Effect of freezing as pre-treatment prior to pulsed electric field processing on quality traits of beef muscles

Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

INNFOO-01227; No of Pages 10

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Innovative Food Science and Emerging Technologies

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Effect of freezing as pre-treatment prior to pulsed electric field processing on qualitytraits of beef muscles

Farnaz Faridnia a, Qian Li Ma b, Phil J. Bremer a, David John Burritt c, Nazimah Hamid b, Indrawati Oey a,⁎a Department of Food Science, University of Otago, Dunedin, New Zealandb School of Applied Sciences, Faculty of Health and Environment Sciences, Auckland University of Technology, Auckland, New Zealandc Department of Botany, University of Otago, Dunedin, New Zealand

⁎ Corresponding author at: Department of Food Scienc56, Dunedin 9054, New Zealand. Tel.: +64 3 479 8735; fa

E-mail address: [email protected] (I. Oey).

http://dx.doi.org/10.1016/j.ifset.2014.09.0071466-8564/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Faridnia, F., et al.,muscles, Innovative Food Science and Emergin

a b s t r a c t

Article history:Received 3 April 2014Revised 15 August 2014Accepted 30 September 2014Available online xxxx

Editor Proof Received Date 30 Oct 2014

Keywords:Pulsed electric fieldsFreezing pre-treatmentLipid oxidationVolatile compoundsFatty acid profileMeat quality

The purpose of this research was to study the effects of freezing as pre-treatment prior to pulsed electricfield (PEF) on the quality of beef semitendinosus muscles. Fresh and frozen-thawed samples were treated usingsquare -wave bipolar pulses at electric field strength 1.4 kV/cm, pulse width 20 μs, frequency 50 Hz and totalspecific energy 250 kJ/kg. PEF caused significant microstructural changes of meat tissue compared to freezing.Combined freezing–thawing and PEF resulted in improved tenderness indicated by reduced shear force, butnot PEF alone. PEF significantly increased purge loss but not cooking loss. A two log-unit increase in aerobic mi-crobial counts during log phase of frozen-thawed PEF-treated samples was positively associated with increasedpurge loss. PEF itself did not affect the ratios of polyunsaturated/saturated fatty acids and omega 6/omega 3 northe free fatty acid profiles. Freezing with and without PEF greatly affected the volatile profile of meat.Industrial relevance: PEF treatment provides an alternative tomechanical, thermal and enzymatic cell disintegra-tion of animal raw materials, providing a short duration (milliseconds) and energy efficient treatment. In thisstudy, the possible relationship between freezing prior to PEF and changes in beef tissue microstructure thatinfluence storage stability and safetywas investigated. The results of this study contribute toward understandinghow PEF induced changes in beef microstructure influence the important quality attributes of beef.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Pulsed electric field (PEF) is a non-thermal food processing technol-ogy that permeabilizes cell membranes by delivering high-voltage briefpulses (μs) through a food product placed between two conductiveelectrodes (Puértolas, Luengo, Álvarez, & Raso, 2012). The applicationof PEF processing has been demonstrated to induce changes in thestructure and texture ofmeat, potentially improving its functional prop-erties or aiding in the development of new products (Topfl & Heinz,2007). Therefore, PEF technology could be applied as a relatively newmethod for cell disintegration (Knorr et al., 2013). Studies on the effectof electropermeabilization on protein-based foods such asfish andmeatare limited and different experimental setups and processing parame-ters make them difficult to compare. PEF treatment of duck meat orbeef and subsequent curing storage for 12 h has previously beenshown to increase tenderness (Töpfl, 2006). PEF (3.5 kV/cm, 20 Hz,5 s) treatment of beef triceps brachii muscles has also been reported to

e, University of Otago, P.O. BOXx: +64 3 479 7567.

Effect of freezing as pre-treatg Technologies (2014), http:/

decrease the average maximum shear force to cut meat by 21.5% andhence enhanced tenderness (Lopp & Weber, 2005). In contrast, otherstudies have shown that tenderness of beef semitendinosus muscles iseither not affected by PEF (1.1–2.8 kV/cm, 5–200 Hz, 12.7–226 kJ/kg)(O'Dowd, Arimi, Noci, Cronin, & Lyng, 2013), or PEF treatment causesa decrease tenderness after cooking (Hoffmann et al., 2009). Recently,we have shown that tenderness and colour stability of beef longissimusthoracismuscles are not affected by PEF (0.2–0.6 kV/cm, 1–50Hz, 20 μs)treatments and a subsequent vacuum ageing (Faridnia, Bekhit, Niven, &Oey, 2014). The apparent discrepancy may be associated withdifferences in the experimental conditions such as the use of differentprocessing parameters of PEF treatment, treatment chamber, samplesize, muscle types and sample preparation prior to PEF treatment suchas freezing and thawing before PEFwhich complicate the interpretationof previous experiments. Due to these contradictory findingsdemonstrating PEF impacts on meat, it is apparent that further studiesare required to investigate the effects of freezing as pre-treatment andPEF on animal tissues.

In addition to tenderness, other quality parameters of meats shouldbe considered since biochemical reactions involved in post-mortemprocess influence the generation of volatile compounds throughenzymatic oxidation of unsaturated fatty acids, and further interactionswith proteins, peptides and amino acids (Huang&Ho, 2001). Fatty acids

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

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in muscle tissue affect meat quality, including its tenderness, colour,lipid stability, odour and flavour (Wood et al., 2004) and are essentialto determine the nutritional value ofmeat. Since PEF treatment changesthe cell permeability, it makes themeat component such as lipids moresusceptible to oxidation or facilitates the reaction between enzymes andtheir substrates. It could induce alteration of fatty acid composition andvolatile profile of themeat and ultimately influencemeat shelf life. Up tonow, no/limited study on the effect of PEF on these quality traits ofmeathas been conducted.

Therefore, the purpose of this research was to study the effects offreezing as a pre-treatment prior to PEF treatment on the quality traitsof semitendinosus (ST) beef muscle. In this study, semitendinosusmusclewas selected because it is retailed as a relatively low value steak due toits high connective tissue content and this muscle is more resistant tothermal, mechanical, chemical, and enzymatic method interventionsto increase the tenderness of muscles (Istrati, Vizireanu, & Dinică,2012) in contrast to longissimus muscle. In this study, the treatmenteffects on tenderness, microstructure, microbial shelf life, fatty acidcomposition, lipid oxidation, and volatile profile after cooking wereevaluated.

2. Materials and methods

2.1. Raw materials and sampling procedure

Beef semitendinosus (ST) muscles from 9 animals (mean coldcarcasses weight of 245–285 kg) were obtained at 24 h post-mortemfrom a local commercial slaughter-house (Silver Fern Farms Ltd.,Finegand Plant, Balclutha). Upon arrival, all visible fat was removed bytrimming and for each animal, eachmusclewas divided into two halves.The first half of the muscle was further divided into two portions; onewas immediately PEF treated (‘fresh-PEF’ (P)) and the other retainedas a ‘fresh-control’ (C)). The second halfwas vacuumpacked in polyeth-ylene plastic bags (~500 g) and immediately frozen at−18 °C in a dig-itally temperature controlled thermostat freezerwith static flow cold airtemperature of −20 °C for 7 days before PEF treatment (the samplesfrom this half are called ‘frozen-thawed PEF’ (FP) and ‘frozen-thawedcontrol’ (FC)). The frozen samples were thawed overnight at 4 °Cprior to PEF treatment.

On the day of PEF treatment, the muscles for both control and PEFtreatment were cut parallel to the fibre direction into triangular piecesusing a guided chopping board fitted with a tailored-made stainlesssteel triangular blade. The form and dimension of the triangularblade were the same as those of PEF batch treatment chamber (6 cmheight × 4 cm width × 6 cm length). The weight of the sample was~70 g for each piece and thefibre directionwas arranged so as to be per-pendicular to the electric current.

2.2. Pulsed electric field (PEF) treatments

The beef samples were processed in a pilot plant scale PEF system(Elcrack-HVP 5, DIL Quakenbruck, Germany) using a batch modeconfiguration. The selection of PEF processing parameters was deter-mined based on the resulting visual quality of samples immediatelyafter PEF treatment and the stability of current delivery (no electricarching) during PEF treatment. Our preliminary work showed thatthe visual quality of samples was severely affected after the treat-ment at electric field strength of 1.7−2 kV/cm (the edges of themeat were cooked). Therefore, the operating variables used in thisexperiment were as follows: constant pulse width of 20 μs, electricfield strength of 1.4 kV/cm, constant frequency of 50 Hz, pulse num-ber of 1032, and total specific energy input of 250 kJ/kg. Pulse shape(square wave bipolar) was monitored on-line with an oscilloscope(Model UT2025C, Uni-Trend Group Ltd., Hong Kong, China) duringtreatment. The pulsed electrical energy, also known as specificinput energy (Wspec) applied to meat samples at square-wave

Please cite this article as: Faridnia, F., et al., Effect of freezing as pre-treatmuscles, Innovative Food Science and Emerging Technologies (2014), http:/

pulse was calculated according to Zhang, Barbosa-Cánovas, andSwanson (1995) using Eq. (1).

Specific energy input; WspeckJkg

� �¼ V2 � nτð Þ

R � Wð1Þ

V is the pulse peak voltage (in kV), n is the number of pulses applied(dimensionless), τ is the pulsewidth of square pulses (inmicrosecond),R is the effective load resistance (in ohm) andW is theweight of sample(in kilogramme) to be treated in the PEF treatment chamber. The tem-perature of samples before and after treatment was monitored using atemperature logger (Grant Squirrel SQ800, Cambridge, UK) and type Tthermocouples. The initial temperature was maintained at 4 °C andthe pH of each meat sample was measured by inserting a calibratedpH probe (HANNA HI 98140, Woonsocket, USA) directly into themeat. Duplicate pH and temperature readings were taken for each sam-ple before and after PEF treatment. A hand held meat conductometer(LF-STAR, R. Mathäus, Germany) was used to determine the change inelectrical conductivity (σ) of beef samples prior to and after treatment,by inserting the twin probes directly into the samples at three differentpositions.

After treatment, all beef samples for shear forcemeasurement, purgeloss, and cooking loss, were vacuum packed in polyamide polyethylenebags and stored at 2 °C for 7 days. After assigned storage time, samplesfor volatile compounds and fatty acids profilewere snap-frozen in liquidnitrogen and stored at –80 °C for less than a month until analysed. Intotal, 6 independent samples coming from different animals wereused (n = 6).

For microbiology testing and lipid oxidation determination, thebeef samples after PEF treatment were placed in a sterile stomacherbag (15 × 23 cm, 0.102 mm thick, 710 ml capacity) and stored at 4 °Cfor different predefined ageing times (up to 16 days). The samplingfor microbiology test and lipid oxidation was taken every 2 days ofstorage (n = 4).

2.3. Study on changes in physical properties of meat

2.3.1. Determination of purge lossThe samples were weighed immediately after PEF treatment (initial

weight). On the day of sampling, the meat samples were removed fromthe vacuum packed bags, blotted dry with paper towels and weighed(final weight). The purge loss was calculated using Eq. (2) andexpressed as %.

Purge loss %ð Þ ¼ Initial weight−Final weightð Þ=Initial weight½ � � 100 ð2Þ

2.3.2. Determination of cooking loss and shear forceThe cooking loss was determined by weighing samples before and

after cooking in a water bath set to 80 °C. Thermocouples were insertedto the centre of each meat sample to monitor the temperature duringcooking. After reaching the desired internal temperature of 75 °C, thesamples were immediately cooled down in an ice-water bath, blotteddry and weighted. The loss of weight due to cooking was calculated ascooking loss and is expressed as a percentage of the original sampleweight.

For shear forcemeasurement, the same sample used for the cookingloss test was employed. After cooling, 10 × 10 × 25 mm cross sectionsamples (ten replicates for each sample) were cut parallel to the fibredirection and the peak shear force (kgF)was determined, perpendicularto the fibre direction using aMIRINZ Tenderometer (Chrystall & Devine,1991).

2.3.3. Electron microscopy analysis using TEMThe microstructure of meat samples was evaluated using Transmis-

sion Electron Microscopy (TEM). For TEM observation, samples were

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fixed with glutaraldehyde (2.5% dissolved in 0.1 M-cacodylate buffercontaining 5% sucrose), before secondary fixation with osmium tetrox-ide and uranyl acetate. Dehydrationwas achieved using a series of grad-ually increasing concentrations of ethanol (50–100%) and propyleneoxide (100%). Samples were then infiltrated with resin and embeddedin epoxy resin blocks. After polymerization at 60 °C, ultra-thin sections(70–100 nm) were cut and placed on TEM grids for viewing. Sampleswere observed using a Philips CM100 BioTWIN transmission electronmicroscope (Philips/FEI Corporation, Eindhoven, Holland), LaB6 emitterfitted with Mega View III digital camera (Olympus Soft Imaging Solu-tions GmbH, Münster, Germany).

2.4. Determination of microbiological quality

For each treatment (C, P, FP and FC) and storage time, four meatsamples coming from independent PEF treatments were tested. Thenumber of total viable counts (TVCs) of the beef samples was assessedevery 2 days by adding known amount of diluent (0.1% w/v peptone,0.9% w/v NaCl) in a ratio of 1:5 to stomacher bags containing the beef(~70 g). The sample was then homogenised using a stomacher for2 min. After preparing appropriate dilutions, the resulting suspensionwas diluted, plated on plate count agar (Oxoid, Basingstoke, UnitedKingdom) plates in triplicate by spread plating and incubated at 25 °Cfor 72 h. All counts were expressed as log colony-forming units pergramme of meat (log CFU/g meat).

2.5. Study on lipid oxidation

2.5.1. Determination of lipid oxidation using TBARSLipid oxidation was evaluated using the 2-thiobarbituric

acid method (Nam & Ahn, 2003). Briefly, 5.0 g minced beefwas homogenised in 15 ml of deionised water using a polytron(model PT-MR 2100, Polytron®, Kinematica, AG, Littau-Lucerne,Switzerland) at 14,000 rpm for 30 s. The beef homogenate (1 ml)was transferred to a disposable test tube. A 50 μl aliquot of butylatedhydroxytoluene (BHT) (7.2% w/v in ethanol) and 2 ml thiobarbituricacid/trichloroacetic acid solution (20 mM TBA and 15% (w/v) TCA)were added. The mixture was vortexed for 30 s, incubated in a90 °C water bath for 30 min to develop colour, and then cooled inan ice-water bath for 10 min. The samples were centrifuged at1500 g and 5 °C for 15 min and the absorbance of the resultingupper layer was measured at 531 nm (Ultraspec 3300 Pro spectro-photometer Amersham Bioscience, Cambridge, England) against ablank prepared with 1 ml deionised water and 2 ml TBA/TCA solu-tion. The results are expressed as 2-thiobarbituric acid reactivesubstances (TBARS) in mg malondialdehyde (MDA) per kg of meatusing a standard curve constructed using tetraethoxypropane(TEP) and mean values are given for triplicate samples (n = 3).

2.5.2. Fatty acid analysisSamples were lyophilized for 48 h until dry and then a 25 mg sub-

samples were placed in 10 ml test tubes. Ten microliter of a tridecanoicacid in toluene (2 g/l) was added as an internal standard followed by490 μl toluene and 750 μl freshly prepared in 5% methanolic HCl. Thesamplewasmixed and the headspace of each tubewas filledwith nitro-gen. The tubes were sealed and placed in a water bath at 70 °C for 2 h.Tubes were cooled down to room temperature and afterwards 1 ml6% aqueous K2CO3 and 500 μl toluene were added and gently mixed.The mixture was centrifuged (1100 g for 5 min) and the organic phasewas removedwith a glass Pasteur pipette for analysis of fatty acidmeth-yl ester (FAME) content. The fatty acid peaks were identified and quan-tified by comparing with the retention time and peak area of fatty acidstandards (Supelco 37 comp. FAME Mix 10 mg/ml in CH2CL2, SupelcoCo., Bellefonte, USA). An internal standard was included as an internalreference before the extraction to determine the recovery of the fattyacids from each sample.

Please cite this article as: Faridnia, F., et al., Effect of freezing as pre-treatmuscles, Innovative Food Science and Emerging Technologies (2014), http:/

For fatty acid analysis, Shimadzu GC-17A gas chromatographyequipped with a FID and a FAMEWAX column (30 m × 0.32 mm ×0.25 μm, RESTEK, Inc., Austin, USA) was used. Nitrogen was used as acarrier gas. The pressure was set to 43 Pa, the flow rate was 7 ml/min,and the oven temperature was held for 5 min at 140 °C, increased to245 °C at 3.5 °C/min, and held for 3 min at this temperature.

2.6. Determination of volatile profile using headspace/HS-SPME analysis

Beef samples (1.5 ± 0.1 g) were placed in 10 ml flat bottom head-space vials fitted with a PTFE/silicone septum and crimp cap (SupelcoCo., Bellefonte, USA). The headspace vialwas heated using a plate heaterat 80 °C for 5 min. Volatile extraction by HS-SPME was carried out ac-cording to the modified method of Ma, Hamid, Bekhit, Robertson, andLaw (2013). The SPME fibre was preconditioned prior to analysis at250 °C for 30min. After equilibration at 45 °C for 5min, the volatile com-ponents in the samples were adsorbed onto a 50/30 μm layer ofdivinylbenzene–carboxen–polydimethylsiloxane (SupelcoCo., Bellefonte,USA) fibre that was exposed to the sample headspace for 30 min.

The Trace GC Ultra (Thermo Scientific, Waltham, MA, USA) wasequippedwith a DSQ seriesmass spectrometer (Thermo Scientific,Wal-tham,MA, USA). The GC–MSwas installed with a VF-5ms low bleed/MSfused-silica capillary column (5%-phenyl-95%-dimethylpolysiloxanephase, 60 m × 0.25 mm × 0.25 μm) (Phenomenex, Torrance, CA, USA).Helium was used as carrier gas with a constant flow rate of 1.5 ml/minin the GC–MS. Chromatographic conditions were as follows: the ovenwas held for 3 min at 40 °C, heated to 250 °C at 5 °C/min, and held for3 min at this temperature. The mass spectrometer was operated in theelectron impact mode with a source temperature of 200 °C, an ionizingvoltage of 70 eV, and the transfer line temperature was 250 °C. Themass spectrometer scanned masses from 48 to 400 m/z at a rate of3.41 scan/s.

Peak identification was carried out by comparison of mass spectrawith spectra in the NIST/EPA/NIH Mass Spectral Database (National In-stitute of Standards and Technology, Gaithersburg, MD, Version 2.0a,2002, USA), or NIST web book (http://webbook.nist.gov/chemistry/).Ten microlitre of 1,2-dichlorobenzene (in methanol, 0.01 ppm) wasused as an internal standard for qualitative identification of the volatilecompounds. To confirm the identity of volatile compounds, the reten-tion index (RI) was calculated for each volatile compound using the re-tention times of a homologous series of C7 to C30 n-alkanes (1000 μg/mlin hexane from Supelco, Bellefonte, USA) and comparing the RI withcompounds analysed under similar conditions. The approximate quan-tities of the volatiles were estimated by comparing their peak areaswith that of the 1, 2-dichlorobenzene internal standard using a responsefactor of 1.

2.7. Statistical analysis

Analysis of variance (ANOVA) was performed on the experimentaldata using the Minitab Statistical Software Version 16 (Minitab Inc.,State College, PA, USA). For microbiological quality and lipid oxidation,datawere evaluated by a Student's t-test. ANOVAandDuncan'sMultipleRange Tests, at the 0.05 level of significance, were used for comparingthemeans to find out the effect of storage period. The chromatographicdatawere collated usingMicrosoft Office Excel 2007. A twoway analysisof variancewas carried out on fatty acid profiles and volatile compoundsfor each treatment.When theANOVAwas significant (p values less than0.05), means were separated by a pairwise comparison using theFisher's least significant difference test. The volatile compounds for alltreatments were further analysed by a principal component analysis(PCA) using the XLSAT MX software release 2010. Individual carcasseswere used as the experimental units and the reported values are themean of 6 replicate carcasses (n = 6).

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

Table 1Effects of PEF treatment on purge loss, cooking loss and shear force of fresh and frozen–thawed beef semitendinosusmuscles.

Treatments Purge loss(%)

Cooking loss(%)

Shear force(kgF)

Fresh control (C) 2.61 ± 0.77a 24.35 ± 1.70a 5.85 ± 0.80b

Fresh PEF treated (P) 4.66 ± 1.02b 24.58 ± 2.74a 5.77 ± 0.93b

Frozen–thawed control (FC) 4.84 ± 2.22b 25.43 ± 2.64a 6.01 ± 0.61b

Frozen–thawed PEF treated (FP) 6.67 ± 2.09c 25.14 ± 2.41a 4.50 ± 0.44a

Within each column, means that have different superscripts are significantly different atp b 0.05.The values are least square means ± standard deviation (n = 6)

4 F. Faridnia et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

3. Results and discussion

3.1. Effect of PEF on the physical changes of meat muscle

In this study, the temperature, electrical conductivity and pH of themeat samples before and after PEF treatment were monitored. The ini-tial temperature of meat was kept at 4 °C as this temperature is com-monly used in the meat industries to avoid quality deteriorationduring the process. However, it should be taken into account that PEF

A

z-lines

IAH

Fig. 1. TEMmicrographs of beef semitendinosus (ST) muscles showing rupture of myofibrils alobefore PEF processing; intact myofibrils. (b) Pulsed electric field (PEF) treated: magnificatiofrozen–thawed PEF treated samples are connective tissues around muscle cells indicating jagg

Please cite this article as: Faridnia, F., et al., Effect of freezing as pre-treatmuscles, Innovative Food Science and Emerging Technologies (2014), http:/

treatment at 4 °C could lower the electroporation sensitivity since thephospholipids in the cell membrane becomes more packed or orderedstate at low temperatures (lower fluidity) making the cells less suscep-tible to pore formation by PEF. PEF treatment leads to a temperature in-crease of meat samples ranging between 10 and 12 °C. Previous workon PEF-treated beef ST muscles has also shown a 5 to 30 °C increase intemperature depending on the electric field strength and the frequencyused (O'Dowd et al., 2013). On average, the electrical conductivity andpH of the untreated beef sample before subjected to PEF treatmentwere 10.63 ± 1.5 mS/cm and 5.70 ± 0.16, respectively. The electricalconductivity of beef samples generally increased after PEF treatment(13.01 ± 0.22 mS/cm), while the pH decreased to 5.62 ± 0.11. Themeasurement of electrical conductivity has been used as an indicationfor the transport of ionic species in the cell membrane material andwas related to PEF induced permeabilising effect on cell membrane(Lebovka, Bazhal, & Vorobiev, 2002). Changes in conductivity after ap-plication of PEF treatmentmight be related to osmotic flow and redistri-bution of moisture inside the sample (Lebovka, Bazhal, & Vorobiev,2001).

Frozen/thawed beef (FC) samples had higher purge loss than fresh(C) samples (p b 0.01). PEF treatment significantly (p b 0.001) in-creased the purge loss after 7 days of vacuum ageing for both fresh

B

Jagged edges

ng with the z-lines. (a) Control: magnification 17,500×. Frozen–thawed beef ST musclesn 17,500×. Frozen–thawed beef ST muscles after PEF processing. Visible white areas ined edges and myofibril separation from z-lines.

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

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and frozen-thawed samples but did not affect the cooking loss (Table 1).It has been reported that about 85% of the muscle moisture is locatedwithin the myofibrils. The increased purge loss due to freezing andPEF in the present study can be associatedwith changes of themyofibrilstructure that ultimately reduces their ability to hold moisture(Huff-Lonergan & Lonergan, 2005; Pearce, Rosenvold, Andersen, & Hop-kins, 2011).

No significant effect on cook loss was observed as a result of freezingand thawing (Table 1), which is in agreement with the results reportedby Pietrasik and Janz (2009) and Smith, Spaeth, Carpenter, King, andHoke (1968). Smith, Carpenter, and King (1969) also showed thatcooking losses did not increase significantly as a result of freezing at –23 °C. Loss of moisture due to cooking has been reported not to differsignificantly between fresh and frozenmeat samples, aswell as for sam-ples frozen and thawed at different rates (Leygonie, Britz, & Hoffman,2012a; Vieira, Diaz, Martínez, & García-Cachán, 2009).

Numerous studies have shown that post-rigour muscle changes intenderness during freezing and frozen storage are dependent on severalfactors including species, pH, ageing, rate of freezing, and frozen storagetime and temperature (Hopkins, 2004; Pietrasik & Janz, 2009). It hasbeen claimed that freezing improved tenderness in beef without ageing,and hypothesized that improved tenderness was a direct consequenceof physical disruption of muscle cells caused by intracellular ice crystalformation. Histological analysis also showed that tenderness improve-ment was proportional to intracellular ice formation that functionedto rupture muscle fibres and to break and stretch connective tissue(Hiner, Madsen, & Hankins, 1945). Freezing and thawing cause damageto the ultrastructure of the muscle cells with the ensuing release of mi-tochondrial and lysosomal enzymes, haem iron and other pro-oxidants.However, the present study found no differences in shear force valuesbetween frozen–thawed (FC) and fresh (C) samples (Table 1). No signif-icant changes in shear force may have been due to the loss of fluid dur-ing thawing that resulted in less water available to hydrate the musclefibres leading to a greater quantity of fibres per surface area and morecompact matrix that required more force to shear.

In contrast to the effect of PEF treatment on the tenderness of freshbeef samples, PEF treatment of frozen-thawed beef samples (FP) result-ed in tenderisation, as shown by a significant decrease in the averagemaximum shear force by 20.13% (p b 0.01) compared to frozen-thawed control (FC) (Table 1). The PEF treatment after freezing−thawing process would lead to additional physical damage of themuscle. Fig. 1 shows transmission electron micrographs for beefST muscles. Frozen–thawed samples (FC) had clear district A-bands, I-bands and Z-lines and the ultrastructure of samples had intactmyofibrils as shown in Fig. 1A whereas myofibrils are ruptured alongthe z-lines due to PEF treatment (Fig. 1B). Frozen–thawed PEF treatedsamples showed significantly less myofibril organization, exhibiting Z-line fractures and degraded myofibril structure, and perhaps an

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indicative of tenderization. In many regions, Z line–I band junctionswere ruptured and amorphous. Thesemicrostructural changes could af-fect the tenderness, water binding andwater holding capacities ofmeat.From the chewiness point of view, it is beneficial asmyofibril fragmentsare able to move along with the ruptures (Siró et al., 2009). It has beenstated that ruptures inmyofibrils and the breakdownof cells can also in-crease the activities of proteolytic enzymes in meat (Dolatowski, 1988).Klonowski, Heinz, Toepfl, Gunnarsson, and Þorkelsson (2006) investi-gated the influence of PEF treatment on fish muscles and found thattreating frozen cod loins with PEF (2.9 kV/cm, 90 pulses) resulted inmore porous tissues which had greater water uptake andwater holdingproperties compared to untreated cod. A PEF treatment of 1.36 kV/cmand 40 pulses has also been reported to cause a reduction in cell sizeand visible gapping between cells of chicken and salmon muscles(Gudmundsson & Hafsteinsson, 2005). Our previous work on PEF(0.3–0.6 kV/cm, up to 35 kJ/kg, 1–50 Hz) treated beef muscles hasalso suggested significant microstructural changes in meat tissue.Cryo-SEM results showed that themeat structure becomesmore porousas the electric field strength increased. In that study, however, no effectof PEF on tenderness of beef LT muscles was observed (Faridnia et al.,2014).

Beside the tenderization due to physical disruption of the sarcomereand the breaking of the myofibrils, PEF treatment in combination withageing seems to have an additional tenderising effect on meat due toan increased rate of proteolysis. PEF treatment could contribute tomyo-fibril degradation by creating the conditions where protease enzymes(e.g. cathepsins B and L) can be feasibly released from the lysosomesand also potentially accelerate the release of Ca++ ions leading to acti-vation of the calcium-activated protease μ-calpain early postmortem aswell as stimulating the glycolysis process that are required for accelerat-ed proteolysis and meat tenderisation (Bekhit, van de Ven, Fahri, &Hopkins, 2014). It is hypothesized that PEF-induced cell perme-abilization enhances the release and the proteolytic activity of endoge-nous enzymes responsible for textural changes in meat that need tobe further investigated. PEF can lead to direct interactions between en-zymes and their substrates leading to faster rates of reactions. Therefore,thawed meat would have provided a more susceptible environment toPEF treatments, and could explain the observed increased tendernessof frozen-thawed PEF treated meat samples (4.50 kgF) compared totheir respective untreated controls (6.01 kgF).

3.2. Effect of PEF-induced changes on the microbiological quality ofbeef muscle

Thenumber of bacteria on the untreated or treated beefmuscleswasdetermined immediately following the treatment and during 16 days ofstorage at 4 °C. Pseudomonas, Enterobacteriacea and lactic acid bacteriaare reported to be mainly responsible for meat spoilage (Coma, 2008).

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ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

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0.4

0.6

0.8

1

1.2

TBAR

S (m

g/kg

mea

t)

Storage time (Days)

Control

PEF

Fig. 3. Evolution of lipid oxidation marker (TBARS) in frozen-thawed PEF treated andfrozen–thawed control beef samples during storage. Different letters at a sampling pointdenote significant differences of means.

6 F. Faridnia et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

In the current study the initial total viable count (TVC) was 2.69–2.77 log CFU/g, which indicated a very good meat quality. Bacterialnumbers did not significantly increase over thefirst 7 days at 4 °C. How-ever an increase in growth occurred from day 8 and reaching7.2 log CFU/g and 6.89 log CFU/g after 16 days of storage for PEF treatedand non-treated fresh meat samples (P and C) respectively (Fig. 2a). Arapid increase in bacterial numbers after 8 days of storage at 4 °C wasalso found for frozen-thawed (FC) and its PEF treated samples (FP)(Fig. 2b). It is evident that sufficientmicrobial reduction can be achievedusing PEF as an alternative to traditional preservation techniques. How-ever, the degree of inactivation is dependent on the intensity of thepulses in terms of field strength, energy and number of pulses appliedon the microbial strain and on the properties of the food matrix underinvestigation (Toepfl, Heinz, & Knorr, 2007). Furthermore, as real meatsamples are heterogeneouswith areas of different electrical resistivities,the effects of PEF treatment can be varied, as some areaswill be untreat-ed while others over-treated in such materials (Barsotti & Cheftel,1999). As shown in Fig. 2, the beef samples which had been frozen (FCand FP) had a 2 log unit increase in bacterial numbers by the end ofthe storage period (Fig. 2B) compared to the non-frozen samples(Fig. 2A). Although frozen storage is a general preservation methodused to control or reduce biochemical changes inmeat that occur duringstorage, it does not completely inhibit chemical reactions that can leadto quality deterioration (Fan et al., 2009). The moisture lost duringthawing provides an excellent medium for microbial growth becauseit is rich in proteins, vitamins and minerals (Leygonie et al., 2012b). Inthis study, frozen/thawed PEF treated samples (FP) during subsequent4 °C storage showed significantly higher microbial counts during logphase than untreated samples (FC) (5.02 log CFU/g compared to3.10 log CFU/g). This phenomenon can be explained by the increasedpurge loss observed for these samples after 7 days of refrigerated stor-age. Apart from the log phase, no differences in the TVCs were detectedbetween PEF treated (FP) and non-treated samples (FC) at the end ofstorage. It has been claimed that the fermentation of raw sausages (sa-lami type) can be accelerated by improving the availability of intracellu-lar liquid for fermenting cultures and that the time required to lower thepH, by lactic acid formation, was reduced after PEF treatment (2 kV/cm,10 kJ/kg) of the minced meat (Raso & Heinz, 2006).

3.3. Effect of PEF-induced changes on lipid oxidation during storage

The TBARS assay was used in the present study to measure the levelof lipid oxidation, which is one of the main factors affecting processedmeat quality. Akamittath, Brekke, and Schanus (1990) and Hansenet al. (2004) have been reported that freezing−thawing could acceler-ate lipid oxidation during shelf-life study, as also found in this study.When PEF treatment is applied to frozen-thawed beef samples, lipid ox-idation of FP samples significantly enhanced as indicated by higherTBARS values (p b 0.05) than non-PEF treated (FC) samples (Fig. 3).The frozen-thawed PEF treated beef samples had the largest accumula-tion of lipid oxidation (0.96 mgMDA/kgmeat) at the end of storage pe-riod. A TBARS value more than 1.0 mgMDA/kg meat has been reportedas an indication of increased rancid flavour and odour determined bysensory panellists (Kim, Frandsen, & Rosenvold, 2011; Zakrys, Hogan,O'sullivan, Allen, & Kerry, 2008). Oxidative deterioration of musclefoods can be promoted during frozen storage due to the formationand the growth of ice crystals by producing mechanical damage inmembrane and other rigid cell structures that would facilitate the expo-sure of pro-oxidants with susceptible molecules and by increasing sol-ute concentration, including pro-oxidants in the unfrozen phase(Utrera, Parra, & Estévez, 2014; Zaritzky, 2012). Benjakul and Bauer(2001) also found that freezing and thawing of muscle tissue resultedin accelerated TBARS accumulation and attributed this finding to thedamage of cell membranes by ice crystals and the subsequent releaseof pro-oxidants, especially the haem iron. There is evidence indicating

Please cite this article as: Faridnia, F., et al., Effect of freezing as pre-treatmuscles, Innovative Food Science and Emerging Technologies (2014), http:/

that lipid oxidation takes place primarily at the cellular membranelevel (Thanonkaew, Benjakul, Visessanguan, & Decker, 2006).

These results suggest that under the described experimental condi-tions, PEF treatment makes frozen–thawed meat more vulnerable to-ward lipid oxidation during ageing due to the fatty acids exposed topro-oxidants such as iron released from muscle cells. In previous stud-ies, increased TBARS values for pressure treated (≥400 MPa at 20 and40 °C) beef M. longissimus dorsi muscles (Ma, Ledward, Zamri, Frazier,& Zhou, 2007) andminced pork (Cheah& Ledward, 1996) have been re-ported, and attributed to pressure-induced protein denaturation whichleads to the release of the free-radicals, that catalyse oxidation(McArdle, Marcos, Kerry, & Mullen, 2010). Increases in lipid oxidationhave also been attributed to the release of metal ions from iron com-plexes, promoting auto-oxidation of lipids in pressurised meat(Chevalier, Le Bail, & Ghoul, 2001). However, to our knowledge, this isthefirst paper reporting the effect of PEF treatment on the oxidative sta-bility of beefmuscles. Nevertheless, in general, aseptic packaging is con-sidered as the most appropriate way of packaging for PEF processedfoods. The modified atmosphere packaging (MAP) which limits oxygenin the headspace could be applied as a complement to PEF to reduce ox-idation of PEF processed food products (Galić, Ščetar, & Kurek, 2011).

3.4. Effect of PEF-induced changes on fatty acid composition

Since PEF treatment enhanced lipid oxidation during ageing, thefatty acid composition of meat after PEF is questioned because it is ofimportance for the sensory and nutritional characteristics of the meat.The fatty acid composition ofmuscle affects its oxidative stability duringprocessing, ageing and retail display, the polyunsaturated fatty acids inphospholipid being liable to oxidative breakdown at this stage (Woodet al., 2008). To the best of our knowledge, no research has been doneso far to study the effect of PEF on beef fatty acid profiles.

The fatty acid composition of frozen and fresh beef, with and with-out PEF treatment is summarized in Table 2. The most abundant fattyacids in beef were palmitic acid (C16:0), stearic acid (C18:0) and oleicacid (C18:1n9c). It has been reported that beef samples have highercontents of C16:0 and C18:0 fatty acids and less C18:2 and C18:3 fattyacids (Mohamed, 2005). The concentrations of myristic acid (C14:0),palmitic acid (C16:0), palmitoleic acid (C16:1n7), oleic acid(C18:1n9c) and linoleic acid (C18:2n6c) were significantly lower in FPsamples (p b 0.05) compared to the control sample. The total monoun-saturated fatty acid content of phospholipids in buffalo meat has beenreported to decrease during refrigerated and frozen storage, with a sig-nificant decreases in oleic and linoleic acids (Rao& Kowale, 1991). Salih,Price, Smith, and Dawson (1989) also reported that the proportion of

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

Table 2The fatty acid composition of beef with and without PEF treatments (mg/100 g dry meat)after 7 days of storage.

Fatty acids C P FC FP

C14:0 16.39b 15.08ab 14.68a 14.52a

C16:0 88.37b 72.44ab 70.69ab 67.46a

C17:0 7.25 6.84 6.69 6.45C18:0 57.40 51.03 47.64 44.40C16:1n7 15.30b 12.17a 11.86a 12.40a

C18:1n9c 143.86b 112.20ab 111.47ab 103.30a

C18:2n6c 34.14b 32.95ab 34.40b 31.00a

C20:2n6 4.86 4.90 5.21 5.12C20:3n6 6.14 6.14 6.19 6.12C18:3n3 10.44 10.27 10.27 9.84C20:5n3 10.43 10.18 10.42 10.48SFA 169.41b 145.38ab 139.70ab 132.82a

MUFA 159.17b 124.37ab 123.33a 115.69a

PUFA 44.58b 43.22ab 44.67b 40.84a

Total 394.58b 334.18ab 329.52ab 311.08a

Total n-6 45.13 43.99 45.79 42.24Total n-3 20.87 20.45 20.69 20.33n-6/n-3 2.17 2.16 2.22 2.09PUFA/SFA 0.31 0.31 0.33 0.32

C: control sample; P: PEF treated sample; FC: frozen control sample; FP: frozen-thawedPEF treated sample.SFA = C14:0 + C16:0 + C17:0 + C18:0; MUFA = C16:1n7 + C18:1n9c;PUFA = C18:2n6c + C18:3n3.n − 6 = C18:2n6c + C20:2n6 + C20:3n6; n − 3 = C18:3n3 + C20:5n3.a,bMean of fatty acids with different treatments within the same row differs significantlyusing Fisher's least significant difference (p b 0.05).

7F. Faridnia et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

linoleic acid in turkey breast meat decreased significantly during frozenor refrigerated storage (Salih et al., 1989). During frozen storage of legmuscle of ready-to-cook chicken meat products, the unsaturated fattyacids decreased, with the levels of linolenic and palmitic acids, veryquickly (Miteva & Bakalivanova, 1987).

In view of nutritional guidelines, the ratios of polyunsaturated fattyacids and saturated fatty acids (PUFA/SFA) and omega 6/omega 3 (n−6/n−3) are important. Nutritional guidelines have recommendedthat the PUFA/SFA ratio should be above 0.4–0.5 (Wood et al., 2008).However, Habeanu et al. (2013) reported the PUFA/SFA and n−6/n−3 ratios for semitendinosusmuscle of Normand cows fed a diet sup-plementedwith linseed or rapeseed, ranging from 0.18 to 0.22 and from3.13 to 4.19 respectively. Calabrò et al. (2014) also reported that thePUFA/SFA ratios for semitendinosusmuscle in ItalianMediterranean Buf-falo young bulls to be 0.36. The combined effects of high pressure pro-cessing and temperature on meat quality attributes of bovineM. pedtoralis profundus were also assessed and the PUFA/SFA ratioswere reported to be 0.35 or 0.37, at 20 °C and 40 °C respectively(McArdle et al., 2010). The PUFA/SFA ratios in the present study werevery similar to those found in these studies, ranging from 0.31 to 0.33,for all samples. No differences in intramuscular fat content (p N 0.05)among treatments were detected (data not shown). The n6/n3 ratio isalso considered important to human health and has a recommendedratio of less than 4 (McArdle et al., 2010). Thus, in relation to the effectof PEF on fatty acid composition, the analysis performed showed no sig-nificant differences in the PUFA/SFA and n6/n3 ratios of the beef meat.Moreover, the n6/n3 ratios in all treated samples remained within therecommended levels, thus indicating no alteration of the nutritionalvalue of beef fatty acids as a consequence of PEF.

3.5. Effect of PEF-induced changes on volatile profile

As mentioned earlier, the secondary products of lipid oxidation thatis measured using the TBARS test can cause rancidity and off-flavours.Therefore it is necessary to investigate the effect of freezing and PEF

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treatment on the volatile compounds in beef. In order to illustrate differ-ences between each treatment on the basis of individual volatile com-pounds, a PCA was carried out (Fig. 4). The PCA described 48.75% and18.32% of the total variation of factor 1 (F1) and factor 2 (F2), respec-tively. All FC and FP samples (except FC5) had positive scores andwere separated from C and P samples that had negative scores alongF1. FC and FP samples corresponded to high positive loadings of n-pentanal (3), hexanal (4), heptanal (5), benzaldehyde (6), octanal (7),and nonanal (8). These volatile compounds were significantly higher(p b 0.05) in frozen samples (FC and FP) as shown in Table 3. Lipid ox-idation is the major form of deterioration seen in frozen meats and re-sults in the formation of toxic compounds, such as malondialdehydeand cholesterol oxidation products, as well as the accumulation of vola-tile carbonyls, alcohols, and acids, which are responsible for off-flavours(Utrera & Estévez, 2013). Min, Nam, Cordray, and Ahn (2008) reportedthat beef meat wasmore prone to lipid oxidation when frozen beef pat-ties were thawed and subsequently subjected to cooking. Hexanal,which originates mainly from linoleic and arachidonic acids (Martın,Timon, Petrón, Ventanas, & Antequera, 2000), can lead to a faintly rancidaroma and it is considered as a good indicator for oxidation. Hexanallevels are directly proportional to TBARS levels and are inversely pro-portional to flavour acceptability (Calkins & Hodgen, 2007). In additionto rancid fragrance, hexanal has also been described as the source offruity or fatty aromas (Kang et al., 2013). Other volatile aldehydessuch as heptanal, octanal, and nonanal, arise mainly from the oxidationof oleic acid, which is themain fatty acid in beef fat (Machiels, Istasse, &Van Ruth, 2004).

It was also found that 3-methylbutanal (1) and 2-methylbutanal(2) were significantly higher (p b 0.05) in all FC samples (except FC5)and had high positive scores along F1. 3-Methylbutanal and 2-methylbutanal can be formed from Strecker degradation (Mottram,1998), which contributes leucine for the formation of 3-methylbutanal. It has been hypothesized that 3-methylbutanal is prob-ably one of the compounds responsible for the roasted beef flavour(Machiels et al., 2004). Koutsidis et al. (2008) reported that freeamino acids, such as leucine, isoleucine, serine, threonine, valine andphenylalanine, increased during conditioning, and are important forthe formation of Strecker aldehydes, such as 2- and 3-methylbutanal,and other aroma compounds such as pyrazines.

Dimethyl disulfide (11)was found at higher levels in FC and FPmeatsamples. Mottram (1998) reported that sulphur containing volatilecompounds were derived from the degradation of sulphur-containingamino acids. Koutsidis et al. (2008) concluded that the concentrationof cysteine increased threefold during storage and that this might alsobe responsible for the increased flavour intensity in aged meat.Sulphur-containing compounds can be formed from the reaction be-tween cysteine and ribose when meat is cooked and these compoundsplay an important role in the flavour of cooked beef, since some hetero-cyclic sulphur compounds have been described as possessing meat likearomas (Mottram, 1998). In addition, the 2,3-octanedione (16) contentof the FP and FC samples were significantly higher (p b 0.05) than thatof C and P. Stetzer, Cadwallader, Singh, Mckeith, and Brewer (2008) re-ported that 2,3-octanedione had a warmed over, oxidized fat flavourwhich is derived from lipid oxidation. In this study, the observed higherlipid oxidation in FP samples could explain the higher 2,3-octanedione(16) content of these samples.

2-Acetylthiazole (12) was significantly lower (p b 0.05) in the FPsamples. Yu, Wu, and Ho (1994) reported 2-acetylthiazole as the pre-dominant volatile interaction products of alliin and glucose. The contentof 2-ethyl-1-hexanol (14) also decreased significantly (p b 0.05) afterPEF treatments. 2-Ethyl-1-hexanol was reported in Irish beef meats(Machiels, Van Ruth, Posthumus, & Istasse, 2003) and described asresin, flower and green aromas (Calkins & Hodgen, 2007).

These findings clearly indicate that PEF and freezing affect the vola-tile profile of meat. The degradation products derived from lipid andprotein oxidation were evident in this study. Hence, the changes in

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

Fig. 4. Bi-plot of volatile compounds in cooked fresh, frozen, PEF treated and frozen-thawed PEF treated beef samples. The volatile compounds are numbered the same as in Table 2, C:control; P: PEF treated; FC: frozen control; FP: frozen-thawed PEF treated (1, 2, 3, 4, 5, 6 denote different animals used in this trial).

8 F. Faridnia et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

volatile profiles could affect the cooked meat flavour. Since flavour andaroma are the attributes most easily detected and assessed by con-sumers as either being acceptable or not, further study using sensoryanalysis needs to be undertaken to determine whether these changescan be detected by consumers.

Table 3Volatile compounds in cooked fresh and frozen, untreated and frozen-thawed PEF treated bee

No. Volatile compounds RId Identificatione Types Fresh

Treatmentf C

Aldehydes:1 3-Methylbutanal 650 MS + RI 0.075a

2 2-Methylbutanal 659 MS + RI 0.058a

3 n-Pentanal 695 MS + RI 0.096a

4 Hexanal 801 MS + RI 0.180a

5 Heptanal 901 MS + RI 0.088a

6 Benzaldehyde 968 MS + RI 0.122a

7 Octanal 1005 MS 85% 0.135a

8 Nonanal 1106 MS 85% 0.722a

Nitrogen and sulphur compounds:9 Carbon disulfide b600 MS 85% 0.083a

10 2-Propanethiol b600 MS 85% 0.155a

11 Dimethyl disulfide 719 MS + RI 0.190ab

12 2-Acetylthiazole 1024 MS + RI 0.189b

13 Methional 911 MS + RI 0.035a

Alcohols:14 2-Ethyl-1-hexanol 1032 MS + RI 0.058b

Ketones:15 2-Pentanone 683 MS + RI 0.091a

16 2,3-Octanedione 986 MS + RI 0.021a

Alkanes:17 Toluene 763 MS 85% 0.056a

18 Decane 998 MS + RI 0.097ab

a,b,cDifferent letterswithin the same row (different treatmentswith the same volatile compoundcolumn, was calculated in relation to the retention time of n-alkane (C7-C30) series. eMS, tentatMS + RI, mass spectrum identified using NIST mass spectral database and RI agree with liteC: control; P: PEF treated; FC: frozen control; FP: frozen-thawed PEF treated. g: ns, non-signific⁎ p b 0.05.⁎⁎ p b 0.01.⁎⁎⁎ p b 0.001.

Please cite this article as: Faridnia, F., et al., Effect of freezing as pre-treatmuscles, Innovative Food Science and Emerging Technologies (2014), http:/

4. Conclusions

This study provides valid evidence that pulsed electric field (PEF)processing affects microstructure of beef tissue, which considerably in-fluences its water holding capacity and textural properties. PEF

f samples after storage for 7 days.

Frozen Significance of effectg

P FC FP Treatments Types Treatment ∗ Types

0.152ab 0.299c 0.229bc ns ⁎⁎⁎ ⁎⁎⁎

0.090ab 0.202c 0.141bc ns ⁎⁎⁎ ⁎⁎

0.093a 0.279b 0.236b ns ⁎⁎⁎ ⁎⁎⁎

0.160a 0.389b 0.409b ns ⁎⁎⁎ ⁎⁎⁎

0.089a 0.183c 0.135b ns ⁎⁎⁎ ⁎⁎⁎

0.107a 0.209b 0.198b ns ⁎⁎⁎ ⁎⁎⁎

0.139a 0.295c 0.207b ns ⁎⁎⁎ ⁎⁎⁎

0.711a 1.154b 0.811a ns ns ⁎⁎

0.143ab 0.234b 0.140ab ns ⁎ ⁎

0.131a 0.887c 0.717b ns ⁎⁎⁎ ⁎⁎⁎

0.131a 0.253b 0.238b ns ⁎⁎ ⁎

0.205b 0.203b 0.131a ns ns ⁎

0.034a 0.068b 0.037a ns ns ⁎

0.030a 0.093c 0.059b ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎

0.080a 0.335b 0.295b ns ⁎⁎⁎ ⁎⁎⁎

0.019a 0.054b 0.063b ns ⁎⁎⁎ ⁎⁎⁎

0.048a 0.057a 0.101b ns ⁎ ⁎⁎⁎

0.083a 0.137c 0.113bc ns ⁎⁎⁎ ⁎⁎⁎

s) differ significantly using Fisher's least significant difference (p b 0.05). dRI on a VF-5 msive identification by comparison of mass spectrumwith NIST library spectrum (over 85%);rature values (Ma, Hamid, Bekhit, Robertson, & Law, 2012). fRatio to internal standard;ant.

ment prior to pulsed electric field processing on quality traits of beef/dx.doi.org/10.1016/j.ifset.2014.09.007

9F. Faridnia et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

treatment leads to a significant decrease in shear force values of frozen–thawed beef samples after 7 days of storage while no differences werefound for the fresh PEF treated samples. These results imply that bothapplied PEF conditions and sample pre-treatment (fresh or frozen–thawed) should be considered when determining the effect of PEF onmeat tenderization. The results of this study could help to explain thecontradictory findings of PEF effects on meat tenderization currentlyavailable in the literature. It is clear that PEF treatment also affects theoxidative stability of frozen–thawed meat and would enhance lipid ox-idation. This study also clearly shows that even though PEF treatment offrozen-thawed samples results in different volatile profiles of beef sam-ples, this processing technology does not change the fatty acid compo-sitions of treated meat samples. Finally, total viable counts of samplesPEF treated and stored at 4 °C for 16 days further demonstrated that ap-plied PEF caused neither significant inactivation nor favourable condi-tions (except log phase) for themicrobial proliferation post-processing.

Declaration of conflicting interests

The authors declare that there is no conflict of interest.

Acknowledgements

The research leading to these results has received funding fromPriming Partnership University of Otago. The authors acknowledge theUniversity of Otago Doctoral Scholarship towards the PhD study ofFarnaz Faridnia. The technical assistance of Ian Ross, Nerida Downes,Michelle Petrie, and Sarah Henry, from the Department of Food Science,University of Otago; and Jennifer Kwan, Stephanie Smithson, GrantPearson, Rebecca Coote, Philip Shuker, and Jason O'Connell, from SilverFern Farms is greatly appreciated. The authors also acknowledge the fa-cilities as well as the scientific and technical assistance from the staff atthe Otago Centre for Electron Microscopy (OCEM).

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