrupole ICP-MS mass spectrometer was used for the analysis of minerals in meat samples. Meat samples were freezedried, ground and 0.5 g of each sample weighed into a MARSXpress (CEM Corporation USA) digestion tube, followed by addition of 10 mL of concentrated quartz distilled (QD) HNO3. The samples were digested individually in a CEM MARS6 microwave reaction system (CEM Corporation USA) using the manufacturer's directions. Dilutions of the sample digests and blanks in 2% (v/v) HNO3 were spiked offline with a cocktail of 7 reference elements to compensate for any drift or possible matrix effects. Calibration standards were prepared by serial dilution of a SPEX CertiPrep multi-element standard (Spex Certiprep, Metuchen, NJ). The ICP-MS was tuned according to the manufacturer's recommendations for robust conditions to minimize interferences and instrumental drift. Where possible multiple isotopes of the analyte elements were measured to confirm the absence of interferences. The accuracy of this single step measurement was established with a Certified Reference Material (bovine muscle powder, reference material 8414, Agriculture Canada, Distributed by The National Institute of Standards and Technology). 2.3. Statistical analysis The data for change in temperature (ΔT), conductivity change (Δσ), change in pH (ΔpH), pH, shear force (analyzed on the loge transformed scale), cooking loss (%), purge loss (%), color parameters, TBARS value and concentrations of minerals (P, K, Fe and Zn) were analyzed as general linear model (GLM) using Minitab (version 16.2.4) using split-splitplot design (Montgomery, 2013). Random effects included in the model were terms associated with the split plot nature of the experimental

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designs, these being animals; sides within animals; blocks within sides; slices within blocks and finally random error. The fixed effects in the model were effects (main and interactions) associated with voltage (2.5 and 10 kV) × post-treatment aging period (1 or 14 days). Extra fixed terms were used for color and lipid oxidation (display time) and minerals (raw/cooked) to account for the effects associated with these factors. Data for heated control samples and their respective control was analyzed using 2-way ANOVA to examine the effect of heat incubation on the measured parameters. 3. Results and discussion 3.1. Effect of PEF parameters on post treatment meat conductivity, temperature and pH In the previous studies, a lower temperature change was found in beef semimembranosus muscle under these conditions compared to that obtained for LL in the present study. The increase in pulse frequency in the present study compared to those reported earlier (Bekhit et al., 2014; Suwandy et al., 2015a, 2015b) led to the high temperature change of the meat samples during PEF treatment. The pulse number may also have contributed to that increase, since O'Dowd et al. (2013) found that increasing the pulse number from 152 to 300 resulted in an increase in the temperature change from 5 to 30 °C in beef M. semitendinosus muscle. In the present study the value of actual electric field strength (calculated from pulse peak voltage divided by the space between the electrodes) was 0.23 and 0.68 kV/cm (Table 1). Effective electroporation of sarcolemma requires an electric field surrounding the cell to exceed the critical transmembrane potential which was reported to be 0.5 kV/cm (Töpfl, 2006). The LPEF treatment was therefore below the required level for electroporation, whereas that employed in the HPEF treatment exceeded the critical transmembrane potential value and thus a much higher change in the conductivity (9.0 mS·cm−1) was found in HPEF treated samples (Table 1). The mean conductivity change values were larger at the higher voltage (9.0 mS·cm−1) compared with the low voltage (1.3 mS·cm−1) (Table 1) which is in agreement with studies reported for pork (Töpfl, 2006), beef ST muscles (O'Dowd et al., 2013) and LL and SM muscles (Bekhit et al., 2016, 2016; Suwandy et al., 2015a, 2015b, 2015c). It is worth noting that the actual electric field strength of LPEF-treated samples (0.23 kV.cm−1) was much lower than the critical transmembrane potential value (0.5 kV.cm−1) of muscle cells, the change in conductivity was within the range found for PEF treatments (voltage of 5 and 10 kV and frequency of 20– 90 Hz) reported for beef LL (Bekhit et al., 2014) and was similar to that found in hot-boned beef LL treated with 10 kV, 90 Hz and 20 μs pulse time (Bekhit et al., 2016). HPEF-treated beef samples were exposed to higher energy density and pulse peak power values compared to LPEF samples (Table 1), which were translated into larger temperature changes in the samples. This significant difference in temperature change can consequently affect protein structure and endogenous enzyme activity and consequently can affect beef quality attributes. The change in pH (ΔpH) in LPEF and HPEF samples was not significantly different (P N 0.05). 3.2. Effect of PEF treatments on the meat conductivity and pH at different aging times Heated control samples had similar pH values to non-treated control samples (Table 2A). The pH of HPEF samples was higher at day 1 posttreatment than non-treated control and at day 14 compared to LPEF samples (P b 0.01) (Table 2B). The heated control samples had similar conductivity to non-treated control samples at 1 and 14 days of treatment (Table 2A). There were no statistical differences among the means of conductivity of the samples at day 1 and 14 of post-treatment

Table 2 Mean pH and conductivity of beef LL muscles; A) heated control and control samples; B) control and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF samples, at 1 and 14 days post treatments. Post treatment aging A Day 1 Day 14

B Day 1

Day 14

Treatment

pH

σ (mS·cm−1)

Control Heated control Control Heated control SEM

5.61a 5.64a 5.51b 5.49b 0.03

4.60a 6.81ab 9.8b 8.2b 1.2

Control Low High Control Low High SEM

5.65b 5.67ab 5.77a 5.40cd 5.38c 5.49d 0.02

6.28c 6.62bc 9.17abc 10.6ab 12.0a 11.3a 0.94

a–d

Within each column, means that have different superscripts are significantly different at P b 0.05.

although a numerically higher conductivity value was found in HPEF samples (Table 2B). 3.3. Effect of PEF on the shear force of beef LL muscle The shear force of heated control samples were not different from non-treated control samples (P N 0.05) and the shear force values of both treatments decreased with aging (P b 0.01) (Fig. 1A). Generally, the shear force of beef LL muscle was affected by PEF treatment (P = 0.024) and post treatment aging time (P = 0.007) but not by the interaction of these two factors (Fig. 1B). Beef LPEF samples had generally a lower shear force compared with HPEF treated samples, and non-treated control samples were not different from either. The mobility of intracellular constituents by permeabilization of plant or animal tissues was shown to be enhanced by low intensity PEF treatment (Barsotti & Cheftel, 1999). This may cause earlier activation of biochemical events (such as proteolysis) and early release of cellular material. However, proteolysis is not an instantaneous process and time is required to achieve tenderness that can be perceived on consumption, which may explain the advantage of including an aging step, resulting in lower shear force. Reported studies on PEF treatment of beef suggest low intensity PEF treatment to be more beneficial compared with higher intensity PEF (Bekhit et al., 2015, Bekhit et al., 2016; Suwandy et al., 2015a, 2015b, 2015c) due to enhanced proteolysis of some structural proteins. High PEF treatment intensity such as that reported by O'Dowd et al. (2013) in ST and in hot-boned SM and LL (Bekhit et al., 2016), as well as in the present study, can lead to high temperature that can cause protein denaturation, including proteases responsible for protein degradation postmortem, which may hinder tenderness development. Also, LPEF treatment did not result in a significant tenderizing effect that we found previously (Bekhit et al., 2014). 3.4. Effect of PEF on the purge loss (%) and cooking loss (%) of beef LL muscle Heating the beef samples at 53 °C for 25 min resulted in higher purge loss compared to non-treated control samples at day 1 (P b 0.05), but there was no significant effect between the two treatments at day 14 (P N 0.05) (Fig. 2A) that is likely due to aggregation of myosin that occurs at temperatures N 45 °C and has reached a maximum between 54 and 58 °C (Tornberg, 2005). Purge loss (%) was affected by PEF treatment (P b 0.001) and the interaction between treatments and posttreatment aging (P b 0.001), but post mortem aging did not significantly influence the purge loss (Fig. 2A). LPEF beef had similar purge loss to non-treated control samples and both were lower than HPEF samples.

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treatment (Suwandy et al., 2015a). It is not clear why this cooking loss was not affected by PEF treatments in the present study. 3.5. Effect of PEF treatment on the lipid oxidation of beef muscle Lipid oxidation, as determined by TBARS values, was affected by post treatment aging time (P = 0.014), display time (P b 0.001), PEF treatment (P = 0.013) and display time × PEF treatment interaction (P = 0.011) (Fig. 3). HPEF samples had higher lipid oxidation at the end of display time compared to LPEF treated beef samples, but both of these groups of samples were not different from the non-treated control samples (Fig. 3). HPEF treated samples aged for 14 days post treatment had the highest increase in MDA concentration, from 0.15 ± 0.12 mg·kg−1 at initial display time to 1.02 ± 0.12 mg·kg−1 at the end of the display time (after 11 days). The generation of reactive oxygen species is a major pathway for lipid oxidation and it has been reported that factors that cause cellular damage facilitate the formation of free radicals and lipid oxidation (Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Given that low PEF (the present study) and modest PEF treatment (Suwandy et al., 2015b) did not affect lipid oxidation, it is likely that the high heat generated during the HPEF treatment diminished the antioxidant capacity of the meat and allowed lipid oxidation to progress at higher rates during storage (Bekhit, Hopkins, Fahri, & Ponnampalam, 2013). Therefore, HPEF appears to promote lipid oxidation and negatively affect the quality of meat (Morrissey et al., 1998; Pearce, Rosenvold, Andersen, & Hopkins, 2011). 3.6. Ultrastructure changes in beef muscles due to PEF treatments

Fig. 1. Predicted means of shear force (N) of beef LL muscles, A) heated control and nontreated control samples; B) control and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF samples, at 1 and 14 days post treatments. a–bBars with different superscripts are significantly different at P b 0.05.

The increase in purge loss with PEF treatment in the present study is in agreement with earlier reports from our lab (Bekhit et al., 2015; Suwandy et al., 2015a, 2015b) where the purge loss (%) was increased by the increase in the voltage. The majority of the observed significant change was found with HPEF treated samples that had a high temperature increase during PEF treatment causing protein denaturation that resulted in low water holding capacity. It was observed that some slight cooking effect occurred on the edges of HPEF treated samples and the average temperature of these samples was 51.4 °C. The higher purge loss and shear force in the HPEF samples suggests denaturation of proteins had occurred as a result of the higher temperature achieved in the meat, that has likely resulted in a negative impact on proteases in these samples. The results of the present study are contrary to those reported by O'Dowd et al. (2013) for beef ST samples that suggested that the water holding capacity was not affected by PEF treatment. This may be related to the lower temperature change (30 °C) reported in their study compared with the HPEF samples in the present work. The results of cooking loss percentage in relation to heating at 53 °C, PEF treatments and post-treatment time showed no significant differences (Fig. 2B). Earlier work showed higher cooking loss with PEF

Transverse sections of non-treated control, heated control and PEF treated beef muscles examined by light microscope imaging at different magnifications (×20 and ×40) are shown in Fig. 4. Generally, spaces between the muscle bundles in beef samples heated at 53 °C tended to be wider (Fig. 4A). According to Tornberg (2005), visual changes in myosin due to heating at temperatures N 50 °C can lead to large myosin aggregates that probably lead to slight changes in the muscle bundle size. Muscle fiber bundles of beef treated with LPEF were elongated compared with non-treated control samples, suggesting that physical change had occurred as a result of LPEF treatment. The figure with ×40 magnification (Fig. 4) shows clear gaps between muscle bundles. High intensity PEF treatment caused shrinkage in the muscle fiber bundles compared with the non-treated control and LPEF samples (Fig. 4). This may be due to a greater water loss from HPEF samples as shown by the higher purge loss (%) in these samples, leading to a lower volume occupied by the bundles. A similar observation of shrinkage was reported by Gudmundsson and Hafsteinsson (2001) for chicken and by O'Dowd et al. (2013) for beef. 3.7. Effect of low and high PEF treatments on the color of beef LL muscle Lightness (L*) values were affected by treatment, display time and their interaction (P b 0.001 all). After allowing 24 h of blooming, the lightness values (L*) of HPEF treated beef samples were significantly (P b 0.001) higher than that of control and LPEF treated samples (Supplementary Fig. 1A). This can be explained by the higher purge loss (Fig. 2A) in these samples as a result of high heat generated during treatment. Low water holding caused by high temperature in this treatment may cause higher moisture on the meat surface that lead to more light being reflected from the surface and hence the lighter color (Bekhit, Geesink, Morton, & Bickerstaffe, 2001). The lightness of control and PEF low treated samples increased up to 3 days of display time, but gradually decreased with further time display. The lightness value of 1 day post-mortem treated samples with high PEF intensity remained the same throughout the display time. The redness (a*) of the meat samples was significantly affected by treatment, post-treatment aging, display time and their interactions

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Fig. 2. Predicted means of A) purge loss (%) and B) cooking loss (%) of beef LL muscles, A) heated control and control samples; B) control and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF samples, at 1 and 14 days post treatments. a-dBars with different superscripts are significantly different at P b 0.05.

(P b 0.001 for all, Supplementary Fig. 1B). The meat samples from 1 day post treatment time had higher redness values at 1 day. Both LPEF treated and non-treated control samples exhibited higher a* values

Fig. 3. Predicted means for lipid oxidation (MDA mg·kg−1) of beef LL muscles, control and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF, and aged for 1 and 14 days post treatments at 1 and 11 days display time. a-d Bars with different superscripts are significantly different at P b 0.05.

compared to HPEF treated samples (Supplementary Fig. 1B). A very fast rate of discoloration was observed in HPEF 1 day post treatment samples. This supports the contention that the antioxidant capacity of these samples was negatively affected by the relatively higher heat generated from HPEF treatment, and led to higher oxidation of myoglobin to metmyoglobin. The yellowness (b*) values were affected by the different treatments and the display time and are shown in Supplementary Fig. 1C. The PEF intensity affected the yellowness of the meat. Samples treated with HPEF at 1 day of post treatment showed a greater value of yellowness compared to other samples. The yellowness of meat samples decreased with further display time. A similar trend was found with HPEF samples after 14 days of aging but there was a delay in reaching a maximal b* value as was the case with a* values. The chroma (C) value of the meat samples was significantly (P b 0.001) affected by display time and treatments (Supplementary Fig. 1D). Chroma values showed a declining trend with the passage of display time. One day post-treatment samples showed highest chroma values at 1 day of display time, but decreased with longer display time. Samples treated at 14 days of post-mortem time showed greater values at day 3 of display time than other times of display. LPEF-treated samples after 1 day post-treatment had the highest C values during the 11 days of display time and no differences were found between the 1 day posttreatment control and HPEF samples. This trend was reversed after 14 days of storage where HPEF-treated samples had the highest C values at the end of the storage period, while C values of control and LPEF-treated samples were not different.

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Fig. 4. Cross-sectional view for ultrastructure analysis of beef LL muscles, control, heated control, and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF, and aged for 1 day post treatment at different magnification power (×20 and ×40).

In agreement with the results for a* values, the hue value (h) of meat samples was significantly (P b 0.01) affected by treatment and display time (Supplementary Fig. 1E). The hue values (h) increased as the display time increased and significantly (P b 0.001) higher h values were found in HPEF treated samples. The results obtained for a* and h values were reflected in the Browning index (630/580 nm and 630–580 nm) with LPEF treated and non-treated control samples at 1 day post treatment having better color stability (higher Browning value) whereas HPEF treated samples had the most significant Browning during the display time (Supplementary Fig. 1F). 3.8. Effect of PEF treatment on the mineral profile of beef muscles One of the objectives of the present study was to evaluate the impact of PEF treatment on the mineral content in beef since there is currently no information available in the literature. Four nutritionally important minerals (P, K, Fe and Zn) were chosen to be evaluated in the present study (Table 3) and their concentrations were determined on a dry weight basis to avoid any impact for moisture content, which is affected by PEF and cooking. The P concentrations in beef LL muscles ranged from 7288 to 8036 and 5480 to 6137 mg·kg− 1 dry basis in raw and cooked samples, respectively. The concentration of P was decreased by cooking (P b 0.001), treatment (P b 0.01) and post-treatment aging

(P b 0.01) but not by their interactions (Table 3). Raw beef had a higher mean P concentration compared with cooked (7619.2 and 5743 mg·kg−1 dry basis, respectively). Beef at 1 day post treatment had a higher P concentration compared to that of aged for 14 days post treatment (6824.0 and 6538.6 mg·kg−1 dry basis, respectively). LPEF treated beef samples had higher (6893.3 mg·kg− 1 dry basis) than control and HPEF samples (6598.8 and 6551.9 mg·kg− 1 dry basis, respectively). Similar trends were found with the concentrations of K. The K concentrations in beef LL muscles ranged from 15,358 to 17,608 and 10,046 to 11,674 mg·kg−1 dry basis in raw and cooked samples, respectively. The concentration of K was decreased by cooking (P b 0.001), treatment (P b 0.01) and post-treatment aging (P b 0.01), but not by their interactions (Table 3). The decrease in K and P concentrations as a result of cooking is in agreement with the results of Gerber, Scheeder, and Wenk (2009) where a 24.6% and a 55.4% decrease in K concentration and a 19.1% and a 33.6% decrease in P concentration were reported in grilled beef ribeye and boiled beef brisket. LPEF treated beef had higher K concentrations (overall mean concentration = 14.101.8 mg·kg−1 dry weight) than HPEF beef samples (overall mean concentration = 12.983.1 mg·kg−1 dry weight) and control samples (mean concentration = 13.327.5 mg·kg−1 dry weight) were not different from either of these two treatment groups. The lower concentrations of K and P in cooked meat suggest that proportions of these

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Table 3 Concentrations of phosphorous (P), potassium (K), iron (Fe) and zinc (Zn) (mg·kg−1 dry weight) in raw and cooked beef LL muscles; A) heated control and control samples; B) control and treated with low (2.5 kV, 200 Hz and 20 μs pulse time) and high (10 kV, 200 Hz and 20 μs pulse time) PEF samples, at 1 and 14 days post treatments. Minerals (mg·kg−1 dry basis) treatments A 1 day posttreatment

14 days posttreatment

Fresh

Control Heated control Cooked Control Heated control Fresh Control Heated control Cooked Control Heated control SEM

Reference material Experimentally obtained value Recovery (%) Limits of detection B 1 day posttreatment

14 days posttreatment

Reference material Experimentally obtained value Recovery (%) Limits of detection

Fresh

Control PEF low PEF high Cooked Control PEF low PEF high Fresh Control PEF low PEF high Cooked Control PEF low PEF high SEM

P

K

Fe

Zn

7438a 7215a

16,123a 15,771a

95.5 94.1

122.9 124.1b

5521b 5536b

10,435b 10,121b

99.1 97.8

130.6 131.9

7100a 7027a

15,883a 14,860a

90.6 88.3

128.1 130.1

5220b 5317b

10,025b 9937b

98.0 96.6

131.0 133.1

159.2 8360 7530 ± 16.0 90 0.02

623.2 15,170 15,003 ± 512 99 0.02

3.8 71.2 71.8 ± 0.6 101 0.01

5.5 142 130.2 ± 4.2 92 0.02

7677a 8036a 7515a 5847b 6137b 5732b 7391a 7807a 7288a 5480b 5593b 5674b 163.7 8360 7459 ± 21.0 89 0.02

16,407a 17,608a 15,917a 10,952b 11,674b 10,319b 16,068a 17,080a 15,358a 9883b 10,046b 10,334b 511.2 15,170 14,926 ± 554 98 0.02

94.2 95.4 92.5 98.4 91.8 83.0 89.9 91.7 84.9 96.7 100.9 82.2 4.8 71.2 72.9 ± 0.4 102 0.01

118.7ab 124.8ab 130.1ab 129.5ab 130.1ab 141.9a 113.2b 122.3ab 133.0ab 135.2ab 128.5ab 143.4a 5.8 142 124.1 ± 5.1 87 0.02

a–b

Within each column, means that have different superscripts are significantly different at P b 0.05.

minerals are present in the sarcoplasmic fraction and are lost during cooking. This may explain the lower concentration of these minerals in HPEF samples as a result of the greater purge loss occurring from the samples. The concentration of Fe was affected by PEF treatment only (P = 0.011). Fe concentration was lower in HPEF beef samples (85.6 mg·kg− 1 dry basis) than LPEF and control samples (95.0 and 94.8 mg·kg−1 dry basis, respectively). The concentration of Zn in cooked meat was affected by cooking only (P = 0.037) with an overall mean Zn concentration of 162.9 mg·kg−1 dry basis in cooked beef samples compared to 123.7 mg·kg−1 dry basis in raw beef samples. The apparent increase in the concentration of Zn present in meat following moisture loss during cooking has been documented in the literature. For example, Lombardi-Boccia, Lanzi, and Aguzzi (2005) found 61% and 40% increase Fe and Zn concentrations (on a wet sample basis) in beef sirloin following pan-cooking. The apparent increase in Fe and Zn concentrations were 42% and 40% in beef fillet under the same cooking conditions. Also, grilled beef ribeye and boiled beef brisket had a relatively much lower increase in concentration of these minerals due to cooking (16.6% and 25.4% for Fe and 2.2% and 17.9% for Zn, respectively) (Gerber et al., 2009). The reported increase in mineral concentrations as a result of cooking is clearly related to the reduction in the moisture content of meat during cooking. In the present study, the samples were boiled in individual bags as described in Section 2.2.4 and the difference between the raw and cooked beef samples appears to be caused

by leaching out of minerals during cooking since the minerals were determined on dry weight basis.

4. Conclusions The results presented here and those published by others indicate that there is an optimal PEF treatment for beef cuts within a range of processing parameters. This study indicates that low and high PEF treatments can lead to ultrastructural changes in beef LL with contrasting outcomes on beef quality. The high PEF treatment negatively affected the shear force, color stability and resulted in lower levels of mineral (P, K and Fe) concentrations primarily due to the high temperature generated from that treatment that probably led to a negative impact on proteases and generated a higher degree of shrinking. These negative effects were not found with LPEF. From the results of the present study and those reported previously, it is important that the PEF parameters are optimized for different muscle types. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ifset.2017.03.002.

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