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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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.
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
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
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.
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.
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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