PASTEURIZATION OF RAW SKIM MILK BY PULSED ELECTRK FIELDS AND

ANTIMICROBIALS

A Thesis

Presented to

The Ficulty of Graduüte Studies

of

The University of Guelph

In partial fulfilment of requirements

for the degree of

Master of Science

November, 2000

O Keith H. Smith, 2000

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ABSTRACT

PASTEURIZATION OF RAW SKIM MlLK BY PULSED ELECTRIC FIELDS AND ANTIMICROBIALS

Keith Smith University of Guelph, 2000

Advisor: Professor G. S. Mittal

This thesis isan investigation of the microbial inactivation in raw skim milk by pulsed

electric field trc;itment and iintiinicrobisls nisin ;ind lysozyme.

The innovativc icçhnology oliising a high-voltage pulsedelectric ficld (PEF) for pastcuiizirig

milk offers advantages of low processing temperatures and low energy utilization, whilo

inactivating pathogenic microorganisms especially when combined with other preservation

methods. A 7.0 log reduction of microorganisms found in raw skim milk h a been achieved

through ii combination of pulsed elcctric field trciitment (80 kV/cm. 50 pulses) . mild Iieat

(52°C) rind addition of the natural antimicrobi;il peptides nisin (3000 iU/inl) iind lysozyiiic

(1000 pg/ml). The combinaiion of PEF, mild heat and antirnicrobiüls resulted i n a much

higher microbial inactivation than the sum of the individual reductions ûchieved from each

treatment alone. indicating synergy. Varying the pH from 6.7 to 5.0 had noeffect on rnicrobial

inactivation.

Acknowledgements

1 would like tri thank Dr. klrinsel W. Grit'fiths ;incl Dr. Puoirf;id;is Piyuselia whci

provided direction on my thesis as members of my advisory coinniittec, Dr. C.D. Kulkaini

from Gay k a Foods for supplying me with raw skim milk, Feng Li from the University of

Waterloo for his technical expertise, Bill Verspagen for his assistance in the modification of

the ireatrnent chamber. Joanne Ryks for her advice on laboratory equipment sclection. Diane

Wood for her instruction on microbiologic;il procedures, Haifeng W;tng for his assistance in

the microbiology laboratory, and Cinally, 1 would like to extend my gratitude ro Dr. Gauri S.

Mittal for his guidance throughout the cornpletion of my thesis.

Table of Contents

Abstract

Acknowledgements

Table of Contents

List of Figures

List of Tables

Nomenclature

1 .O Introduction

2.0 Review of Literature 2.1 PEF System 2.2 Antimicrobials

2.2.1 Nisin 7.2.2 Lysozyme

2.3 Food Preservation by Combined Methods 2.4 Microbial Inactivation by PEF 2.5 Mechanisms of Inactivation of PEF 2.6 PEF Treatment of Milk

3.0 Research Objectives

4.0 Materials & Methods 4.1 Skirn Milk 4.3 PEF Systern 4.3 Sanitation Procedure 4.4 Microbial Enurneration 4.5 Statistical Treatment of Results 4.6 Effect of Temperature 4.7 Effect of pH 4.8 Effect of Antimicrobial Addition 4.9 Effect of Pulse Number 4.10 Clarification of Milk

5.0 Results & Discussion 5.1 Preliminary Experiments 5.2 Effect of Temperature

v i i

5.3 Effect of pH 5.4 Effect of Antimicrobials 5.5 Effect of Pulse Number

6.0 Conclusions Sr Recomrnendations

References

Appendix A: Statistical Analysis

Appendix B: Experimental Data

... I I I

List of Figures

Figure 4.1.

Figure 4.2.

Figure 4.2.

F i p - c 4.4.

Figure 4.5.

Figure 5.1.

Figure 5.2.

Figure 3.3.

PEF treatment System.

Treatment chamber (unassembled).

Drawing of treatrnent chamber.

Instant-charge-rcvcrslil pulse wavcforni for skini itiilk at 74°C wlien 80 kV/cm were iipplierf. I Syiiare = 5000 V (vertical), 500 qs (horizontal

Block diagram of PEF treatrnent system (adapted from Ho et al., 1995).

Effect of temperature on microbial population in r iw skim milk.

Effect of pH on rnicrobiril population in niw skim milk.

Effect of pulse nuniber on rnicrobiril popiillition in raw skirn

List of Tables

Table 2.1.

Table 2.2.

Trible 5.1.

Table 5.2.

Table 5.3.

Table 5.4.

T;ible 5.5.

Table 5.6.

Table 5.7.

Table 5.8.

Trible 5.9.

Tiible 5.10.

Table 5.1 1.

Table 5.12.

Trible 5.13.

Table 5.14.

Surnrnary of Microbial Inactivation with PEF Treatment of Milk

Surnrnary of Microbial Inactivation with PEF and Antimicrobials

PEF Treiitment (90 kV/cm. 20 pulses) of skim milk inoculateci with Psecc<lornoricrs f 1iiorescen.s.

Injection of sarnple into treatrnent charnber without application of pulses.

Effect of clarification procedure on raw skim milk.

Heat ireatment of raw skim milk.

ANOVA for the effect of temperature uii rriicrobial in;ictivstion.

Duncan's LSD for the effect of temperature on microbial inactivation.

PEF treatment (80 kVlcrn, 50 pulses) of raw skim milk at varying pH levels.

Addition of lysozyme to rriw skim milk (4000 pg/ml).

Addition of nisin to rnw skim milk (4000 iü/ml).

Addition of Lyso:Chrisin to raw skim milk ( 1000 pg lysozyme/ml; 3000 [U nisinlml).

ANOVA for the effects of antimicrobials and temperature on microbial inactivation in raw skim milk.

Duncan's LSD for the effects of antimicrobials and temperature on microbial inactivation in raw skim rnilk.

PEF treritment (80 kV/cni. 50 pulses) of niw skim milk contriiiiing lysozyme (4000 pgiml).

PEF treatment (80 kV/crn, 50 pulses) of rliw skim milk containing

Table 5.15. PEF treatment (80 kV/cm, 50 pulses) of ïilw skim milk containing 48 Lyso:Chrisin (1000 pg lysozymelml; 3000 IU nisin/ml).

Table 5.16. GLlM for the effect of antimicrobials on PEF rrenctnent (80 kV/cm, 48 50 pulses) of raw skim milk.

Table 5.17. Duncan's LSD for the effect of antimicrobials on PEF treatment 48 (80 kV/cm, 50 pulses) of raw skim milk.

Table 5.18. PEF treatment (80 kVIcm, 50 pulses) of rriw skim milk containing 50 Lyso:Chrisin (1000 pg lysozyme/tnl; 3000 iü nisintml) with varying pulse nuniber.

Tiible 5.19. GLM for the eft'ect of pulse number on PEF treritment (80 kV/cin) 5 1 of raw skim milk containing Lyso:Chrisin (1000 pg Iysozymelml: 3 0 IU nisinfml).

Table 5.20. Duncan's LSD for the effect of pulse number on PEF treatment 5 1 (80 kV1cm) of raw skim miik containing Lyso:Chrisin (IO00 pg lysozyme/ml; 3000 TU nisinlml).

Nomenclature

ce11 radius (m) electrode surface areri (ml) effective capacitance (F) cnpacitance (F) distance between electrodes (m) ce11 diameter (m) electric field strength (Vlrn) external field strength (V/m) shear elasticity modulus membrane thickness (m) maximum peik current (A) cell length (m) number of piilses final micrc biiil populution (cfu/tml) initiiil microbial population (cfiilml) energy ( J I resistance (Q) standard deviation treritment tirne (s) electric potential difference (V) voltage (V) criticrd brc;ikdown potential (V) induccd potential (V I elastic modulus

membrane thickness (m) permittivity of free space (8.8542 x IO*" F/m) membrane relative permittiviiy (Flm) relative permittivity (Flm) resistivity (mcm) conductivity (Slm) pulse width (s)

vii

1.0 Introduction

Bovine milk has been consumed by humans for thousands of years because of its

nutritional value, however, r iw milk also happens to be an ideal mediiiin for niicrobial growtti

because of its high water activity, moderate pH (6.4-6.6) and ample supply of nutrients

(Bramley and McKinnon, 1990). A number of human pathogens have been found in milk

which include Escliericliia coli, Stctpit-vlococcirs niireirs, Strl~nonrlla, Listerici

niortugrogcries, Mycobacreriuni havis and Mycobacteri~rnt tirbercdosis (Adams and Moss,

1995).

[n the 1860's. Louis Pasteur fouiid thiit wine kept bettcr if ii was Iield ;it a Iii$

temperature for some time then cooled. Soon atierwards. the srime treatment was king

applied to milk in order to make it safer for human consumption. Pastcuriziition is deîïned by

the International Dairy Federation (DF) as "a process applied to a product with the object of

minimizing possible health hazards arising from pathogenic microorganisms rissocilited with

milk, by heat rreatment which isconsistent with minimd cherniciil mIor~anoleptic chiinge to

the product" (Vamam and Sutherland, 1994).

Early pasteurization took the form of a batch process, called low temperature holding

(LTH), where milk was heated to62.8"C for 30 min. In most countries this technique bris been

superseded by high temperaturelshort time (HTST) pasteurization. which is a continuous

systsm whcrc milk is piimped throiigh a heat rxchanp;ind Iie;lted ro 7 1.7"C and hrld at t h ~ i t

temperature for 15 s before being tripidly cooled. A commercial sterilizrition process. ul t r l i

high temperature (UHT), involves heating milk to 135-150°C for 1-4 s (Harding 1995).

Although thermal processing is effective at eliminating pathogens, the sensory and nutritional

properties of milkdecline because of protein denaturation and the Ioss of vitamins and volatile

flavours. In addition, it is an energy intensive process.

The ciesire to produce milk witli ;i ti.esh-like raste has genmtild interest in non-therni;il

processes which offer the tidvantages of low processing rernperiitui-es. low energy iitilization

and the retention of nutrients and organoleptic qualiries, while inactivating pnthogenic

microorganisms. The innovative technology of using a high-voltage pulscd electric field for

food preservation iippeiirs promising, especially when combined with other preservation

inethods.

2.0 Review of Literature

2.1 PEF System

A Pulsed Electric Field processing system consists of a power source, a capacitor, a

high-voltage switch and a treatment chamber, anda pump toconduct food through the treatrnent

chamber. An oscilloscope is used to observe the pulse waveform. The power source, a high

voltage DC generritor. converts volta_ir from ;in uiility line ( I IO V) into high voltage AC. then

rectifies to ri high voltage DC. Energy from the power source is storetl in the capricitor :inci

is discharged through the treatment chamber to generate an electric field in the food material.

The maximum voltage across the capacitor is equal to the voltage across the generator (Ho et

al.. 1995).

The cnergy stored in a ciipncitor is given by (Ho et al. 1905):

where Co is the capacitance and V is the chürging voltage.

The effective capacitance, C, can be calculated by (Ho et al., 1995):

where T (s) is the pulse duration, a (Sm) is the conductivity of the food, A (.m2) is the m a of

electrode surface, and d (rn) is the distance between electrodes (Barbosa-Canova et al.,

1998).

Assuming the food mriteriül has homogenous dielectric and electricd properties, the

effective capacitance can dso be calculated using (Zhang et al.. 19954:

where E,, is the permittivity of free space and E, is the relative perrnittivity. i.e. dielectric of

the food material.

The energy stored in the ccipacitor is discharged using a high-voltage switch which

must be able to operate at a high power and repetition rate. The type of switches which may

be used include gas spark gaps. thyristors, thyrrttrons, ignitrons, and vacuum tubes. The switch

must be able to resist the maximum voltage present ricross the capacitor, ris weil ris the peak

currcnt, resulting primarily tiorn the food sample'selectrical rcsistivity. Tlie maxiniurn peak

current, I,,,,,, is defined by (Zhang et al.. I995a):

The actual peak current will be smaller thm I,,,,, due to inductance in the circuit.

Treatment chambers are designed tocontiiin the food material during PEFprocessing

and to hoiise the discharging electrodes. They can be either batch-type or continuous. with

batch being more suitable for lab-scde experimentation. A typical treatment chümber consists

of two parillel electrodes encased in insulacing material. The chamber volume is adjusted

using spacers made with insulating material of varying thickness.

Sale and Hamilton (1967) designed one of the first chambers for PEF treatment using

carbon electrodes supported on brass blocks. with a U-shaped polythene spncer placed

between the twoelectrodes . The maximum field strength for their design was limited to 30

kVlcm due to the electrical breakdown of air above the food. The chamber used by Dunn and

Pcürlmrin ( 1987) consisted of two stiiinless steel electrodcs, with an effective electrods area

of 78 cm', and a 2 cm thick cylindrical nylon spacer. Barbosti-Ciinows et ;il. (1998)

reviewed other treatment chambers based on these two designs. Sample tllling and removril

adds CO the complexity in chamber design since the chamber needs to be degassed before

treatment in orderto prevent the dielectric brakdown of the foodsample (Zhanget al. 1995a}.

An oscilloscope is tised to observe the pulse wavcform :ind ro meLisure the voltage

bcrwecn the slzctrodes.

2.2 Antimicrobials

The poputarity of minimally processed foods has brourght increased attention to

preservation through thc use of iintimicrobirils. which inhibit or tlestroy rhc ~rowrh of

rnicroorg;inisms. Antirnicrobitils rire produced by iinimlils. pl;itits and iiiicrot>rganisms.

Among the most widely investigated antimicrobials are nisin and lysozyme.

Lactic acid bacteria produce ri wide range of antimicrobial proteins known ris

biicterioci ns. Nisin. ptodiiced by Ltrcrococcii.~ irlcti.~ subsp. lcicsi.~ is ;i inernber of tlie cl;iss

of bacteriocins known as Lantibiotics, which contain the amino acid lanthionine.

Nisin exhibits inhibitory activity against Gram-positive bacteria, including L. lmis

subsp. laciis, and subsp. cremoris, L. biilgarictis, Saiphylococctts nirrais, and Listerin

monocytogeties and prevents the outgrowth of spores of many Closrrkliiini and Bacillus spp.

(Abee et al., 1995; Harris et al.. 1992: Hurst and Hoover. i 993).

Although Gram-neglitive bacteria rire normally resistant to nisin, they have been shown

to be sensitized by subiethal injury due to heat treatment (Kalchayanand et al., 1992),

hydrostatic pressure and electroporation (Kalchayanand et ai., 1994) and i n the presence of

chelating agents, siich as EDTA (Stevens et rd.. 1991: Stevens et al.. 1992). Nisin hiis ;ils0

been show to t1est;ibilize the outer inembmne vesicles ofGr;rni-negiitive b;icteria (Gao r r ;il..

1991).

Nisin functions by interacting with the phospholipids in the cytoplasmic membrane

Jacket al.. 1995). The action of nisin proceeds through insertion and pore formiation, resulting

in an efflux of low molecular weight compounds and the depolarization of the mernbrrine (Hill.

1995). Gao et id. ( 1991 ) studied the interiiction of nisin with liposomes cind concludecl thrit

a membrane potenlia1 (negative inside) andlor a pH gradient (alkaline inside) enhiinces the

activity of nisin. They also found that the activity of nisin is intluenced by the phospholipid

composition of the membrane, which mayaccount fordifferences in nisin sensitivity bctwccn

bacterial species or strains. The effectiveness of nisin depends on the biicterial loiid. As the

number of orynisms inci.eases. the inhibitory effectivcncss of iiisin decrc:iscs (.Scott ;ind

Taylor, 198 1).

Nisin is heat stabile (Hurst, 1981), however, it becomes increasingly ineffective in

solutions that approach pH neutrality (Daeschel, 1993). The antimicrobial action of nisin is

most powerful at pH 6.5 to 6.8 (Luck and Jager, 1995), aithough its stability in this pH range

is very poor. Jung et al. (1992) investigated the rictivity of nisin agiiinst Lisrericl

nlonoqtogenes in milk and observed that the efficacy of nisin decreases with increasing fat

content. They hypothesized that nisin absorbed to milk-fat globules, reducing its availability

tu inhibit cells.

2.2.2 Lysozyme

Naturallyoccumingcompounds which have the ability to inhibit microor_eanisms have

attracted attention in theiruse as food additives. Lysozyme, which is an enzyme found in foods

of animal origin, occurs naturally in milk at a concentration of approximately 0.13 p@ml

(Reiter. 1978).

Lysozyme lyses bacteria by hydrolyzing the P ( 1,4) linkiiges between N-acetylmurrimic

acid and the N-acctyl glucosamine of the peptidoglycan layer of bacteriril çell tvalls. The

cleaving of this linkage results in the osmotic uptake of water leading to the expansion and

eventual rupture of the cytoplasmic membrane (Board, 1995). Gram-positive bacteria are

niore susceptible to the action of lysozyme because of the relatively simple structure of their

cell wall which contains iip to908 peptidoglyc;in. Gram-negative bacteria ai.? more resistaiit

to lysozyme because of the smaller umount of peptidoglycan containcd in the ccll wall. III

addition, the cell wall is surrounded by an outer membrane, which consists of a lipoprotein-

lipopolysaccharide layer, that limits access of lysozyme to polysaccharide layers of the cell

wall (Conner, 1993), although susceptibiiitycan be induced following certain environmental

stresses that weriken the outer membrwc (Beuchrit and Golden, 1989).

Lysozynie liiis been shown to be effective a~ainst several Gram-positive bacteria.

including Micrococciis, Smcincr, Lactobacillus and Bacillus as well 11s sevenl Gnm-

negative bacteria, such as Sulmonrlli, Pseticlomonas, Auroniontis and E. coli (Proctor and

Cunningham, t988). Hughey and Johnson ( 1987) found lysozyme was able to effectively

inhibit C. butitlirium and Listerici ntonoqtogeries. Ibrahim et al. (1996) found that the hcat

denaturation of lysozyme resulted in an increase in activity against Gram-neptive bacteria

such as E. coli and Staph. airreus. The activity rate of lysozyme is Iiighest in the pH range of

7

around 3-7 (Proctor and Cunningham, 1988).

Nisin and lysozyme have both been granted GRAS (generally regarded as süfe) status

by the U.S. Food and Driig Administration (Luck and Jaser. 1995). Tlicy ;ire riwaiting

iipproval by Health Canada.

2.3 Food Presewation by Combined Methods

The concept of hurdle technology was introduced to improve the sensory and nutritive

properties of processed foods through the use of combinations of mild preservation techniques

(Leistner. 1992). The hurdle concept ciin also be üpplied to antimiçrobial agents whrre two

or more rit suboptimal levels are more effective than one iit the optimal lcvel wirhout iiffecring

the acceptance quality of the food (Leistner and Gorris, 1995).

Moisnier-Patin et al. ( 1995) reported that nisin reduces the heat resistance of Lisreriri

mortoqtogenes in milk. It hüs also been demonstrated that a combination of heat and nisin

w a more effective againsr spores ot'hacilli ancl closrr-idici t l m nisin alonc (Scott and Tii~ior.

1981: Wurst, 198 I : Wandlinget al., 1999). Zapicoet iil. ( 1998) invt.stip;ired rlicet'kct til'nisiti

addition toultn-high temperature processed(UHT) skirn rnilk. With the addition of 100 iü/ml,

no effect on counts of Listeria tnonocvtogenes were observed, however, when nisin was

combined with the Iactoperoxidrise system (LPS), which occurs naturally in rnilk. a 5.5 log

reduction wris richieved, indicating a synergistic effect.

Garcia-Graells et al. (1999) studied the inactivation in milk of four E. coli striiins by

hydrostatic pressure, alone or in combination with the antimicrobial peptides lysozyme and

nisin and at different temperatures. They found that a process temperature of 50°C and high

concentrations of lysozyme (400 pg/ml) and nisin (400 W/ml) improved the efficacy of the

pressure treatrnent. In their experiments. nisin and lysozyme wcre added before hydrostritic

pressure treatment. PEFcan be combined with moderately high temperatures (<60°C) orother

processing aids such as antimicrobials io increase the extent of microbial inactivation

(Hulsheger et al, 198 1 ; Jayaram and Castle, 1992; Kalchayand et al., 1994; Liu et al ., 1997).

Severi1 nutliors investigated the effect of combinin; nisin with other antimicrobial

agents. Synergistic effects were reported for nisin combined with the bacteriocin pediocin

AcH (Hanlin et al.. 1993), with plant essential oils, thymol (Ettayebi et al., 2000) iind

carvacrol (Pol and Smid, 1999) and with the naturally occurring enzymes lysozyme (Hauben

et al., 1996; Garcia-Graells et al., 1999) iind lactoperoxidase (Zapico et ;II.. 1998). Hin l in

et al. (1993) proposed that these synergistic effects are a rcsult of nisin binding with the ceil

wnll and impiiring the barrier funcrions. which rillows forother molscules to pus through thc

wüll, come in to contact with the cytoplasmic membrane, and destabilize its functions.

2.4 Microbial Inactivation by PEF

When an electric field is generated between two prirallel-plate electrodes, the electric

field strength, E (Vlm) is defined as:

where U (V) is the electric potential difference and d (m) is the distance between the two

electrodes. Field intensity has amuch greatereffect on microbial inactivation than either pulse

numberor pulsedunition (Hulshegeretal., 198 1; Jayaram and Castle. 1992; Ho et al.. 1995).

When subjected CO a PEF, polarization of the dipoles and the bulk movement of ions

induce capacitive and resistive currents (Barbosa-Canovas et al., 1999). Assuming the food

9

has homogeneous dielectric and electric properties, the resistance, R (Q), of the food sarnpie

is defined by:

where A (m2) is the electrode a m , d (rn) is the distance between electrodes. a (S/rn) is the

conductivity of the food, and p (RIcrn) is the resistivity of the food. The dielectriç constant

of a food increnses with increasing water content and decreases with increasing tempemturc.

Food conductivity increases with an increase in temperature.

The treatment tirne is given by:

t = n ?

where n is the number of pulses and r is the pulse width.

I'he surviviil raie depcnds more on the physiologictil properties of the testeci cell

popiiltition thtiii on the type ofmicroorganisrn bcing treatrci (Hiilslicgcr et ;il.. I9S3). Thew

include the microbial growth stage, initial innoculum s ix , preciilture condition, ionic

concentration andconductivityof the suspension fluid (Wouters and Smelt, 1997; Barsotti and

Chefttel, 1999). Cells from the logariihmic growth phase are more susceptible to PEF

trcatment than cells harvested from the stationary growth phase (Hulsheger et ;il.. 1953).

Hulshegeret al. ( 1983) alsoobserved that Grain-positive bticteri;i and yerists tire less scric;itive

to electric pulse treatment than Gram-negative bacteria, when low pulse numbers are applied.

There are two major wavefoms which are generally used in PEF technology, the

exponential decay pulse and the square pulse. Other researchers (Zhang et al., 1994;

Pothakarnury et al., 1995; Qin et al.. 1995. Zhang et al.. 1995b: Calderon-Miranda et 31..

10

1999a; and Pol et al., 2000) used these waveforms where the energy per piilse was generdly

high, resulting in heatingof the treated food. While this excess heat ha been reported to have

no bactericidal effect when the tempemtureof the ftuid food is maintained below 55°C (Shang

et al, 19941, cooling of the food sample is required during processing.

An insttint-charge-reversal puIse wtiveform hüs been devcloped (btitt;il, 1998) wliich

generrites no Iieüt due to the low energy lipplied per pulse. Uniike a bipollir pulse, wherc the

polar@ of the pulses is reversedalternatety the charge reversal is instantaneoiis with no time

lag and an oscillating field is applied. The amplitude of the negative peak is not large enough

to provide cell membrane brerikdown, but a high alternating stress on the ce11 membrane is

produceci resulting in srnictural fiitiguc ( Ho et al.. 1995).

Pothakamury et a!.. (1996) founrl that sqüiiïc wiivc pulses werri 9 '2 more effecrivc

against E. coli. and S. attreiis inoculated in simulated milk ultnil?ltrnte (SMUF) than

exponential decaying pulses. The use of alternating polarity and very short duration pulses

reduces the risk of undesirable electrochemical reactions, as weil as the formation of deposits.

iIC the electrodes (Barsotti et al., 1999). Qin et al. (1995) compared the effect of bipolar.

oscilli~tory, exponentiallydecaying and square-waw pulses and concluded that square-w;ive

pulses generate the smallest survival fraction for identical peak voltrige and energy delivered.

2.5 Mechanisms of Inactivation of PEF

A biological cell subjected to a pulsed electric field of sufficirnt strength underocs

membrane permeabilization. The magnitude of the rransrnembrrine potential determines

whether the formation of pores is reversible (electrical) or irreversible (mechanical). The

exact mode of action for pore formation is unclear. Some theories suggest that a large

populiition of porcs is nlwnys present,expanding rnpidly in response to large potentials. wliile

others hypothesize that pores are rapidly created by large potentials, followed irnmediately

by rapid pore expansion (Weaver and Powell, 1989).

Sale and Hamilton ( 1968) considered theeffect of the potential difference developed

ricross the cell membrane. They proposed that damage to the membrane occurs wlien the

trmmembrane potential is greater than I V . rcsiilting in tlie loss of intrinsic properties, suc11

as electrical resistance, membrane potential and barrier function.

Hulsheger et al. (1983) give an equation to calculate the induced potential, V,, for

spherical cells surrounded by nonconducting membranes:

where ;t is the ceIl radius (m) and E, is the external field strength (V) .

Zimmerman et al. (1974) expanded on this equation to include non-spherical cells

using the assumption chat the shape of a cell can be considered as two hemispheres on either

end of a cylinder:

where 1 is the cell length (m) and d is the cell diameter (m).

Sale and Hamilton (1967) investigated the effects of high electric fields on bacteria

and yeasts and described membrane damage as the lysis of erythrocytes and protoplasts, as

well as the leakage of intracellular contents. They also observed the loss of the ability of E.

' d i to plasmolyze in a hypertonie medium (20 mM phosphate butter, pH 7.2 + 10% sucrose)

and the release of P-galactosidase rictivity in ri permease-negative mutant of E. coli.

Zimmeman et al. (1974) presented the dielectric rupture theory which suggests that

rupture of the cell membrane accurs when the overall membrane potential excceds the cell

membrane's natural potentitil (- I V) resulting in pore formation and increased permeabil ity.

Using transmission eiectron microscopy (TEM), Harrison et al. ( 1997) studied the

effects of PEF treatment on SriccCiaromyces cerevisicw. They observed cytologicül disruption

(damaged organelles and lack of ribosomes) indicaiing that electroporation may not be the

only inactivation mechanism.

Coster uncl Siinmerman ( 1975) drsçriberl the tllectromcchaiiicnl instiibil ity theory on

electroporiitioii as il consequencr of electro-cornpi-essivc rorcts i v I i ic l i c1ecic;isc thc rtiickriess

of the ce1 l membrane. Since the bilayer volume is incompressible, the membrane undergoes

an increase in area per lipid. This results in a destabilized bilüyer. The critical potentiül

difference for electro-mechanical breakdown of the membrane, V,, is givcn by:

where Y is the elastic modulus of the membrane, 8, is the unstrained membrane thickness, md

E is the relative electric permittivity and E, is the electric permittivity of free space. This

theory fails CO distinguish between non-reversible membrane rupture and reversible membrane

discharge (Ho and Mitttil, 1996).

Dimitrov (1984) developed an equrition to express electric potentiiii. V,,,, based on a

simple viscwlastic film model which he developed using experimental data. The critical

breakdown potential ,V,, is given by:

where a is the surface tension, G is membrane shear elasticity rnodulus. h is membrane

thickness (m). E, is membrane relative perrnittivity. niid E,, is the clectric permittivity offrer:

space.

Unlike the model proposed by Zimmerrnim et al. ( 1974), this model tlikes into ;iccount

the surface tension and viscosity of the membrane.

2.6 PEF Treiitment of Milk

Inactiviition oi'microorgrinisrns in liquid foods using PEFllas been stiitlird extensivdy.

A summary of the research conducted on milk and simulated milk ultra-filtrritc is given in

Table 2.1.

According to Martin et al. ( 1996) the inactivation of E. coli using PEF is more Iimited

in skim mi& than in a buffer solution when exposed to similar treatment conditions of field

intensiry and number ofpulses due to thecornplex coniposition of skiin milk and the presence

of proteins.

The influence of milk fat on the PEF inactivation of microorganisms is unclcar. Reina

et (il. (1998) observed no significant differences in the inactivation of L. monocytoyrnrs in

whole milk, 2% milk, and skim milk. Less than I log difference between inactivation of E.

coli in milk and in phosphate buffer was reported by Dutreux et al. (2000), However.

14

experimentsconducted by Grahl and Markl(1996) indicate that the fat content of the medium

is inversely related to microbial inactivation.

The maximum field strength which can be applied to a fluid food is oot only limited

by the PEF apprirlitus but also by the electric and dielectric properties of the food itself. The

goal in PEF treatment is to induce the dielectric breiikdorvn of the cell nicmbrrine whilc

preventing the dielectric breakdown of the tluid food. Dielectric brrakdown, wtiicli is

observed as a spark, can be reduced by using a smooth electrode surface to minimize electron

emission; employing round electrode edges to prevent field enhancement; ensuring that the

xtual applied field strength does not exceed the dielectric strength of thc tluid food; and

clzgassing rhr: trcarinent clilimbci- prior CO treatment tocIimin;ite gtis bubble foimition (Zliang

et al., 1995a).

Zhanget al. ( 1994) performed dielectric strength experiments with miik and observed

local dielectric brerikdown when gas bubbfes were present. They found gas bubble formation

to be directly related to the energy input ofeach pulse which limits the electric field thlit can

be applied. Since there is a lirnit to the electricid parameters which rnay iiscd for PEF

inactivation of microorganisms. other variables have been studied which improve the r f f i~icy

of PEF treatment.

Hulshegeret al. (198 1) found that increasing the initial temperature of the medium in

which cells are suspended lead to an increase in microbial inactivation. Coster and

Zirnmerrn;in (1975) suggested thnc the increase in the rate of inrictivrition with increasing

temperature mily be due to the increase in the electric brerilidown potcntid of the b;icttli-id cc11

membrane.

The temperature of the milk simple has a significant influence on the membrane fluidity

15

properties of the bacteria. At low temperiitures, the phospholipids in the cell membrane are

closely packed in a rigid gel structure, while at high tempentures they are less ordered and

the membrane has a liquid-crystalline structure. Jayaram and Castle ( 1992) proposed that

temperature related phase transition of the phospholipid molecules from gel to liquid-

crystalline phase is associated with a reduction in bilaycr thickness which makes the ce11

membrane more susceptible to breakdown at higher temperatures.

Juy;irarn and Castle ( 1992) réportcrl that increasirig the metliiim teiripcr:itiirc from 24

ta 60°C rcsulted in an increase in PEF inüctivrition 0f~ucrobucifiu.s brevis. Pothak;itiiury et

al. (1996) found that the rate of inactivation of E. d i , subjected to PEF rit selected

temperatures ranging between 3 and 40°C. increased with an increase in the temperiture.

Sensoy et al. ( 1997) studied the temperature effect on Sdniorielli cliiblitl in skim milk between

the range of IO to 50°C and found thiit increasing the tcmper;iture incrc;iscs the sensitivity of

microorganisms to PEF treatment. Grrihl and Marke1 ( 1096) perforrned scnsory evrilliations

on milk that had been treated by PEF ai tempemtures ranging from 45 to 50°C and concludcd

that there was no significant deterioration.

The effect of initial microbial concentration on PEF inactivation appears to depend on

the type of org:inism. Zhung et al.. (1994) founcl thiit lowcring the initial concentration of S.

cerevisitic. resulted in an increase in the inactivation rate while no siich increase wtis observecl

with E. coli. Alvarez et al. (2000) studied the inactivation of S. senfienberg and concluded

that microbial inactivation was not a function of the initial cell concentration.

Recently, mearchers have started combiningantimicrobials with PEF treatment. Tible

2.2 summarizes the expetiments which have been conducted. Pol et al. (1999) subjected

Bacillus cerelis to low doses of nisin (0.06 pp/ml) and PEF treatment. The combination of

16

treatments resulted in a reduction of 1.8 log units more than the sum of the individual

treatments which indicates that PEF is able to enhance the bactericidal action of nisin.

Calderon-Mirandaet al. ( 199%) investigrited the inactivation of Lisreriri innocira in skim milk

by pulsed electric Field and nisin. They noticcd a synergistic effect with the addition of nisin

Followiiig PEF treatment on the inactivation of L. iri~ir~ciin. They obtriined a 3.8 log rcduction

for an electric field intensity of 50 kV/cm and 32 pulses followed by exposure of the

microorganism to 100 IU nisin/ml. Kalchayanand et al. ( 1994) observed thrit nisin was able

to enhance the PEF inactivation of L. rnoriocyrogenes, E. coli and S. rvphitriuriiirrr. Using

tr;insmission etrctron microscopy, Calderon-Miranda et al. ( I99b) observed that the

combinatioii of PEF rind nisin exhibited an ridclitive ef'fect in the niorphulogicrit daiii;ige on

Listeria inrrociru.

While research on PEF ireritmeni of milk has indicated some potential for use as a

pasteurization technique, more is required before this process can be considered as a viable

alternative to HTST pasteurizaion. Almost dl of the resenrch on PEF treritment of milk has

been performed on skim milk or simulared milk ultratiltrate (SMUF) which has been

inoculated with one or more microorganisms. This is useful in determining the susceptibility

of microorganisrns to PEF treatment relative toothermicroorganisms, but it does not indicate

the effectiveness of this process for milk with a natudly occuning microbiül population. The

nnturai micro floraof milk would be much more resilient than the inoculated microorganisms

which would be under much ge;iter stress due to the ctianging environmciits rissociatecl with

growing, harvesting and inocutating the milk samples. The only reported research on PEF

treatment of raw skim milk was by Raso et al. (1998a). They were only able to achieve a 2

logreduction in the microbial population. The next logical step in determining the feiuibility

17

of pasteurizing milk by PEF is toconduct more inactivation studies on raw milk iind to try und

increuse the Ievel of microbial inactivation.

The principal advantage of PEF treatment over heat pasteurization is the low energy

requirements associated with this process. The energy input for the HTST process (assurning

100% efficiency) is 364 Jlml compared to 28 J/ml for a 6 log reduction using il PEF system

IBarbosa-Canova et al.. 1999) The electricril energy iised by a PEF system depends on the

type ;ind size of the piilse wriveform beins applietl to the tliiitl food s;iiiipk. The developmcnt

of instant-charge-reversal pulse appears promising as it can drristicalIy reduce enorgy

requirements to as low as 1.3 Jlml (Barbosa-Canovas et al., 1999). however. this typeof pulse

waveform hûs received limited attention relative to both square-wave and exponential decuy

pulses.

3.0 Research Objectives

The objective of this research is to improve the efficacy of PEF ireatment by

combining this process with othzr treiitments. Microbial inlictiv~ttion by PEF is a resiilt of

pore formation in the cell membrane, therefore. combining PEF rreatmeiit w ith other

treatments which affect the cell membrane may result in synergistic cffects.

Higher processing temperatures result in an increase in microbial inactivation by

PEI: since an increase in temperature increases the t'lu idity properties of the ceil

membrrine. however, ro inriintain PEF treatmcnt ris a non-thermd tcchniclile. it is i i ewssxy

ro determine thiit the processing temperatlire is not responsible t'or microbial inactiviitioii.

The effect of milk temperature on microbial population will be determined for

temperatures ranging from 50 to 55°C. A processing temperature with a low fevel of

microbial inactivation due to thermal effects will then be selected from these results for

use i n future PEF cxperimcnts.

Lriwei-ing the pH of raw skim milk mriy providc an cnvironmcrit;il strrs.+ on rfie

rnicroorganisrns and possibly weaken the cell mernbriine of the microorgrinisrns miiking

them more susceptible to PEF treatment. The effect of pH on microbial population will be

determined with and without PEF treniment for raw skim milk with pH values rringing from

its nriturril pH of 6.7 to 5.0, which is above the isoelectric point of milk ( p H 4.6).

PEF treatment of raw skim milk con~aining iintimicrobials will be investigated.

Since nisin and Lysozyme are more effective against Gram-positive bacteria while PEF

treatment is more effective against Gram-negative bacteria, combining these two treatments

should provide a synergistic effect on microbial inactivation in n w skim milk which

contains both Grlirn-positive and Griitn-negativc bncrsria. The efkcr of nisin and Iysozyrne

on the microbial population of milk will be deterniined with and without PEF treatment.

Microbial inactivation resulting from PEF treatment increases as the number of

applied pulses are increased. With the addition of antimicrobials, the effect of varying the

pulse number will be observed at a constant electric field and pulse rate. The effect of

piilse niimber on microbiül inactivation wil1 be dcterrnined by applying 10. 50 and Y0

pulses to raw skim milk containing nisin and lysozyme.

The low level of microbial inactivation achieved by PEF treatrnent of milk has

been postulated to be due to an inhibitory effect caused by milk proteins. A clarification

procedure will be used to suspend the casein micelles which should negate this inhibitory

effect. The k:isibility of PEF tlutmenr of cl;irificrl niilk will flicil bc clctrrminrct.

4.0 Materials & Methods

4.1 Skim Milk

Early experiments were performed on autoclaved skim milk inoculated with Lisreritr

rnonocvtogenes, Escherichiri coli, and Pseirdomonc~s jiirorescens (ATCC # 1 5456). The

procedure iised to hrirvest these microorganisms is given below:

Preparation of Bacterial Culture

1 . Mix 500 ml of Tryptic Soy Broth (TSi3) and autoclave at 12 1°C for 15 min.

2. Innoculate 250 ml of TSB with bacterial culture and maintain at room temperature for

24 h.

3. Transfer0.25 ml ofcultiire to 230 ml of frtsli bt-oth rtntl maint;iin ;it room tempcrriture

for rinother 24 h.

4. Harvest microbial cells usingacentrifuge (Beckinan J2-MC. New York) broth for 10

min at 4°C and 6000 rpm.

5. Wüsh the cells twice with sterile distilled water and resuspend theni in 1 L rrutoclwrd

skim milk. This should provide a concentrrition of 10'- 10~clls/nil .

Raw skim milk wüs obtained frorn Gay Lra Foods, Guelph. ON. To achievc microbial

levels in the range of 10' cells/ml, the milk wris left for approximately two weeks at 4°C.

Since initial microbial levels varied frorn batch to batch, it was easier to grow bacteria at this

temperature while avoiding spoilage of the milk. This resulted in the microbiül population

being largely represented by obligrite psychrophiles and psychrotrophs. with mesophiles and

thermophiles remaining rit their initial lrvels (Garbutt. 1997).

4.2 PEF System

The PEF processing system used for these experiments (Fig. 4.1) consists of a 30 kV

DC high voltage pulse generritor, aO. 12 pF capacitor, a series of 6 iMQ resistors. r i thyratron.

and a circular treatment chamber (Fig. 4.2) consisting of two polished stainless

steel electrodes (16.5 cm in diameter) surrounded by Delrin, which is an insulating material

(Ho et al., 1995). A drawing of the treatrnent chamber is given in Figure 4.3. A 3 mm Delrin

gasket is inserted between the two electrodes resulting in a process volume of 49.5 ml.

Gaskcrs ut'inciensecl thickness caii bt. iriscrterl tci iiicicisi: tlic total ircitinent voluinc. howcvcr.

this reduces [lie peak electric tïeld strength. Thc circuit configiiration gerierated instant-

charge-reversal pulse waveforms (Fig. 4.4) with a pulse width of 2 ps and a rise time in the

range of nanoseconds.

Unlike exponential decay or square wave pulses which increase the temperature of

the treated srimple throiigh ohmic heciting, instant-charge-reversal piilses do not cause rin

increase in the sample temperature. For an input voltage of 24 k V , the pulse energy is 34.6

Jipulse (Ho et al., 1995). The pulse rate can be controlled manually, using single pulses,

or automatically with pulse periods ranging from 2 s to 1 1 s, however, a pulse period of 2

seconds was used for all experiments.

The resistors are imrnersed in oil to prevent coronri and x c i n ~ . The sample is

drawn into the treatmcnt chamber using a laboratory scrile v:icuum piiitip. The tipplied

pulse waveforrns were measured in the treatment chamber with a high voltage electrode

connected to an osciiloscope (Tektronix, TDS-340). A block diagram of the PEF treatment

system is given in Figure 4.5.

Figure 4.1. PEF treatment system.

Figure 4.2. Treatment chamber (unassembled).

26

Figure 4.4. hstant-charge-reversal pulse waveform for skim milk at 80 kV/cm and 24°C. Scale: I square = 5000 V (vertkü: j, 5 ps (horizontal).

Figure 4.5. Block diagram of PEF treatment sysiem (adapted from Ho et al., 1995).

The foilowing method was used to apply puises to the skim milk sample:

PEF Treatment Procedure

Turn on filament switch locatedon pulse console and dlow thyratron filament io heat

up for approximately 10 minutes.

Turn on main switch located on power supply.

Place sample in treatment chamber.

Attach electrodes to treatnient charnber.

Depress "Lock" and "Hold" biittons siinultrineously on pciwer supply.

Adjust voltage on power supply to 24 kV using course and fine çontrol knobs.

Adjust pulse period to 2 s by turning pulse rate knob counter-clockwise.

Flip trigger switch on pulse console to "auto''.

Apply desired number of pulses to treritrnent chiimber.

Depress "Off' button located on power sripply,

Discharge any remaining voltage using "grounding rod".

Remove electrodes from treatmen t chamber.

Remove sample from treatment chamber.

The treatment chamber developed by Ho et al. (1995) required modification in order to be

compatible wilh milk ris the fluid food sample: probterns with the existins setup wcse

encountered while trying to achieveconsistent volumes of milk. Excessive foiimin_o of the niilk

sample during filling resulted in the presence of air inside the treatrnent chamber. This caused

sparking and lead to dielectric breakdown of the sample. In order to reduce foaming, the

ripplied vacuum was vriried; an operating pressure of 20-25 kPü proved to be optimal. The

effect of increrising the diameter of the tubing used to fiIl the treatrnent chamber from 4 mm ID

to 6 mm ID was also investigated. The 6 mm ID tubing allowed for consistent treatment

charnber volumes, however, foaming still posed a problem and special care was required to

iivoid allowing iiny air inside the chamber. The entrmcc and exit poiats of the treiitrnent

chamber were cnlarged to rillow for the laigrr tubing and the rlircatl Icngth ~ ) f the v;ilvr plugs

was increased to eliminate leakage of the sample.

Another method was developed to fil1 the treatrnent chamber using a sterile 60 cc

syringe with a hypodermic needle (Becton-Dickinson & Co., New Jersey). A hole drilled

through the tretltment chamber's gasket to facilitate injection was plugged by insening a sealed

plastic piece of tubing of the same dirimeter as the hypodermiç needle. This metliod provecl

ineffective since even a gradua1 depression of the syringe plunger resulted in leakiige of the

sample between the electrodes and the insulation.

Different rnethods for heating the skim milk samples before PEF treatmcnt were

investigated. A tubular circulation heater (Chromalox. CVCHS- 15 1. Pittsburgh) in

conjunction with aperistriltic pump wris evaluated in ntitlition to usin? eithcra niicrowavt: or

a water bath to heat the skim milk sample to the desired temperature. The main criterin wm

to avoid heating the milk sample above the desired temperature to avoid thermal destruction

of the microbial population. The system incorporating the circulation heater required large

amountsof sampledue to itscontinuous nature. Since PEFexperiments were to be performed

in batch mode, it was decide to use a heating system which hcilitated the use of smsller

volumes of milk. Microwave heating was the most efficient heating method, however. even

when the initial temperature of the milk was held constant, the same heating times did not

always result in aconsistent final temperature. While a water bath required more time for the

30

sample to come up to the desired processing temperature (approximritely 8 min.), it was the

method of choice since there wris no risk of reinperiiture over-shoot rind the time required was

much lower than the 30 minute treatment times used to determine the processing temperiturc

as reported in Section 4.5.

4.3 Sanitation Procedure

It was necessary to tlevelop a ~tinitiiiion procerilire which t.nsuretl a sterilc: processing

environment while preventing any microbial inactivation due to the presence of sanirizin2

agents. The following method was developed:

Each sample was injected into the treütment chamber using the following method:

Procediire for Injecting Sample into Treatment Chamber

Draw 100 ml of a SO% chlorine blench saluiion tlirough the trc;rtitient climbei usin;

a vacuum pump.

Rinse treatment chamber with 600 ml of distilled water.

Pre-heat treatment chamber using I L of distilled water at 90°C.

Draw sample into treatment chamber. Allow ripproximately 300 ml of sarnple to run

tlirough chamber, then close the upper valve.

Allow vacuum pump to remain on for an additional 30seconds. in order to remove riny

remaining air bubbles, and then close the bottom valve.

Attach electrodes to treatment chamber.

Apply PEF treatment.

Clean top and bottom ports of treatmentchrimber by applying chlorine bleach solution

with an autoclaved cotton swab.

Rinse top and bottom ports of treatment chamber with sterile distilled water with an

autoclaved cotton swab. Repeat three times to ensure removal of any residual chlorine

bleacti solution.

Insert 7 cm section of autoclaved tubing into bottom port ro facilitate rrn1ov;il oi

sample.

Remove sample, by gravity, into sterile sample containers.

Pre-heating the treatment chamber was required in order to maintain the temperature of the

milk sample at 52°C during processing. The 7 cm section of tubing insertetl into the bottom

port prevcnted the samplc from dribbling across the bottom of the chiimbcr riiiring retnowl.

To ensure that this procedure was not responsible for any microbial inactivrition. an

experiment was perforrned without the application of PEF treatment white includingatl of the

remaining steps.

4.4 Microbial Eniinieratioii

Enurneration of colony forming units (cfu) was pcrformed according to procedutes

outlined in the Compendium of Methods for the Microbiological Examination of Foods ( L 942).

Samples were serially diluted in sterile O. 1 % peptone water and plated using the Surface Plrile

technique. Dilutions were made in the riinge IO0to IOX with O. 1 ml from e x h dilution being

plrtted. Plates were made using tryptic soy a p (TSA). which is a nonselrctive iigrir. The

plates were incubated at 37°C for 40-48 hours, and cfulml was calculated using plate counts

between 25 to 250. The plate counts of al1 dilutions were recorded to ensure that each

successive dilution of srimple resulted in ri 1 log reduction of cfiilrnl. therefore iivoiding the

use of riny erroneous pliites. When therc were no colonies on the plate with the iintlilii~ecl

sarnple ( IOU), the plate count was recorded as c I O cfulml. This w u to account for the 0. I ml

sample plating volume. The microbial procedures employed for the preparation of agar plates

and dilution tubes are outlined below.

Procedure for Preparation of Agar Plates

1 . Wrigh out 39.5 g of TSA (tryptic soy agar) and ~idd ta I L of distiilcd watcr.

2. Mix thoroughly and bring solution to a boil.

3. Autoclave agar solution at 121°C for 15 minutes.

4. Pour approximately 15 ml of the agar into sterile petri plates.

5. AlIow plates to dry in a 1arnin:ir tlow haod.

Procediire for Preparation of Dilution Tubes

1 . Weigh out I .O g of Peptone and add to I L of distilled writer.

2. Mix thoroughly.

3. Pour 9 ml of Peptone Water into test tubes.

4. Autoclave test tubes at 121°C for 15 minutes.

4.5 Statistical Treatment of Results

The collected data were analysed using the SAS' (SAS, 1990) General Linerir Mode1

(GLM) procedure for unbalanced data sets and Analysis of Variance (ANOVA) procedure for

balanced data sets. Duncan's LSD was used to determine the statistical significance of the

meüns. The rnean and standard deviiition. o. were calculritecl for the log reduction of

microorganisms for each set of experiments. A 95% confidence interviil was used for a11

procedures. The results of the statistical anaiysis are provided in Appendix A.

4.6 Effect of Temperature

Previous work has indicated that temperatures rangin2 from 50-55°C are the most

effective for PEF inactivation (Grahl and Markl. 1996: Sensoy et al.. 1997: and Reina et al..

1998). Higher processing temperatures increase the PEF inactivation of microorganisms,

however, to maintain PEF treatment as anonthermal method toputeurize milk, it is importrint

to vet-ify thrit inactivation is not aresultof thermal effects. Processina temperatures of 50,5 1 ,

57 ,53.54, and 55°C were evaliiated by pliicing 100 iiit saniples ot'riiw skiin rnilk in ri water

bath for thirty niinutes. Srimple temperatures were measured a microprocessor bused

thermocouple accurate to + O.I°C (Oakton, 35628-00, Quebec) Four replicarions were

perforrned foreach temperature with the samples king plated before and after heat treatment.

4.7 Effect of pH

The pH of raw skim milk, which is approximately 6.7, was adjiisted to viiliies of 5.0,

5.5,6.0 using hydrochloric acid (HCI). Since the isolelectric point of milk is 4.6, the pH

could not be lowered any further without causing the precipitation of casein. Thcse pH

adjusted samples were subjected to PEF treatment at 4°C and rilso at P C . A peak electric

field strength of 50 kV/cni and 50 pulses were applied to e x h sample. Thrce replications

were carried out for al1 pH experiments.

4.8 Effect of .4ntirnicrobisrl Addition

Nisin with an activity of 1061U/g and lysozyme from hen's egg white wiis obttiined

from Sigma-Aldrich Canada Ltd., Orikville, ON. Lyso:Chrisin, which is a 1 :3 combin;ition

of lysozyme and nisin, was obtained from Canadian Inovatech Inc., Abbotsford, BC.

Lyso:Chrisin was used at aconcentration of O.W;/ml of milk which gave an activity level of

1000 pg lysozyme/ml and 3000 iü nisinlml. Nisin and lysozyme were also used alone at a

concentrition of 0.04 @ml which gave activity levels of JO00 IUIml and 4000 pglinl

respectively. The antimicrobials were mixed into the raw skim milk samples at 4°C using a

magnetic stirrer. prior to heat treatment.

The effect of lysozyme, nisin, and Lyso:Chrisin were determined in raw skim milk rit

4°C and rit QaC, Four milligrrims of each lysozyme. nisin. iind Lyso:Clirisin were addecl to

100 ni1 ofraw skim milk at the concentrations mentioned iibove ancl held at tht.cori.espoiidin~

temperature for 30 minutes before being plated. Thecombinedeffect of heat treatment (52°C).

the addition of antimicrobials, and PEF treatment was investigated. A petik electric field

strength of 80 kV/cm and 50 pulses were applied to each sample. Four replications were

performed for each experiment.

4.9 Effect of Pulse Number

Lysozyme (1000 pglml) and nisin (3000 Wml) were added to raw skim milk. Milk

samples were subjected to 20,50, and 80 pulses with a pulse frequency of 2 S. Each sample

was processed at 52OC and the peakelectric field strength was maintained at 80 kV/cm. Four

replications were completed for each set of pulse numbers. Niimei-011s studies have been

performed on the effect of pulse number (Hulsheger et al., 198 1 ; Jtiyarrim and Cristle, 1992:

Martin et al.. 1997, Calderon-Miranda et al., 1999a), therefore. it was decided that

experiments without heat treatrnent or the addition of antimicrobials would not be necessary.

4.10 Clarification of Milk

It was postulated that the inhibitive effect of milk protein on PEF inactivation çould

be determined by clarifying skim rnilk. Owen and Andrews ( 1984) developed the followin;

procedure to clarify milk:

Procedure for Clarification of Milk

1. Prepare EDTA stock solution: 50% (wlv) solution of tetrasodium EDTA acljiistsd to

pH 7.5-8.0 with concentrated HCI.

2. Prepare Triton X-100 stock solution: 50% (vlv) solution of Triton X- 100 in absolute

ethanol.

3. Add 8 ml EDTA stock solution and 6 ml Triton X-100 stock solution to 60 ml skim

milk.

4. Incubate solution for 60 minutes at 37°C.

Clarification of milk causes the casein micelles to dissociate resulting in the

formation of a translucent solution. Raw skim milk was plated before and afterclaritïcrition.

The existing procedure of using a vacuum pump to draw the sample into the treatment chamber

was inadequate with the clarified nw skim milk sarnples. The vacuum punip caused

excessive foaming of the sarnple resulting in low liquid volumes combined with air in the

treatment chamber. Other rnethods were attempted, such as injecting the sample into the

treatrnent charnber using a syringe, however, these too proved ineffective.

While it would be intercsting to determine the exx t role rhat psoteiii plays in PEF

treatment of skim milk. it was decided that clürified rnilk was not compatible with the PEF

treatment system.

5.0 Results & Discussion

3.1 Preliminary Experiments

A Gram stain was perfomed on the Lisrericr monocpugerirs and Psrrr~fo~ttunas

fluorescens cultures. The gram stain on the Listeria moriocytogenw culture indicated chat

there were two species present, however. neither bacteria apperired to be Gram-positive or

to be rod-shaped which is a characteristic of Listeria species. This culture was abandoned

in fiivoirr of the P.s<iirtlo~rzoricis~~i~r~.sce~~s culture which containeri Gram-negritive rods. us

êxpected.

Skim milk, at 4 ° C was inwulatecl with Pserrtlurnoncisflt~~)rcscens and subjected to

PEF treatment. A peak electric field strength of 90 kV/cm and 20 pulses wns applied to each

sample. A microbial reduction of slightly more than I log cycle (Table 5. t ) was achieved

indicating that a higher treatment temperntiire would be necesswy in order to achievc

significant microbial inactivation. Since limited reseürch hiis beeii conductsd on PEF

treatment of raw milk, it was decided that future experirnents would be ccirried out using rtiw

skim milk.

Table 5.1. PEF treatment (90 kV/cm, 20pulses) ofskim milk inoculated wiih Pserrclomonas jlwresceus.

- - -- . -

Trial # N,, (ch/ml) N (cfuirnl) log ?UN,,

1 4.8 x 10" 2.9 x 107 -1.2 2 6.7 x lo7 4.6 x IO6 - 1.2 3 3.1 x 10" 1.3 x IO7 - 1.4 4 4.3 x IO8 9.7 x IO6 - I .O

No= initial rnicrobial population, N = final microbial population

Table 5.2 gives the results from the experiment which was designed to verify that the

sanitation procedure used on the treatment chamber was not responsible for any microbial

inactivation.

Table 5.2. Injection of samplc into treiitinerit cfii~m ber without iipplicat ion of piilses.

- - . --

Trial # Inlet temp. (OC) Exit tcmp (OC) N , (cfii/rnl) Y I I ) log NIK,,

There wris nodifference between the initial and final counts of the microbiril populiition wliich

proves that the srinitation procedure is effective. The clririt'kation procediire resulted in a log

reduction ofapproximately4 in the initial microbial concentration (TabIc 5.3), ~ i v i i i g :i find

concentration in the range of IOJcfu/ml.

Table 5.3. Effect of clarification procedure on raw skim milk. - --

Triul # N,, (c~ulm1~ N (cf~ilnil j los NIN,,

I 3. I 107 4.1 x 10; -3.9 2 4.0 x IO' 6.7 x IO' -3.8

5.2 Effect of Temperature

The effect of temperatures ranging from 50 to 55°C on the microbid population of rrtw

skim milk are given in Table 5.4. Heriting rnw skim milk to temperrittires of 52°C or lower

had no significanteffect on the microbial population (Fig. 5.1). At temperatures of 53°C and

higher, microbial inactivation starts to occur.

Table 5.4. Heat treatment of rnw skim milk.

Trial # Temp. ( O C ) N,, (cfulml) N (cfulml) log N/N,, Mean u

I 50 4.8 x 107 3.4 x 107 -0.1 -0.1 o. 1 2 50 6.7 x 107 5.0 x 107 -0.1 3 50 3.1 x 107 3.5 x 107 o. 1 4 50 4.3 x 107 2.5 x 107 -0.2

It w u drcided to use a processing temperature of 52°C for the rcmaining experiments. even

though at 54"C, there wris less than a 0.5 log reduction in microorganisms. The rcason for

this was to provide a buffer which would ensure that microbial inactivation would not be due

to thermal effects since it is difficult to experimentally determine the helit resistance of

rnicroorgünisms (Garbutt, 1997). Since the PEF system ernployed i n these experiments

produces instnnt-charge reversal pulses, the temperattire of the raw skim milk could be

maintained throughout the treatment. The statistical analysis of results using the ANOVA

procedure is given in Thle 5.5.

Table 5.5. ANOVA for the effect of temperature on microbial inactivation.

. ---

Soiirce Degrees of Frerciom Melin Squrirc Probability > F

Model 5 O. 138 0.00 15 Error L 8 0.077 Totaf 23

Microbial inactivation at 50°C was statistically significant compared to inactivation at 5 5 ° C

however, increnients of one degree did not result in il stiitisticülly significant change in

tiiicrobial inactivation.

Table 5.6. Duncan's LSD for the effect of temperature on microbitil intictivütion.

Temperature Mean log N/N,, Duncan's LSD

50 -0.1 1 a b 5 1 -0.07 a 52 -0.16 ;i b c 5 3 -0.3 l b c 34 -0.37 c d 5 5 -0.56 d

Temperatures with the samecorresponding letters under the heading 'Duncan's LSD' in Tiible

5.6 are not statistically different from each other, Le., the mean log reduction at 50,5 1 , and

52°C are not statistically different.

50 51 52 53 54 55 Temperature

Figure 5.1. Effect of temperriture on the microbiid population in r w skini iiiilk.

Raw skim milk subjected to PEF treatment at 52'C resulted in ri 1.3 log reduction of

microorganisms. A peak electric field strength of 80 kV/cm and 50 pulses were applied to

each sample. Raso et al. (1998) reported ri reduction of approximntely 2 log cycles for PEF

treated riiw skim miIk usin; an input voltage of s'O kV ancl 50 pulses. The electric Field

strength and treatment temperature were not reported. The increase in sensitivity rit higher

temperatures may bedue tom increase in the fluidity of the membrane phospholipids resulting

in a more fragile plasmatic membrane (Barsotti and Cheftel, 1999). Coster and Zimmerman

(1975) suggested ihat the increase in the rate of inactivation with increasing temperature rnay

5c due to the decrease in the eloctric brerikdown potential of the bacterird cell membrane.

Fernandez-Molinaet al. (2000) found chat combining aprocessing temperriture ofSO0C for 6

42

seconds followed by PEF treatment at 30 kV1cm with 22 pulses was effective at reducing the

spoilage microbial flora in skim milk. Using such a high processing temperature defents the

purpose of combining PEF treatment since the microbial inactivation would be due rilmost

entirely to thermal destruction iind the resulting organoleptic qiitilities of the processed milk

woukl be similar to HTST pasteurized milk.

5.3 Effect of pH

At a temperature of 4°C. PEF treütment had no effect on the pH ~idjusted skim milk

srimpIes (Fig. 5.2).

Table 5.7. PEF treatment (80 kV/cm, 50 pulses) of rnw skim rnilk at varying pH levels.

- - - - - - - -- -

Trial # pH No (cfulml) N (cfulml) log N/N, Mean a

I 5 .O 1.8 x IO8 1.6 x IOX -0.1 0.0 0.1 2 5.0 4.7 x IOX 5.4 x IOX O. 1 3 5 .O 6.9 x IOn 5.7 x IOX -0.1

There was no discernible difference in microbial inactivation between milk at a pH

of 5.0 or at its natural pH of 6.7 (Table 5.7) which indicates that the ceIl membranes of the

rnicrmrganisms present in milk are not adversely affected by changes in the pH.

Figure 5.2. Effect of pH on microbial population in raw skim milk.

This agrees with Hulsheger et al. (198 1 ) who observed no effect on the inactivation

of E. coli frorn varying the pH of phosphate-buffer solutions in the rringe of 5 to 9. A faod

grade acid, such as acetic or citric acid, would have been more appropriate for adjiisting the

pH of milk, however, the type of acid used would not have affected the end resul!.

Heating the samples to 52°C caused precipitation of the caseins when the pH was at

6.0 or below due to the temperature related increase in the isoelectric point of the caseins.

Since slight reductions in pH resiilted in the precipitation of proteins when subjected to mild

heat treatments. lowering of the pH is not compatible with PEF pasteurizrition of skim milk.

5.4 Effect of Antimicrobials

The antimicrobials lysozyme and nisin, either alone or in combination as LysoChrisin.

did not inactivate a signifiant number of microorganisms when iidded to rnw skim milk at

4°C. When the temperature was increased to 52°C. microbial inactivation wcis st i l l tow. with

the 1:3 combination of lysozyme and nisin having the greatest efkct, resultinç in ri 1.2 log

reduction.

Table 5.8. Addition of lysozyme to raw skim milk (4000 pglml).

Trial # Temp. ( O C ) N,, (cfu/ml) N (cfulml) log NIN, bleaii (3

I 4 1.4 x 107 1.5 x 107 0.0 -0.1 0.1 2 4 1.6 107 1.5 x 107 0.0 3 J 5.8 x 107 3.2 x 107 -0.3 4 4 9.2 x 107 3.8 x 107 -0.4 5 52 8.3 x IO6 6.5 x IO6 -0.1 -0.2 0.2 6 52 6.7 x 10" 4.4 x IO6 -0.2 7 52 5.8 x IO7 3.6 x IO7 -0.2 8 52 9.2 x IO7 3.2 x IO7 -0.5

Table 5.9. Addition of nisin to raw skim milk (4000 iülml).

Trial # Temp. ( O C ) No (cfutml) N (cfutml) log N/N, Mean u

I 4 1.4 x IO7 4.8 x IO6 -0.4 -0.5 0. I 2 4 1.6 x \O7 6.7 x IO6 -0.4 3 4 5.8 x 107 3.1 x 107 -0.5 4 -I 9.2 x 107 4.3 x 107 -0.7 5 5 2 8.3 x IO" 4.8 x 105 -1.0 -0.8 0.2 6 52 6.7 x IOh 6.7 x IO" -0.7 7 52 5.8 x lo7 3.1 x lo7 -0.6 8 52 9.2 x IO7 4.3 x lo7 -0.9

'Table 5.10. Addition of Lyso:Chrisin [O rriw skim milk ( 1000 pg lysozyme/ml: 3000 [Li nisin/ml).

Trid # Temp. (OC) No (cfulml) N (cfulml) log N/N, Mean u

1 4 1.4 x 107 4.4 x IO6 -0.5 -0.7 0.2 2 4 1.6 x 107 3.1 x IO6 -0.7 3 J 5.8 x 107 1.5 x 107 -0.6 4 J 9.2 x 107 1.1 x 107 -0.9 5 52 1.4 lo7 1.3 x 10" -1.0 - 1 .7 O. I 6 5 2 1.6 x 107 1.1 x IOh -1.2 7 5 2 5.8 x IO' 3.4 x IO" -1.2 8 5 2 9.2 lo7 5.0 x IOh -1.3

As seen in Tables 5.8,5.9, and 5.10 the addition of antimicrobials to raw skirn milk does not

have much of an effecton the microbial population. The statistical analysis ofresul~s using the

ANOVA procedure is given in Tnblc S. I 1 .

'l%blc 5.1 1. ANOVA for the cffects of antiniiciobids and tt.iiipci;truic ut1 tiiicrobial inactivation in raw skim milk.

Source Degrees of Freedom Mean Square Probability > F

Mode1 5 0.570 0.000 1 A 2 1 .O68 0.000 1 T I 0.540 0.0002

Error 18 0.025 Total 23

Table 5.12. Duncan's LSD for the effects of antimicrobials and temperiture on microbial inactivation in raw skim milk.

Antimicrobial Temperature Mean log N/N, Duncan's LSD

lysozyme 4 -0.16 a lysozyme 52 -0.24 b

nisin 4 -0.48 c nisin 5 2 -0.S I d

Lyso:Chrisin 4 -0.68 t'

Lyso:Chrisin 52 -1.17 f

The addition of lysozyme, nisin, and Lyso:Chrisin at 4 and 52°C resulted in significantly

different effect on microbial inactivation (Table 5.12). When combined with PEF

treatment using a peak electric field strength of 80 kV/cm and 50 pulses. the net effect wss

a significant increase in the microbial iniictivation.

Table 5.13. PEF tre'eatment (80 kVIcm. 50 pulses) ofraw skim milk contliining lysozyme (4000 pglml).

- - - -. . .. -- -

Trial # N,, ( c h / 1111) N t cfii/iiiI) los WN,, Mean u

I 1.5 x 10" 1.3 x 10 -3.1 -3.7 0.4 2 1.6 x IOn 1.5 x 10' -3 .O 3 1.1 x \on 1.9 x 104 -3.5 4 1.3 x 10' 1.7 x ld -2.9

Table 5.14. PEF treatment (80 kV/crn, 50 pulses) of rriw skim milk containin; nisin (4000 IU/rril).

Trial # N , (cfuJml) N (cth/ml) log NIN,, Mean O

I 1.1 x 107 < I O -6.0 -5.9 0.5 2 2.7 x 107 < IO -6.4 3 1.1 x 10" 180 -5.6 4 1.3 x IOR 110 -5.7 5 6.4 x Io7 50 -5.4 6 1.7 x 107 c 10 -6.2 7 3.4 x 10' 130 -5.1 Y 2.0 x 107 90 -4.9

Table 5.15. PEF treatment (80 kVIcm, 50 pulses) of tliw skim milk contriining Lyso:Chrisin (1000 pg lysozyrne/rnl; 3000 IU nisinlml).

Trial # No (cfulml) N (cfu/ml) 1% NN,, Mean O

1 3.2 x IO" < 10 -7.5 -7 .O 0.7 2 1.1 x 10" < IO -7.0 3 1.1 x 10" < IO -7.0 4 1.3 x IOn 30 -5.7 5 1.4 x IOn 60 -5.7 6 1.4 x IOX < 10 -7.1 7 1.6 x 10" 1 O -5.8 s 8.4 x 107 < I O -6.9

The addition of lysozyme (Table 5.13) resulted in a 3 2 log reduction while nisin (Table 5.14)

reduced the initinl microbial level by 5.9 log iinits. Lyso:Chrisin (Table 5.15) provided the

highest inactivation with a 7.0 los rediiction. The statistical aiialysis of resiilts usiiig the GLM

procedure is given in Table 5.16,

Table 5.16. GLM for the effect of antimicrobials on PEF treatment (80 kVIcrn, 50 puises) of MW skim milk.

- - - - - - --

Soiirctt Degiees of Freedoin Merin Square Probubility > F

iModeI 2 15.843 0.000 1 Error 17 0.364 Total 19

Table 5.17. Duncan's LSD for the effect of antimicrobials on PEF treatment (80 kV/cm, 50 pulses) of raw skim milk.

Antimicrobial Mean log NIN,, Duncan's LSD

lysozyme -3.18 ;L

ni sin -5.93 b Lyso:Chrisin -6.98 c

The log reductions achieved by combining lysozyme, nisin, or Lyso:Chrisin with PEF

treatment (80 kV, 50 pulses) were significantly different (Table 5.17). The initial microbial

levels were in the range of IO' to IO*. The fina1 rnicrobiiil levels for PEF treated rnilk

containing nisin and Lyso:Chrisiti were al1 less than 250cf~drnl with approximately half of the

samples forming Ocolony forming units which indicates that nisin alone is almost as effective

as a combination of lysozyme and nisin. The combination of PEF, mild heat and

mtiniicrobials resulted in a much higher microbial inactivation than the sum of the individual

rtlcliictioiis iicliievcrl from cuch rreariiient d«ne. it~dic;iting synrrgy. .-\ ic;ison for tliis is th;it

PEF treritment is more effective iipüiiist Gi;iiri-nrglitive b;icteria [liail Grani-positive bacteria

(Hulsheger et al.. 1983; Pothakamury et al., 1995; Barbosa-Canovas and Swanson, 1998).

while the antirnicrobials nisin and lysozyme are effective against Gram-positive bacteriri

(Hughey and Johnson, 1987; Hanis et al.. 1992). The synergistic effects observed with PEF

treiitment. mild herit treatment and the addition of lysozyme lincl nisin ;ire rilso ris a result of

the weakening of the ceil wall by the antimicrobids dong with the additional stress causcd

by the increase in temperature which makes the membrane more susceptible to pore formation

by PEF treatment.

Calderon-Mirandaet al. ( 1999a) also observed ri synergistic effect with PEF treritrnent

and the ;iddition of nisin on the inactivation of Lisrerilr irittocirti i n skini iiiilk. Thcy obtiiinetl

~i 2.4 log reduction with PEF in combination witli 100 IU iiisinlml t'or :in r1t.cri.i~ field intcnsity

of 50 kV/cm and 32 pulses. Their use of low levels of nisin dong with a processing

temperature of 34°C explains why only a 2.4 log reduction was achieved.

5.5 Effect of Pulse Number

The optimal combination of PEF (80kV/cm, 50 pulses), mild heüt (52"C), and

Lyso:Chrisin(lûûû pg lysozymdml; 3000 iü nisinhl) discussed in the previous section was

also evaluated by applying 20 and 80 pulses respectively. As seen in Table 5.18, increasing

the number of pulses appliecl cluring PEF trciitment fiom 30 tri 50 had a signitlcünt effecr on

rnicrobiril inactivation. Increüsing the pulse number to 80did not h i~ve inuchoEiineffect sincc

50 pulses was sufficient for an rilmost complete inactivation of the initial microbial

population.

Table 5.18. PEF treatment (80 kVlcm) of raw skirn miik contiiining Lyso:Chrisin (1000 pg lysozyme/mI; 3000 TU nisinlml) with viirying pulse iiumbcrs.

Ti*iril # # of Pulses Y,, (ctii/ml) N (cfu/inl) log N/N,, Meiiii o

1 20 3.7 x IOn 1.6 x 10' -5.3 O 0.2 2 20 1 . 1 x 10n 1.6 x 10' -4.8 3 20 1 . 1 x IOH 1 . 1 x 10' -5 .O 4 20 1.3 x IOH 1.4 x 10' -5 .O

3.2 x IOn 1 . 1 x 10" 1 . 1 x 10" 1.3 x I O " 1.4 x 10n 1.4 x 10" 1 . 6 ~ 10" 8.4 x Io7

The microbial population of raw skim milk decreased as the number of pulses increased (Fiç

5.3) ils proven for individual microorganisms (Hulsheger et al., 198 1 ; Jayaram and Castle.

1992; Liu et al.. 1997; Martin et al., 1996; Vega-Mercado et al.. 1997; Caltieron-Miranda et

al., 1999). The statistical analysis ofresults using the GLM procedure is given in Table 5.19.

Table 5.19. GLM for the effect of pulse number on PEF treatment (80 kV1cm) of raw skirn milk containing LysoChrisin (1000 pg lysozyme/rnl; 3000 iü nisinlml).

Source Degrees of Freedom Mean Square Probnbility > F

Model - 7 4.703 0.0026 Error 13 0.430 Total 15

Table 5.20. Duncan's LSD for the effect of pulse nurnber on PEF treatment (80 kV1ct-n) OC raw skim milk containing LysoChrisin (1000 pg lysozymelml: 3000 IU nisinlmi).

- - - -

Pulse Number Mean Io? NN, , Duncan's LSD

20 -5 .O3 a 50 -6.98 b 80 -7.02 b

As seen in Table 5.20, microbial inactivation iit 50 and 80 pulses was significantly

different since 50 pulses wris adequatc t'or i\n ;ilmost coniplete inactivation of

microorganisms, therefore. an increase in the number of pulses would iesult in the needless

expenditure of energy. The optimal pulse number could lie between 20 and 50, however,

no further tests were conducted at intermediate numbers.

20 30 40 50 60 70 80 NumSer of Pulses

Figure 5.3: Effect of pulse number on microbial population in raw skim milk.

Barbosa-Canovas et al. ( 1999) concludcd that lis the nurnbcr of pulses incrcasrs. the

critical potential difference of the ce11 membrane decreues resulting in the higher

susceptibility of microorganisrns to PEF. It appears that there is a threstiold value for the

critical potential difference, below which no funher microbiai inactivation occurs.

Piisteurization of raw skim milk by PEF and anhicrobirils has ri promising future in

the rnrinufiicture ofcheeses currently produced frorn raw rnilk. The risks associrited with any

pathogenic bacteria which may be contained in raw miik are ignored in favour of the flavour

which can only be achievedusing milk whose organoleptic qualities remain unaffected by heat

treatrnents. Cheese produced with PEF treated milk would eliminate the risks currently

associated with consuming unpasteurized dairy products.

6.0 Conclusions and Recommendations

The use of PEF treatment in combination with mild temperature treatment and the

;iddition of the ;intimicrobials lysozynie ml nisin is an effective nictliod for the pasrsurizrition

of raw skirn niilk. The addition of ;intiinicrobirils allows fortlie h m trcatmcnL to rcinnin ini1tl.

thus maintaining PEF treatment as a nonthermal püsteurization method.

A processing temperature of 52 "C was chosen in order to rnaintain microbial

inactivation due to thermal effecis at a low level. Temperatures over the range from 50 to

55°C had a small effect on the microbial population of milk. however, hisher temperatlires

increiise the risk of riffectins the orgiinolepric qurilities of milk.

Varying the pH of raw skim milk from pH of 6.7 to 5.0 rit 4°C had no effect on ilie

rnicrobial level when combined with PEF treatment. Incresing the temperature of pH

adjusted skim milk resulted in the precipitation of milk protein which made the combination

of Iieat trciitment and pH ;idjiistnient unfeasible.

Thc conibiniition of PEF treiitiiiciit ar ;I peak clectric ficlcl stt'en~tli 01'80 kV/cni ml 5 0

pulses with mild heat treatment (52°C) and the addition of lysozyme ( 1000 pg/inl) ;incl riii*iri

(3000 Wlml) provided the highest inactivation with a 7.0 log reduction. When nisin (4000

Wrnl) and Lysozyme (4000 pgirnl) were combined separately with PEF(80 kVlcm, 50 pulses)

and heat (52°C) treatments. log reductions of 5.9 and 3.2 were achieved. respectively. The

combination of PEF. mild heat and antimicrobials resulted in a miich hizher microbial

inactivation than the sum of the individual reductions achieved from e x h treatment alone.

indicating synergy.

Increasing the pulse number for PEF treatrnent from 20 to 50 pulses resulted in a

significant incrciise in microbirit inuctivatioii while a fiirther iricrerise to 80 piilses did not

accomplish any further increase. With the appliclition of 50 piilses, virtiially al1

microorganisms were inactivated (a 7 log reduction), therefore, the application of additional

pulses resulted in the needless expenditure of energy. The optimal pulse number could lie

between 20 and 50. however, no further tests were conducted at intermediate numbers.

Treriting clarified milk with the PEF systern employed in this investigation wtts not

i'easible due to problerns encountered with foaining.

To further improve the efficacy of PEF treatment of raw milk the following areas

require study:

1. The effect of fat content. Studies using I%,2% and whole inilk should be carried out

[O deterinine if t'lit ti;is ari inhibitory ttftecr on niicrobi;il inactiv;itioii.

2. The minimum levelsof antimicrobial addition required for similar levels of microbial

inactivation. Lower levels of nisin and lysozyme addition will result in acost savings

since they are very expensive

3. Srnsory evriliiation and slielf-life stiidies of treriied milk. Triste tests should be curried

out to determine if this treacment affects rhe îiavour andior nutritionid properties of

milk. The shelf-life of PEF treated milk should be determined before regulritory

approval can be obtained.

4. Enzyme inactivation. Valuable enzymes such as lactase, galactase, and phosphatasr

are destroyed during trsdition;iI pasteuriz;ition rnethods but shoiild remain stable in

PEF treated milk.

References

Abee, T., Krockel, L. and Hill, C. (1995). Bacteriocins: Modes ot' Action and Potentials in Food Preservation and Control of Food Poisoning. Int. J . Food Micro., 78: 169- 185.

Adams, M.R. and Moss, M.O. (1995). Food Microbiology. Royal Society of Chemistry, Cambridge. pp. 104- 1 12.

Alvarez, I., Raso, J., Palop, A., and Sala. F.J. (2000). Influence of Different Factors on the Inactivation of Salmonella Senftenberg by Pulsed Electric Fields. Int. J. Food Micro.. 55: 143- 146.

Barbosa-Canovas, G.V.. Pothakriinury. U.R., Paloii. E. ancl Swaiisoii. B. r 1998). Nonthermal Preservation ut' Foods. Marcel Dekker. New York. pp. 73- 1 10

Barbosa-Canovas, G.V. and Swanson, B.G. (1998). Mechanisms of PEF iVIicroorganism and Enzyme Inactivation. Fï 1998 Annual Meeting: Technical Program Abstracts, pp. 91.

Barbosa-Canovas, G.V., Gongora-Nieto, M.M., Pothakamury, U.R. and Swanson, B.G. (1999). Preservation of Foods with Pulsed Electric Fields. Academic Press. London. pp. 4- 164.

Barsotti. L.. Merle, P., rind Cheftel, J.C. (1999). Food Processing by Pulsed Electric Fields. 1. Physical Aspects. Food Rev. Int.. 1 j(2): 163- 180.

Barsotti, L., and Cheftel, J.C. (1999). Food Processing by Pulsed Eieïtric Fields. II. Biological Aspects. Food Rev. Int., l5(2): 18 1-2 13.

Beuchat, L.R. and Golden, D.A. (1989). Antimicrobials Occurring Naturally in Foods. Food Tech., 43: 134-142.

Borird. R.G. ( 1995). Nitiiral Antimicrobials t'rom Animals. in New Methocls of Food Prcservation. edited by Gould, G.W. Chapmrin Sr Hall, New York. pp. 40-37.

Bramley, A.J. and McKinnon, C.H. (199'0). The Microbiology of Raw Milk. In Dliiry Microbiology. V. 1. The Microbiology of Milk, edited by Robinson, R.K. Elsevier Applied Science, New York. pp. 163-208.

Calderon-Miranda, M.L., Barbosa-Canovas, G.V., and Swanson, B. (1999a). Inactivation of Listeria innocua in Skim Milk by Pulsed Electric Fields and Nisin. Int. J. Food Micro., 5 1 : 19-30.

Calderon-Miranda, M.L., Barbosa-Canovas, G.V.. and Swanson, B. (1999b). Transmission Electron Microscopy of Listeria innocua Treated by Pulsed Electric Fields and Nisin in Skimmed Milk. I. J. Food Micro., 5 1 :3 1-38.

Compendium of Methods for the Microbiologiçal Examination of Fods. ( 1992). Edited by Vanderzant. C. iind Splittstoesser, D.F., Americiin Public Health Xssoc., Washington. pp. 75-94.

Conner, D.E. (1993). Naturally Occurring Conipounds. In Antimicrobials in Foods. edited by Davidson, P.M. and Branen, A L . Marcel Dekker, New York. pp. 44 1-468.

Coster, H.G.L. and Zimmerman, U. (1975). The Mechanism of Electric Breakdown in the Membranes of Valonia utricularis. J. Membrane Biology, 273-90.

Daeschel. M.A. ( 1993). Applications and Interactions of Bacteriotins fi-on1 Lactic Acid Bacteria in Foads and Beverages. In Biicteriocins of Lactic Acirl B;ictcriii. cdireci by Hoover. D.G. and Stecnsoii. L.R.. Aciirltmic Psess. NCW York, pp. 63-86.

Dimitrov, D.S. (1984). Electric Field-Induced Brcakdown of Lipid Bilayers and CeIl Membranes: A Thin Viscoelastic Film Model. J . Membrane Bio.. 7853-60.

Dunn, J.E. and Pearlman, J.S. (1987). Methods and Apparatus for Extending the Shelf- Life of Fluid Food Products. United States Patent. US 4 695 472.

Dutreux, N., Notermans. S.. Wijtzes. T.. Gongora-Nieto. M.M.. Barbosa-Canovas, G.V.. iind Swanson. B.G. (2000). Pulsed Electric Fields Inactivation of Attaïhed and Free- Living Escherichia coli and Listeria innocua Under Several Conditions. Int. 5 . Food Micro., 54:9 1-98.

Ettayebi, K., Yamani, J.E., and Rossi-Hassani, B.D. (2000). Synergistic Effects of Nisin and Thymol on Antimicrobial Activities in Listeria Monocytogenes and Bacillus Subtilis. Micro. Let., 183: 19 1- 1%.

Fernandez-Molina, J.J., Barbosa-Canovas, G.V. and Swanson, B.G. (2000) Pasteurization of Skim Milk with Heat and Pulsed Electric Fields. ET 2000 Anniial Meeting: Technical Progrrim Abstriicts. pp. 207.

Gao, F.H.. Abee. T. and Konings, W.N. ( 199 1 ). Mechanisrn of Action of the Pcptidc Antibiotic Nisin in Liposomes and Cytochrome c Oxidase-Containing Proteoliposomes. Appl. Env. Micro., 57(8):2 164-3 170.

Garbutt, J. (1997) Essentials of Food Microbiology. The Bath Press, London pp. 5-58.

Garciü-Graeils, C.. Masschalck, B., and Michiels, C.W. ( 1999). Inactivation of Escheriü coli in Milk by High-Hydrostatic-Pressure Treatment in Combinstion with Antimicrobial Peptides. J. Food Prot., 62( 1 1): 1248- 1254.

Grahl, T., and Markl, H. (1996). Killing of Microorganisms by Pulsed Electric Fields. Appl. Micro. Biotech., 45: 148- L 57.

Gupta. R.P. and Murray, W. ( 1989). Pulsed High Electric Field Sterilization. 7"' iEEE Pulsed Power. Con ferme. pp. 58-64.

Hünlin. M.EL Kalchayanand, Y, Ray. P. and Ray. B. (1993). Bacteriocins id' Lactic A d Bacteria in Combinstion Have Greater Antitiacterial Activity. J. Food Prot.. 56(3):252-255.

Harding, F. ( 1995). Processed Milk. In Milk Qud ity. ediced by Harding F. Chapman & Hall, New York. pp. 1 12-132.

Harris. L.J.. Fleming, H.F. and Klaenhammer. T.R. 11992). Developments in Nisin Research. Food Res. lnt.. 35:57-66.

Harrison, S.L., Barbosa-Canovas, G.V.. and Swrinson, B. ( 1997). Saccharomyces cerevisiae Structural Changes Cnduced by Pulsed Electric Field Treitrnent. Lebensni. Wiss. Technol.. 30:236-240.

Hauben, K.J.A., Wuytack, E.Y., Soontjens, and Michiels, C.W. ( 1996). High-Pressure Transient Sensitization of Escherichia coli to Lysozyme and Nisin by Disruption of Outer-Membrane Pemeability. J. Food Prot., 59:350-355.

Hill. C. ( i99SI. B~icteriocins: Niitiirtil Antimiçrobials t'rom 3Iiïrot)rgmisrns. In Ncw klethods of Food Preservritini~, editeti by Guulrf. G.W. Chapniari 'Y: Hall. Ncw York. pp. 22-39.

Ho, S.Y., Miital, G.S., Cross, J.D., and Griffiths, M.W. (1995). Inactivation of Pseudomonas fluorescens by High Voltage Electric Pulses. J. Food Sci., 60: 1337- 1 340,1343.

Ho, S.Y. and Miital, G.S. (1996). Electroporation of Cell Membranes: A Review. Crit. Rev. Biotech.. 16(4):349-362.

Hughey, V.L. and Johnson. E.A. ( 1987). Antimicrobial Activity of Lysozyme against Bacteria lnvolved in Food Spoilage and Food-Borne Disease. Appl. Env. Micro., 53(9):2 165-2 170.

Hulsheger, H., Potel, J. and Niemann, E.G. (198 1). Kil l ing o f Bacteri with Electric Pulses o f High Field Strength. Radiat. Environ. Biophys., 2053-65.

Hulsheger. H., Potel, J. and Niemrinn, E.G. ( 1983). Electric Field Effects on Bacteria iind Yei~st Cells. Radiat. Environ. Biophys.. 77: 149-162.

Hurst, A. (198 1). Nisin. In Advances in Applied hticrobiology, edited by Perlman, D. and Laskin, A.I.. Academic Press, New York. pp. 85- 123.

Hurst A. and Hoover, D.G. (1993) Nisin. In Antimicrobials in Foods, edited by Davidson, P.M. and Branen, A.L. Marcel Dekker, New York. pp. 369-394.

Ibrahim, H.R., Higashiguchi, S., Koketsu, M., Juneja, L.R., Kim, M., Yamamoto, T., Siigimoto. Y.. Aoki, T. (1996). Partially Unfolded Lysozyme at Neiitral pH Agglutinates and Kills Gram-Negiilive and Gram-Positive Butteris through Membrane Damage Mechanism. J. Agric. Food Chztti., J4:3799-3806.

Jack, R.W., Tagg, J.R. and Ray. B. (1995). Bacteriocins of Gram-Positive Bacteria. Micro. Rev.,59: 17 1-200.

Jayüram, S. and Castle, G.S.P. (1992). Kinetics o f Sterilization o f Lactobûcillus brevis by the Application of High Voltage Pulses. Biotech. Bioens., JO: 14 12- 1420.

Jiing, D.S.. Botlyfelt. F.W. tind Dxschei. M.A. 19921. Infliiençe of Fat ancl Emulsitiers on the Efticuçy of Nisin in Inhibiting Listerh monoçytogcncs i n Fliiicl Mlk. J. D~iisy Sc.. 75337-393.

Jung, K.J., Shin, J.K., Cho, H.Y., and Pyun. Y.R. ( 2 0 ) . Pisteurization of Milk Using Continuous Pulsed Electric Fields System. Proceeedings of 8"' ICEF, Aprii 2000. Pp. 266.

Kaichayanand, N., Hanlin, M.B. and Ray, B. (1992). Sublelhal ln jury Makes Gram- Negative and Resistant Gram-Positive Bacteria Sensitive to the Bacteriwins, Pediocin AcH and Nisin. Lett. Appl. Micro., 15239-243.

Ka1chriy;inrind. N., Sikes, T., Dunnr, C.P., and Ray, B. (1994). Hydrostiitic Presslire and Electroporation Have Incrcased Bactericidal Efîiciency in Combination with Bacteriocins. Appl. Env. Micro., 6 1 :4174-4 177.

Knorr, D., Geulen, M, Grahl, T. and Siczmann. W. (1994). Food Application of High Electric Field Pulses. Trends Food Sci. Tech., 5:7 1-75.

kistner, L. ( 1992). Food Preservation by Combined Methods. Food Res. [nt., 25: 15 I - 158.

Leistner, L. and Gorris, L.G.M. (1995) Food preservation by Hurdle Technology. Trends Food Sci Tech., 6:4 1-46.

Liu X., Yousef, A.E., and Chism G.W. (1997). Inactivation of Escherichia coli O157:H7 by the Combination of Organic Acids and Pulsed Electric Field. J. Food Safety, 16:287-299.

Luck, E. and Jager, M. ( 1995). Antimicrobial Food Additives: Characterisitics, Uses, Effects. Springer, London, pp. 108-22 1.

Maisnier-Patin, S., Tütini, S.R., and Richard, J. (1995). Combined Effect of Nisin and Moderate Heat on Destruction of Listeria monocytogenes in Milk. Lait, 75:8 1-9 1.

Martin, O., Qin, B.L., Chang, F.J., Barbosa-Canovas, G.V., and Swanson, B.G. (1996). lnactivation of Escherichia Coli in Skim Milk by High lntensity Pulsed Electric Fields. J. Food Proc. Eng., 20:3 1 7-336.

Mich;il;ic. S.L.. Alvarez. V.B. ;inci Zhang. Q.H. (1999). Microbial Reduction in Skim Milk Using PEF Technoiogy. IFT 1999 Annual Meeting: Technical Progrsiil Abstracts. pp. 709.

Mittal. G.S. ( 1998). A New Approach to Enhance the Efficiency of PEF Treatments. CFT 1998 Annual Meeting: Technical Program Abstracts, pp. 9 1.

Owen, J.A. and Andrews, A.T. (1983). A Procedure for the Complete Clarification of Milk of Various Species and its Suitability for use with Calorimetric Measurements. J. Dairy Res., 51(1984):307-3 15.

Pol. I.E., and Smid, E.J. (1999). Combined Action of Nisin and Carvacrol on Bacillus cereus anci Lisleria iuonocytogenes. Lett. Appl. Micro., 29: 166- 170.

Pol. I.E., Mastwijk, H.C., Bartels, P.V., and Sniid, E. J. (2000) Piilsed-Electric Field Treatment Enhances the Bactericidal Action of Nisin against Bacillus cereus. Appl. Env. Micro., 66( 1):428-430.

Pothakarnury, U.R., Monsalve-Gonzalez, A., Barbosa-Canovas, G.V., and Swanson, B.G. ( 1 995). Inactivation of Escherichis Coli and Staphylococcus Aureus in Model F d s by Pulsed Electric Field Technology. Food Res. Int., 28(2): 167-17 1.

Pothi1k;imury. U.R.. Vey, H.. Zhiing, Q., Barbosa-Canov;is. G.V.. and Swanson. B.G. ( 1996). Effeït of Crowth Stage and Processing Temperature on the Inactivation of E. coli by Pulsed Electric Fields. J. Food Prot., 59(1 1 ): 1 167- 1 17 1.

Proctor, V.A. and Cunningham, F.E. (1988) The Chemistry of Lysozyme and Its Use as a Food Preservative and a Pharmaceutical. Crit. Rev. Food Sci. Nut., 26(4):359-395.

Qin, B.L., Zhang, Q., Barbosa-Canovas, G.V., Swanson, B., and Pedrow. P. (1994). Inactivation of Microorganisms by Pulsed Electric Fields of Different Voltage Waveforms. E E E Trrins. Dielectrics Electrical insulrition 1 : 1047- 1057.

Qin, B.L., Pothakamury, Vega, H. Martin, O., Barbosa-Canovas, G.V.. and Swanson. B. (1995). Food Pasteurization Using High-Intensity Pulsed Electric Fields. Food Tech., 1257-99.

Raso, J., Gongora, M., Calderon. M.L., Barbosa-Canovas, G.V. and Swanson, B.G. (1998). Pasteurization of Raw Skim Milk by High Intensity Pulsed Electric Fields. [FT 1998 Annuiil Meeting: Technical Program Abstracts, pp. 308.

Reina. L.D.. Jin ZT.. ZRnng, Q.H., and Yousef, A.E. ( 1998). Inactiviitiun of Listeria monocytogenes in klilk by Pulsed Electric Field. J. Food Prot., 6 l(9): 1203- 1206.

Reiter, B. (1978). Review of the Progress of Dairy Science: Antimicrobial Systems in Milk. J. Dairy Res., 45: 13 1-147.

Sale, A.J.H. and Hamilton, W.A. (1967). Effects of High Electric Fields on Micro- Organisrns 1. Killing of Bacteria and Yeasts. Biochim. Biophys. Acta, 148:78 1-788.

Sale, A.J.H. and Hamilton. W.A. (1968). Effects of High Electric Fields on hlicro- Orgimisms 111. Lysis of Erythrocytes iind Protoplüsts. Biochim. Biophys. Acta. 163:37- 43.

SAS (1990). SASISTAT User's Guide, 4Ih cdn. Version 6. SAS Institiite Inc.. C;iry. NC.

Scott, V.N., and Taylor, S.L. (198 1). Temperature, pH, and Spore Load Effects on the Ability of Nisin to Prevent the Outgrowth of Clostridium botulinum Spores. J. Food Sci., 46: 12 1 - 126.

Sensoy, I., Zhang, Q.H., and Sastry, S.K. (1997). Inactivation Kinetics of Salmonella Diiblin by Piilsed Electric Field. J. Food froc. Eng., 20:367-38 1 .

Stevens, K.A., Sheldon, B.W., Klapes, N.A., and Klaenhammer. T.R. (1991). Nisin Treatment for Inactivation of Salmonella Species and Other Gram-Negative Bacteria. Appl. Env. Micro., 57( IS):36 13-36 15.

Stevens, K.A., Sheldon, B.W., Klapes, NA., and Klaenhammer, T.R. ( 1992). Effect of Treatment Conditions on Nisin Inactivation of Gram-Negative Bacteria. J. Food Prot., 5 3 10):763-766.

Vrirnam, A H . , and Sutherland, J.P. ( 1 993). Milk and Milk Products: Technolagy, Chernistry and ~Microbiology. Chapmrin & Hall, New York. pp. 1-YS.

Vega-Mercado, H., Pothakamury, U.R., Chang, F.J., Barbosa-Canovas, G.V., and Swanson, B.G. (1996). Inactivation of Escherichia Coli by Combining pH, Ionic Strength and Pulsed Electric Field Hurdles. Food Res. Int., 29(2): 1 17- 12 1.

Wandling, L.R., Sheldon, B.W., and Foegeding, P.M. (1999). Nisin in Milk Sensitizes Bacillus Spores to Heat and Prevents Recovery of Survivors. J. Food Prot.. 62(5):492- 49s.

Weaver, J.C. and Powell, K.T. (1989). Theory of Electroporation. In Electroporütioii and Electrofusion in Cell Biology. edited by Neumann, E., Sowers, A.E. and Jordan, C.A. Plenum Press, New York, pp. I l 1-126.

Wouters, P.C., and Smelt, J.P.P.M. (1997). Inactivation of Microorganisms with Pulsed Electric Fields: Potential for Food Preservation. Food Biotech.. 1 l(3): 193-229.

Zapico, P., Medinii, M.. Gaya. P., and Nunez. M. ( 1998). Synergistic Effect of Nisin and the Lactoperoxidase System on Lysteria kIonocytogenes in Skim ivlilk. Int. J . Food Micro.. 40:35-42.

Zhang, Q., Monsalve-Gonzalez, A.. Barbosa-Crinovas, G.V., and Swanson. B.G. ( 1994). Inactivation of E. Coli and S. Cerevisiae by Pulsed Electric Fields Under Controlled Temperature Conditions. Trans. ASAE, 37(2)58 1-587.

Zhang, Q., Barbosa-Canovas, G.V., and Swanson, B.G. (1995a). Engineering Aspects of Pulsed Electric Field Pasteurization. J. Food Eng., 2526 1-28 1.

Zhring. Q.. Barbosa-Canovlis, G.V., and Swanson, B.C. ( 199%). 1naçtiv;itinn of E. Coli i'or Food Pasteurization by High Strength Pulsed Electric Fields. J. Food Proc. Pres.. 19: 103-1 18.

Zimmerman, U., Pilwat, G. and Riemann, F. (1974). Dielectric Breakdown of Cell Membranes. Biophys. J., 14:88 1-899.

Appendix A: Statistical Analysis of Data

Effect of Temperature on Raw Sklm Mllk

Trial Y Temperature ('Cl No(CFU1ml) N (CFUIml) log N/No Mean a Duncan's CS0

1 52 4.80E47 3.10E47 4.19 -0.16 O. 1 a b c 2 52 6.70E47 4.70E47 4.15 3 52 3.10E47 2.70E47 4.06 4 52 4.30E47 2.60E47 4.22

Analysis of Variance Procedure

Source OF Sum of Squares Mean Square F Value Pr > F

rnodel 5 0.691 0.138 6.33 0.0015

error 18 0.393 0.022

wirwed total 23 1.084

R-Square c. V. Root MSE

0.638 -56.270 O. 148

Effect of Antimicrobial Addition on Raw Skim Mllk . -- ...

Trial # Anlimicrobial Temperature CC) No (CFUfrnl) N (CFUlrnl) log NINO Mean a Duncan's LSD

lysozyme lysozyme lysozyme lysozyme lysozyme lysozyme lysozyme lysozyme

Anilyris of Variance Procsdura

Source DF Surn ot Squares Mean Square F Value Pr > F

model 5 2.847 0.570 22.58 0.0001

correcled taal 23 3.301

&Square C. V. Root MSE

0.862 -26.95 0.159

Effect of Antimicrobials an Pff Treatrnent of Raw Skim Milk - - -

Trial # Antimicrobial No (CWrnI) N (CFUlrnl) log NINO Mean o Duncan's LSD

1 lysozyme 1.50E+08 1.30~+05 -3.06 -3.18 0.4 c 2 lysozyme l.6ûE+O8 1.50E+05 -3.03 3 lysozyme 1 .lOE+üB 1.90E+04 -3.76 4 lysozyme 1.30E48 1.70E45 -2.88

General Linear Model Procsdum

Source OF Sum of Squares Mean Square F Value Pr > F

model 2 31.686 15.843 43.56 0.0001

error 17 6.t83 0.364

corrected total 19 37.870

R-Square C. V. Root MSE

0.837 -10.850 0.603

Effect of Pulse Number on PEF Treatment of Raw Skim Milk

Trial # # af Pulses No (CFUIml) N (CFUlrnl) log MO Mean o Duncan's LSD

General Umar Modal Prochdute

Source D F Sum of Squares Mean Square F Value Pr > F

mode1 2 8.407 4.203 9.77 0.0026

error 13 5.592 0.430

corrected total 15 13.000

R-Square C. V. Root MSE

0.601 -10.45 0.656

Appendix 6: Experimental Data

Hoat Tnatod Raw Skim Milk

1 100 10' 102 te 10' 1 05 1 o6 10' 1 oa

Heat Tmatrd Raw Skim Milk

Control 1 Contra12

50,l 50.2

51,l 51,2

52,l 52.2

53,l 53,2

54,l 54,2

55.1 55,2

Control 1 2250 Control 2 r250

50.1 >250 50.2 2250

51.1 ~ 2 5 0 51.2 >250

52.1 ,250 52,2 >250

53.1 a250 53,2 >250

54,l >250 54,2 >250

55.1 >250 55,2 >250

>250 a250

>250 >250

>250 >250

>250 >250

>250 >250

>250 ~ 2 5 0

~ 2 5 0 >250

PEF Tmatment (80 kVIcm) and Lyso:Chrisin (0.04 @ml)

PEF Treatrnent (80 kVlcm) and Lyso:Chrisin (0.04 girnl)

Contrai 1 Control 2

20 Pulses,l 20 Pulses.2

50 Pulses,l 50 Pulses,2

80 Pulses,l 80 Pulses,2

20 Pulses 1 >250 113 1 O 1 O O O O O 20 Pulses ~ 2 5 0 143 15 2 O O O O O

1 oo

>250 ~ 2 5 0

>250 1250

O O

O 7

PEF Treatment (80 kVlcm) and Lyso:Chrisin (0.04 glml)

50 Pulses 50 Pulses

PEF Treatment (90 kVlcm, 20 pulses) of Pseudomonas fluorescens at 4 "C.

O O O O O O O O O 3 O O O O O O O O

PEF Treatrnent (9â kVlcm, 20 pulses) of Pseudomonas fluorescens at 4 "C.

PEI: Treatment (80 kVlcm, 50 pulses) and nirin (0.04 glml) at 52%.

Trial1 Trial 2

1 O O O O O O O Trial 2 O O O O O O O O

>250 >250 i250 >250 a250 133 14 1 O s2SO i250 a250 >250 >250 97 10 1 O

Sanitalion Protedurs

PEF Treatment (80 kVIcm, 50 pulses) and Antimicrobials (0.04 glml)

Trial 1 Trial2 Trial 3

Control 1 >250 >250 >250 >250 >250 >250 108 12 1 Conti012 1 S E 0 > 2 M r2X) >22M ,250 r250 130 12 1

,250 >250 >250 >250 r250 >250 8 1 7 2 >250 i25O >250 >250 >250 >250 77 8 1 ~ 2 5 0 >250 >250 r250 2250 ~ 2 5 0 73 8 1

PEF Treatment (80 kVlcm, 50 pulses) and lysozyme (0.04 glml) at 52°C.

lysozyme 1 lysozyme 2

>250 i250 194 21 2 O O O O 2250 a250 169 20 1 O O O O

PEF Treatment (80 kVlcrn, 50 pulses) and Lyso:Chrisin (0.04 glml) at 52°C.

O O O O O O O O Trial 2 O O O O O O O O

Controll Control2

PEF Treatment (80 kVlcm, 50 pulses) and Lyso:Chrisin (0.04 glml) at 52°C.

>250 ,250 s250 s250 ,250 >250 140 15 1 >250 >250 >25C ~ 2 5 0 >250 >250 138 12 1

O O O O O O O O Trial 2 O O O O O O O O

PEF Treatment (80 kVlcrn, 50 pulses) and nisin (0.04 glml) at 52°C.

O O O O O O O O Trial 2 O O O O O O O O

Control 1 Control2

PEF Treatment (80 kVlem, 50 pulses) and nisln (0.04 giml) at 52 OC.

s250 >250 >250 2250 >250 s250 108 15 1 2250 >250 >250 >250 2250 >250 27 4 1

1 O O O O O O O Triai 2 O O O O O O O O

Addition ot Antimicrobials to Raw Skim Mllk (0.04 glrnl) at 52°C.

Addition d Anîimicrobhlr to Raw S M Mllk (0.M g/d) at 4%.

lysozyme, 1 lysozyrne.2

AddiUon of Anttmicrobidi to Raw Skim MHk (034 @ml) aï 2C.

Control t Contrat 2

Addltlon of Antlmicrobiali to Raw Skim Mllk (0.04 ghl) al Sic.

1 100 1 O' t# 103 10' i 05 106 1 o7 1 0.3

Conlrol 1 Conlrol 2

lysozyme,l lysozyme3

nisin, t nisin2

Lyso:Chrisin.l Lyso:Chrisin,Z

Addition of Antimicrobials to Raw Skim Mllk (0.04 $ml) at WC.

Control 1 Contiol2

Lyso:Chrisin,l Lyso:Chrisin,P

PEF Tnatment (80 kVlcm, 50 pulars) and LymxCMsln (0.04 glml) at 52%.

Trial 1 O O O O O O O O Trial 2 1 0 O O O O O O

Control

PEF Treatment (80 kVlcm, 50 pulses) and nisin (0.04 gh l ) at 52C.

100 10' 102 103 1 04 105 i d 10' 1 o8

,250 r250 >250 >250 34 3 O O O

1 O O O O O O O Trial 2 2 O O O O O O O

Control

Addition of Antimicmbials to Raw Skim Mllk (0.04 gfml) et 4°C.

1 O0 10' 102 ioj 1 o4 1 0s 1 O6 1 0' 1 Oa

>250 a250 >250 >250 20 2 O O O

PEF Troatment (80 kVIcm, 50 pulses) and lysozyme (0.04 glml) at 52%.

Control

Trial 1

1 o0 10' to2 1 o3 i 04 1 os 1 o6 io7 1 O=

>250 >250 >250 138 15 2 O O O

>250 >250 >250 85 4 O O O O

PEF Tnitmont (80 kVlem, 50 pulsms) md lysozyme (0.02 gml) at 52%

Trial 1 >250 >250 >250 150 19 4 O O O

Trial 2 1 >250 >250 ,250 > Z N 129 11 O O O

Cantrol >250 r250 r250 >250 >250 151 16 2 O

PEF Treatment (80 kVIcm, 50 pulses) and Lyso:Chrisin (0.04 giml) at 52°C.

Addition of Antimicrobials to Raw Skim Mllk (0.1 qlrnl lysozyme; 0.3 glml nisin; 0.4 g/ml Lyso:Chrisin) at 5Z°C.

Control

Heat

PEF

PEF Treatmrnt (80 kVlcm, 50 pulser) and Lyso:Chriain (0.08 g/ml) at 52°C.

1 o0 1 o1 t oZ 103 1 O' 1 os 1 o6 1 o7 1 o8

>250 >250 >250 >250 >250 84 10 O O

>250 ~ 2 5 0 >250 44 3 O O O O

1 O O O O O O O O

Control

Heat

PEF

Clarification of raw skim milk

i o0 1 O' 102 103 i o4 1 os i 06 1o7 1 08

>250 >250 >250 >250 >250 36 3 O O

>250 127 43 2 O O O O O

1 O O O O O O O O

lysozyme lysozyme

>250 ,250 >250 >250 82 13 O O O r250 >250 >250 a250 106 15 O O O

Trial 1 l >2so >2so 4 1 a O O O O O Trial 2 >250 >250 67 5 1 O O O O

Control 1 Control2

,250 >250 >250 ,250 >250 >250 3 1 1 O >250 >250 ,250 >250 >250 >250 40 3 O