Proteolysis of Cheese Slurry for Pef Milk

ORIGINAL PAPER

Proteolysis of Cheese Slurry Made from Pulsed ElectricField-Treated Milk

Li Juan Yu & Michael Ngadi & Vijaya Raghavan

Received: 27 March 2009 /Accepted: 24 February 2010 /Published online: 31 March 2010# Springer Science+Business Media, LLC 2010

Abstract Raw milk cheeses have unique flavor and texturecharacteristics not obtainable in cheeses made frompasteurized milk. However, cheeses made from pasteurizedmilk are widespread, primarily for public health reasons.Pulsed electric field (PEF) treatment as a non-thermalpasteurization method has shown its ability to keep theflavor and natural characteristics of food samples intact,thus providing advantage over conventional heat process-ing. In this study, PEF treatment was performed in acontinuous treatment chamber, consisting of two parallelstainless steel electrodes separated by a 50-mm-thickinsulator. A 30-kV pulse generator was used to deliver bi-polar square waveform electric field to milk sample. Pulsewidth was 2 µs; pulse frequency was 2 Hz, and up to 120pulses were applied. Cheese curds were made from rawmilk, pasteurized milk, and PEF-treated milk, and theirproteolysis processes were compared using curd slurryincubated at 30 °C for 5 days. The profiles of water-solublepeptides were measured using an reversed-phase high-performance liquid chromatography (RP-HPLC) system.The concentration of free amino acids was measured byCd–ninhydrin method. Results indicated that PEF-treatedmilk has intermediate proteolysis profiles between raw milkand pasteurized milk in terms of peptide and free aminoacid concentration. The results showed the potential ofmaking high-quality cheeses by PEF treatment withoutsacrificing the natural characteristics of the cheeses.

Keywords Pulsed electric field . Proteolysis .

Cheese curd slurry

Introduction

Raw milk cheeses possess unique flavor and texture nottypically obtainable in cheeses made from pasteurized milk.However, raw milk cheeses have been involved in the cheese-related foodborne illness outbreaks. Pasteurization of milk canbe effectively achieved by heating using appropriate temper-ature–time schemes (Fox et al. 2000). However, apart frombacterial inactivation, heat treatment can adversely affect theflavor, taste, and nutrients of the product. In contrast topasteurization, microfiltration of milk eliminates a great partof the indigenous microflora without heat-induced changesin enzymes. However, microfiltration can only be applied toskim milk (Beuvier and Buchin 2004). Non-thermal pasteur-ization, such as pulsed electric field (PEF), is anotherpossible alternative for milk pasteurization.

PEF involves the application of high voltage pulses atrelatively low temperature to a food placed between twoelectrodes for very short time (normally less than 1 s). Agreat number of researches (Dunn and Pearlman 1987; Qinet al. 1995; Martin et al. 1997; Li et al. 2003; Sepulveda etal. 2005; Shamsi et al. 2008; Sampedro et al. 2009;Walkling-Ribeiro et al. 2009) have demonstrated thepossibility of pasteurizing milk by PEF treatment withoutsacrificing its quality. Dunn (1996) reported that milktreated with PEF suffered less flavor degradation whencompared with raw milk. The author proposed thepossibility of manufacturing dairy products such as cheeseusing PEF-treated milk. Sepulveda-Ahumada et al. (2000)evaluated the quality of cheese produced from PEF-treatedmilk in terms of sensory and texture evaluation and

Part of this paper has been presented at the CSBE 2006 AnnualConference, July 16–19, 2006.

L. J. Yu :M. Ngadi (*) :V. RaghavanDepartment of Bioresource Engineering, McGill University,21111 Lakeshore Road,Ste-Anne-De-Bellevue, QC, Canada H9X 3V9e-mail: [email protected]

Food Bioprocess Technol (2012) 5:47–54DOI 10.1007/s11947-010-0341-5

compared with cheese made from heat-pasteurized milk.They claimed that, using milk pasteurized by PEF, theobtained cheese appeared to have improved quality. Xianget al. (2009) reported that PEF induced some structuralmodifications in the structure of whey protein isolateswhich could affect their functionality.

Knowledge of PEF effects on major cheese-making steps,such as coagulation and ripening, are crucial to developinghigher-quality cheeses made from PEF-pasteurized milk.Effect of PEF treatment on rennet coagulation properties ofmilk has been studied by Yu et al. (2009). Results indicatedthat, in most cases, PEF-treated milk showed better rennet-ability compared with thermally pasteurized milk. However,very little work has been reported so far about PEF effects oncheese ripening properties.

Cheese ripening involves a complex series of biochem-ical events, which lead to the characteristic taste, flavor, andtexture of each cheese variety. Major biochemical changesoccurring in cheese ripening include proteolysis, glycolysis,and lipolysis. Many cheese ripening studies have beenfocused on the proteolysis process due to its importance incheese texture and flavor development (Farkye et al. 1995;O’Shea et al. 1996; Albenzio et al. 2001; Benfeldt andSorensen 2001; Verdini et al. 2003).

Proteolysis involves the degradation of the casein matrix toa range of peptides and free amino acids. It has both direct andindirect roles on the formation of texture and flavor of cheeses,although other biochemical changes such as lipolysis are alsoimportant in the development of flavor compounds in cheeses.Peptides may have a direct impact on cheese flavor. However,the major role of proteolysis in cheese flavor is in theproduction of free amino acids which act as precursors forproducingmany important aromatic compounds (McSweeneyand Sousa 2000; Yvon and Rijnen, 2001; McSweeney 2004).

Cheese ripening is a slow and expensive process.Various approaches have been used to accelerate theripening of cheeses, including the use of an elevatedtemperature, addition of exogenous enzymes, and use ofadjunct cultures and cheese slurries made from cheese curd,water, and salts (Fox et al. 2000; McSweeney 2004). Theprincipal attractions of cheese slurries are the short ripeningtime, the low cost and the possibility of including numerousparameters in a single study which is not possible withactual cheese making. Cheese slurry system that allowscheese to ripen at 30 °C for 5 to 30 days has been used as aquick tool to evaluate the contribution of different compo-nents into the cheese (Kristoffersen et al. 1967; Farkye et al.1995; Muehlenkamp-Ulate and Warthesen 1999; Coskun2006; Kumar et al. 2007).

The objectives of this study were (1) to evaluate theproteolysis process of cheese by using cheese curd slurriesmade from PEF-treated milk and (2) to compare the resultswith those from raw milk and heat-pasteurized milk.

Materials and Methods

Raw Milk and Heat-Pasteurized Milk

Raw milk with 3.14±0.02% (w w-1) protein and 3.81±0.06%(w w-1) fat was obtained from the dairy farm of Macdonaldcampus, McGill University (Ste-Anne-de-Bellevue, QC,Canada). The microbial load of raw milk was measuredbased on a standard method (Horwitz 2000), and it was lessthan 5,000 CFU mL-1.

Raw milk was filled in sterile plastic bottles and stored at4 °C for less than 4 h prior to PEF or heat pasteurization at63 °C for 30 min in a water bath.

PEF-Treated Milk

A 30-kV pulse generator (TG-01, Samtech Ltd, Glasgow,UK) with a matched output impedance of 100 Ω and acontinuous treatment chamber system were used in theexperiment. The output voltage profile was bi-polar instantreversal square waveform. The treatment chamber, designedand constructed in our laboratory, consisted of two parallelstainless steel electrodes separated by a 50-mm-thickDerlin® polyoxymethylene insulator, with 102 mm2 ofsurface area. The voltage and current across the treatmentchamber were captured simultaneously using a two-channeldigital oscilloscope (TDS3000, Tektronix, Wilsonville, OR,USA).

The samples were treated following the procedure de-scribed by Amiali et al. (2006). Liquid flow rate through thechamber was set at 6 mL/min using a peristaltic pump(Masterflex 77521-40, Cole-Parmer Instruments Co., VernonHills, IL, USA). After exposure to the desired number ofpulses, the milk sample was transferred to a sterile flask andkept at 4 °C. Treatment temperature was monitored using Ktype thermocouples (OMEGATM, Stamford, CT, USA). Lessthan 1 °C difference was maintained between inlet and outlettemperature for each treatment cycle.

The treatment conditions were selected based onprevious studies in our laboratory which demonstrated thatup to 5 log reduction of pathogenic microorganisms such asSalmonella enteritidis in whole milk could be achievedusing 30 kV/cm electric field intensity and 80 to 120 pulsesat 50 °C (Yu et al. 2005; Yu 2009). Therefore, theparameters and selected levels for this chapter were: electricfield intensity of 30 kV/cm; outlet temperature of 50±1 °C;pulse number of 80, and 120 pulses. The pulse width was2 µs, and the frequency of pulses was 2 Hz.

Preparation of Cheese Curd Slurries

The cheese curd was made following the revised procedureof Farkye et al. (1995). The 2% (v v-1) liquid starter culture

48 Food Bioprocess Technol (2012) 5:47–54

(Institute Rossel Inc., St-Laurent, QC, Canada) and 0.02%(w v-1) liquid rennet (Institute Rossel Inc., St-Laurent, QC,Canada) were added in sequence to 500 mL warm milksamples (30 °C). After brief mixing, the rennet-treated milkwas then left to coagulate at 30 °C and 40 min underquiescent conditions. The coagulum was cut and cooked to39 °C for over 30 min and held at this temperature for15 min. The whey was drained at pH 5.3, and the curd waskept for slurry preparation.

The slurry was prepared by blending 65 g cheese curd,5 g NaCl, and 30 g sterile distilled water in a sterile blenderjar (Hamilton, Beach, model C54252, Mexico). The dilutedslurry (100 g) was then transferred aseptically into steriletubes, capped loosely, and incubated at 30 °C for 5 days.Each slurry preparation was replicated two times usingfreshly made cheese curd.

Extraction of Water-Soluble Fraction

Water-soluble fraction (WSF) of slurries was made basedon the method of Kuchroo and Fox (1982). A sample (10 g)of cheese slurry was homogenized with 20 mL of distilledwater at 1,600 rpm on a vortex mixer (Fisher Scientific, CatNo. 022 15365, USA) for 30 s. Resulting mixtures weretempered in a 40 °C water bath for 20 min. Next, theextracts were centrifuged at 10,000g for 20 min at 4 °C.The fat layer was removed. The aqueous layer was thenfiltered through Whatman No. 1. The permeate waslyophilized in a ULT freezer (Thermo Electron Corporation,Waltham, MA, USA) and stored for further analysis.

Peptides Analysis by RP-HPLC

Peptide profiles of the WSF extracts were analyzed by RP-HPLC using a Varian system (Varian Associates, CA, USA)equipped with a photodiode array detector (model 330), anauto-sampler (model 410), and a quaternary solventdelivery module (model 240). The system is connected toa personal computer, and data was processed using VarianStar Workstation 5.5 software (Varian Associates, CA,USA). Separations were carried out using a 150×4.6 mm,C-18, 90 Ao pore size column (Varian Associates, CA,USA). A guard column (Supelguard LC-18-DB, 20×4.6 mm ID) was used in all cases.

Samples (10 mg) of each lyophilized WSF extract weredissolved in 2 mL 0.1% aqueous trifluroacetic acid (TFA) andfiltered through a 0.45-μm filter. A 100-µL loop was used tointroduce the sample into the high-performance liquid chro-matography (HPLC). The composition of solvent Awas 0.1%TFA in water and that of solvent B was 0.1% TFA inacetonitrile/water (75:25, v v-1). A stepwise gradient elution inthe order of 0% B (100% A) for 5 min; 30% B over 40 min;65% B over 15 min; and 80% B over 10 min at a flow rate of

0.75 mL min-1 was used. The column was then rinsed andallowed to equilibrate for 25 min between injections. Theeluate was monitored at 220 nm.

HPLC test for each WSF extract was performed intriplicate. The total integration area of peptides detected at220 nm during the HPLC run was determined. The UVabsorption peaks observed for the HPLC runs were dividedinto two groups to allow a quantitative hydrophobic–hydrophilic index analysis (Lau et al. 1991). The firstgroup consisted of the peaks with retention times from 5 to50 min, which was considered as the hydrophilic peptideportion. The second group of peptides with retention timefrom 50 to 70 min was the more hydrophobic peptideportion (Guo et al. 1986). The ratio of hydrophobic tohydrophilic peptides was obtained by dividing the totalpeak area of the hydrophobic peptide portion by that of thehydrophilic peptide portion.

Free Amino Acid Analysis by Cd–Ninhydrin Method

The presence of free amino acids in the WSF wasdetermined in triplicates based on the Cd–ninhydrin methodof Folkertsma and Fox (1992). Lyophilized WSF extractswere reconstituted in distilled water (1% w v-1). A portion(200 μL) of the reconstituted extract was diluted to 1 mLwith distilled water, and then 2 mL Cd–ninhydrin reagent(1 g of CdCl2 dissolved in 1 mL of water plus 0.8 gninhydrin, 80 mL of 90% ethanol, and 10 mL of glacialacetic acid) was added. The solution was heated at 84 °Cfor 5 min and cooled, and the absorbance was read at507 nm (A507) on a UV-VIS spectrophotometer (Thermo-Spectronic UV1, Cambridge, Great Britain). The A507 wasconverted to millimolar leucine from a standard curveprepared with leucine (0.1 to 10 mM). The results wereexpressed as milligrams leucine per gram WSF extracts.

Statistical Analysis

Analysis of variance was performed using the general linearmodel procedures of the Statistical Analysis System (SAS,Version 8.02, 2001, Cary, NC, USA). Experiments wereconducted in triplicates, and the means of the three data arepresented.

Results and Discussion

Concentration of Peptides in WSF

The present study used HPLC analysis to compare the peptideprofile in the WSF extracted from cheese curd slurries madefrom raw milk (RM), pasteurized milk (PM), and PEF-treatedmilks at 120 pulses (PEF120), and 80 pulses (PEF80).

Food Bioprocess Technol (2012) 5:47–54 49

Figure 1 shows the total peak areas of HPLC profiles ofcheese curd slurries made from RM, PM, PEF120, andPEF80. In a typical HPLC profile of cheese curd slurry, thetotal area under the peaks on the HPLC profile representsthe light absorbed by aromatic amino acids and peptidebonds present in the WSF of cheese (Lau et al. 1991). Ascheese ages, more caseins and high molecular weightpeptides are broken down into smaller peptides that maybe water soluble. Therefore, as the cheese aged, the totalwater-soluble peptide content increased. As expected, withincreased incubation time, the total peak areas increased.This implies that the total water-soluble peptide contentincreased. When comparing the results on day 0 and day 3,it can be found that RM samples had the largest peak areas,followed by the PEF80, PEF120, and PM samples. Thisresult was consistent with that from normal cheeses ripenedfor 6 months (McSweeney et al. 1993), although the cheeseslurries in the present study were ripened only 5 days.McSweeney et al. (1993) compared HPLC profiles ofhydrophilic and hydrophobic peptides in WSF of cheddarcheeses made from raw and pasteurized milk and reportedthe differences between the two profiles. They deduced thatthe difference could be due to the nonstarter microflorapresent in the raw milk. Although PEF treatment caninactivate the nonstarter microflora, the indigenousenzymes can survive since they require more severe PEFtreatment to yield a significant activity reduction (Ho et al.1997; Vega-Mercado et al. 1997; Van Loey et al. 2001;Bendicho et al. 2003; Shamsi et al. 2008). These enzymescould function in cheese aging and may have resulted inproduction of more water-soluble peptides. Lau et al.(1991) also used HPLC approach in order to quantify

water-soluble peptides in cheese. The authors reported that,although the total amount of water-soluble peptides in RMand PM cheese were the same, the relative proportion ofhydrophobic and hydrophilic peptides were different inthese cheeses.

Less peak areas of PEF120 were found than that ofPEF80, which was due to the fact that increasing the lengthof pulses led to a more severe modification of enzymeactivity (Giner et al 2001; Perez and Pilosof 2004). Thereduced enzyme activity may lead to less production ofpeptides and amino acids. The mechanism of enzymeinactivation by PEF is still under investigation, but it isgenerally considered to be due to unfolding, denaturation,and breakdown of covalent bonds and oxidation–reductionreactions in the protein structure caused by intense electricfield (Barsotti and Cheftel 1999; Shamsi et al. 2008).

On day 5, the trend was slightly different. RM still gave thelargest peak area, while the peak areas of PM, PEF80, andPEF120 were not much different (p<0.05). For RM, the peakarea increased rapidly with the increase in the incubationtime, while the peak area for PEF-treated milk increasedrelatively slowly. The modification of enzyme by PEF mayhave led to changes in the proteolytic properties of milkresulting in reduced formation of peptides and amino acids inPEF-treated milk as compared with raw milk.

Analysis of Hydrophilic and Hydrophobic Peptides

Figures 2 and 3 show the amounts of hydrophilic andhydrophobic peptides in WSF of cheese curd slurries madefrom RM, PM, PEF80, and PEF120.

0.00E+00

5.00E+07

1.00E+08

1.50E+08

2.00E+08

2.50E+08

3.00E+08

0 3 5

Incubation time (day)

Tota

l p

eak

area

Raw milk PEF80 PEF120 Pasteurized

Fig. 1 Total peak areas of HPLC profiles for cheese curd slurriesmade from (1) raw milk (RM); (2) PEF-treated milk at 80 pulses(PEF80); (3) PEF-treated milk at 120 pulses (PEF120); (4) pasteurizedmilk (PM) at the incubation time of 0, 3, and 5 days

0.00E+00

1.50E+07

3.00E+07

4.50E+07

6.00E+07

7.50E+07

9.00E+07

0 3 5


Hyd

rop

hil

ic p

eak

area


Fig. 2 The amounts of hydrophilic peptides in WSF of cheese curdslurries made from (1) raw milk (RM); (2) PEF-treated milk at 80pulses (PEF80); (3) PEF-treated milk at 120 pulses (PEF120); (4)pasteurized milk (PM) at the incubation time of 0, 3, and 5 days


It was found that both hydrophilic and hydrophobicpeptides increased with the increase of the incubation timein all the test samples. This was consistent with the findingsby Lau et al. (1991), who reported that hydrophilic andhydrophobic peptides increased during the cheddar cheeseaging for both raw and pasteurized milk cheeses.

Champion and Stanley (1982) stated that extracts ofbitter cheddar cheese contained a high proportion ofhydrophobic peptides. Lau et al. (1991) found a higherproportion of hydrophobic peptides present in the WSF ofcheddar cheese made from pasteurized milk than in cheesemade from raw milk. The authors believed that a properbalance among the various water-soluble components isimportant for the development of a typical cheddar cheeseflavor. He proposed that the difference in the ratio ofhydrophobic to hydrophilic peptides present in the WSF incheddar cheeses made from pasteurized and raw milk maycause flavor differences.

Figure 4 shows the ratio of hydrophobic to hydrophilicpeptides in RM, PM, PEF80, and PEF120 cheese curdslurries. It was found that on both day 3 and day 5, PMgave the highest ratios of hydrophobic to hydrophilicpeptides compared with those of raw and PEF-treated milk(p<0.05). PEF80 gave similar ratios as RM (p>0.05),whereas PEF120 gave the lowest ratios compared with RM,PEF80, and PM.

Analysis of Free Amino Acids

Figure 5 presents the concentration of free amino acids(FAA) in the WSF extracted from cheese curd slurries made

from RM, PM, and PEF-treated milks at PEF120 andPEF80.

As expected, with increased incubation time, the concen-tration of FAA increased. On day 0, RM and PEF80 sampleshad the same level of FAA (p>0.05), which was a little higherthan that of PEF120 and PM samples. On day 3, theconcentration of FAA in all four samples increased dramat-ically compared with that on day 0. RM samples still had thehighest level of FAA, followed by the PEF80, and PEF120samples; PM samples lagged behind. The result that RM had

0

1

2

3

4

5

6

0 3 5


Rat

io


Fig. 4 The ratio of hydrophobic to hydrophilic peptides in WSF ofcheese curd slurries made from (1) raw milk (RM); (2) PEF-treatedmilk at 80 pulses (PEF80); (3) PEF-treated milk at 120 pulses(PEF120); (4) pasteurized milk (PM) at the incubation time of 0, 3,and 5 days

0.00E+00

3.00E+07

6.00E+07

9.00E+07

1.20E+08

1.50E+08

1.80E+08

2.10E+08

0 3 5


Hyd

rop

ho

bic

pea

k ar

ea


Fig. 3 The amounts of hydrophobic peptides in WSF of cheese curdslurries made from (1) raw milk (RM); (2) PEF-treated milk at 80pulses (PEF80); (3) PEF-treated milk at 120 pulses (PEF120); (4)pasteurized milk (PM) at the incubation time of 0, 3, and 5 days

0.00

0.02

0.04

0.06

0.08

0.10

0 3 5


Fre

e am

ino

aci

ds

(mg

/g)


Fig. 5 Concentration of free amino acids in WSF of cheese curdslurries made from (1) raw milk (RM); (2) PEF-treated milk at 80pulses (PEF80); (3) PEF-treated milk at 120 pulses (PEF120); (4)pasteurized milk (PM) at the incubation time of 0, 3, and 5 days


higher FAA than PM is not surprising. McSweeney et al.(1993) and Shakeel-Ur-Rehman et al. (2000) found a higherconcentration of amino acids in cheddar cheese made fromraw milk which had a higher and more heterogeneousnonstarter lactic acid bacteria population than pasteurizedmilk cheeses. Albenzio et al. (2001) studied biochemicalcharacteristics of Canestrato Pugliese cheese made from rawmilk and pasteurized milk and also noticed consistent higherconcentration of FAA in raw milk compared with pasteurizedmilk cheeses.

On day 5, the increase of FAA in all the samples was a littleslower compared with the beginning of the ripening period.This was consistent with other similar work done byMuehlenkamp-Ulate and Warthesen (1999), who also pre-sented a faster increase in total FAA in cheese slurries at thebeginning of the ripening period and slower afterward. In ourwork, on day 5, RM still had the highest level of FAA;PEF80 samples turned to the second level, followed byPEF120 and PM samples which did not show any differencein FAA level. The FAA in PEF120 was consistently lowerthan that of PEF80, which was probably due to the greaterseverity of that PEF treatment. As mentioned earlier, theincrease in pulses may lead to a more severe modification ofenzyme (Giner et al 2001; Perez and Pilosof 2004). Thereduced enzyme activity may lead to less production ofpeptides and free amino acids.

PEF80 samples gave similar FAA concentration as RMsamples on day 0 (p>0.05) and PEF80 and PEF120consistently gave higher FAA content than PM samples(p<0.05) on day 3 and day 5. This indicated that PEFtreatment could possibly compete with traditional pasteuri-zation process to provide “cold-pasteurized milk” cheeseswith ripening quality similar to that of raw milk cheeses.

The content of FAA for all samples in our results variedfrom 0.018 to 0.091 mg leucine g-1 WSF extracts during the5 incubation days, which was within the reasonable rangeof other researches (Farkye et al. 1995; Muehlenkamp-Ulate and Warthesen 1999; Briggs et al. 2003). Farkye et al.(1995) used starter-free cheddar type cheese slurries toinvestigate proteolytic abilities of some lactic acid bacteriain a model cheese system. Their samples were ripenedanaerobically at 32 °C for 5 days, and the concentration ofFAAvaried from approximately 0.034 to 0.101 mg leucine g-1

WSF extracts. Muehlenkamp-Ulate and Warthesen (1999)evaluated ten strains of nonstarter lactobacilli for theirproteolytic abilities in cheddar cheese slurries by ripeninganaerobically at 30 °C for 12 days. The concentration ofFAA in their studies varied from 0.1 to 0.5 mg leucine g-1

WSF extracts from day 0 to day 6. The higher concentra-tion of FAA in their work was due to an additionalprocedure for enhanced WSF fractionation: the WSF wasfurther fractionated in a micro-concentrator with a 1,000-molecular-weight cutoff. The permeate of the micro-

concentrator was used for FAA analysis. Briggs et al.(2003) used cheese slurries to monitor the cheese ripeningprocess accelerated by PEF-treated lactic acid bacteria.The concentration of FAA in their studies was approx-imately ten times lower than the values obtained in thepresent study and by Farkye et al. (1995). The lowerconcentration of FAA reported by Briggs et al. (2003)may be due to the different types of treatment applied inthe study. The authors treated microorganisms by PEFbefore inoculating them to milk samples, whereas, in thisstudy, PEF-treated milk was mixed with untreated starterculture.

Conclusions

The present study indicated that the proteolytic propertiesof milk treated by pulsed electric field were intermediatebetween raw milk and thermally pasteurized milk. So thereis a potential of producing safer cheese through PEFprocessing.

The results of this study were based on cheese slurriesripened at 30 °C for 5 days. It could be considered as asimplified and accelerated model for normal cheeseripening. So scale-up of cheese ripening process usingPEF-treated milk is needed for future studies. Lipolysisis equally important in the development of flavorcompounds in cheeses. Evaluation of the lipolysisprocess in cheese made from PEF-treated milk is alsoneeded.

Acknowledgment The authors would like to acknowledge thefinancial support of the National Science and Engineering ResearchCouncil of Canada (NSERC).

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