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Evaluation of conventional and microwave heating systems for food processing based on TTI kinetics

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EVALUATION OF CONVENTIONAL AND M IC R O W A V E HEATING
SYSTEM S FOR FOOD PROCESSING BASED ON TTI KINETICS
BY
ZHEN TONG
D epartm ent o f Food Science and Agriculture Chem istry
M acdonald Cam pus o fM cG ill University
M ontreal, Canada
July 4, 2002
A thesis submitted to the Faculty o f G raduate Studies and Research in partial fulfillm ent
o f the requirem ent fo r the degree o f M aster o f Science
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ABSTRACT
Therm al
kinetics
o f enzym atic
tim e-tem perature
integrators
(TTIs)
were
experim entally evaluated under both conventional and m icrowave heating systems in the
pasteurization tem perature range (50 to 90°C). Recent developm ents o f process
evaluation methodologies have shown that standardized enzym atic tim e-tem perature
integrators (TTIs) could be successfully used for fast and correct quantification o f
therm al processes. Promising results have been reported for the a-am y lase based TTI
from Bacillus subtilis (BAA), which was chosen in this study as the TT I to compare the
effectiveness o f continuous-flow heating system s w ith microwave and conventional
heating modes. Therm al inactivation kinetics o f a-am y lase w as studied by measuring the
residual activity o f heat treated samples in isothermal conditions in a tem perature range
o f 50 to 95°C and pH range, 5.0 to 6.9. Based on a first order rate o f inactivation kinetics,
kinetic param eters, decimal reduction tim e, D, and tem perature sensitivity indicator, z,
w ere calculated. The time corrected reference D value (at 70 °C) and z values were: 72.5
m in and 30.3°C at pH 6.9, 5.5 min and 33.6°C at pH 6.5, 2.3 min and 32.7°C at pH 6.0,
1.1 min and 29.5°C at pH 5.6, 0.4 min and 21.1°C at pH 5.0. The enzym e w as m ore
sensitive to thermal inactivation at lower pH. The broad range o f sensitivity o f B A A to
therm al inactivation indicated their suitability as TTI for pasteurization and cooking
processes.
For obtaining kinetic param eters under m icrowave heating
conditions,
a
continuous-flow microwave heating system w as set up using 2 m icrowave ovens w ith
1000 W output pow er each at 2450 MHz. The outlet tem perature w as determ ined as a
II
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function o f flow rate, initial tem perature and heating volume. H eating characteristics o f
the system as tim e-tem perature curves, temperature rise vs. flow rate/residence time,
heating rates, absorbed power, Reynolds and Dean numbers for w ater and enzymes
solutions w ere determined and the average residence tim e was calculated by dividing the
volum e o f heated sample in the helical coils situated inside the m icrowave oven by the
volum etric flow rate.
TT I dispersed in a buffer solution (pH 5.0 and 5.6) initially at 20°C was
continuously circulated through two helical coils connected in series. The sample flow
rates w ere adjusted to result in specific exit tem perature (65 - 80°C). A short fully
insulated helical coil at the exit o f the second oven w as used as holding tube. Timetem perature profiles obtained during heating and cooling o f the sam ples w ere used to
correct com e-up times. The corrected D-values under m icrowave heating condition
ranged from 16.0 s to 4.9 s at pH 5.0 between 65 and 75 °C and 27.0 to 5.7 s at pH 5.6
betw een 65 and 80°C, respectively. D -values during the thermal hold period varied from
39 s to 18 s between 65 and 75°C at pH 5.6. Com paring w ith the D -values betw een
continuous flow M W and thermal hold, as well as from the batch kinetics, enzym e
destruction occurred m uch faster under continuous-flow m icrow ave heating condition
than under conventional thermal heating condition.
I ll
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RESU M E
Les cinetiques therm iques des integrateurs enzym atiques de tem ps et tem perature
(TTI) ont ete experim entalem ent evaluees dans des systemes conventionnel et par
chauffage a m icro-ondes dans la gamme de tem peratures de pasteurisation (50 a 90°C).
Les developpem ents recents des methodes d ev alu atio n des procedes ont prouve que des
integrateurs enzym atiques standardizes de temps et tem perature (TTIs) pourraient etre
avec utilises succes pour la quantification rapide et precise des processus therm iques.
D es resultats prom etteurs ont ete obtenus pour le TTI base sur des a-am ylases de
Bacillus subtilis (BAA), qui a ete choisi dans cette etude comme T T I pour com parer
l'efficacite des system es de chauffage a debit continu au chauffage par m icro-onde ainsi
qu’aux m odes conventionnels de chauffage. L a cinetique therm ique d'inactivation de l'a am ylase a ete etudiee en mesurant l'activite residuelle des echantillons soum is a un
traitem ent therm ique en conditions isothermes dans une gam m e de tem peratures entre 50
et 90°C et dans un pH de 5.0 a 6.9. Les parametres cinetiques, le tem ps decim al de
reduction, D et l'indicateur de sensibilite a la temperature, z, ont ete ainsi calcules en se
basant sur un ordre d ’inactivation cinetique du premier degre. La valeur referencielle de
D corrigee pour le tem ps (a 7Q°C) et les valeurs de z etaient: 72.5 m inutes et 30.3°C sous
pH 6.9, a 5.5 m inutes et a 33.6°C sous pH 6.5, 2.3 minutes et 32.7°C sous pH 6.0, 1.1
m inutes et 29.5°C sous pH 5.6, 0.4 m inute et 21,1°C sous pH 5.0. L'enzym e etait plus
sensible a 1'inactivation therm ique qu'au basses valeurs de pH.
L a large gam m e de
sensibilite de B A A a 1'inactivation therm ique ont ainsi indique leur convenance com m e
T T I pour la pasteurisation et des procedes de cuisson.
IV
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P our obtenir des param etres cinetiques dans des conditions de chauffage par
m icro-ondes, un system e a debit continu de chauffage par m icro-ondes a ete installe en
utilisant 2 fours d ’une puissance de 1000 W chacun a 2450 megahertz. L a tem perature
obtenue a ete determ inee en fonction du debit, de la tem perature initiale et du volume de
chauffage. Les caracteristiques de chauffage du systeme incluant les traces de temps en
fonction de la tem perature, Felevation de la tem perature avec le temps en fonction de
l'ecoulem ent ou du tem ps de residence,le taux de chauffage ainsi que les valeurs
Reynlods et D ean po u r l’eau et les solutions ont ete determines et le tem ps de sejour
m oyen a ete calcule en divisant le volum e de l'echantillon de chauffage dans les
enroulem ents helicoidaux situes a l'interieur du four a m icro-ondes par le debit
volum etrique
Le TT I disperse dans une solution tam pon (pH 5.0 au 5.6) initiallem ent a 20°C a
ete continuellem ent circule par deux enroulem ents helicoi'daux relies en serie. Le debit
d'echantillons a ete ajuste pour obtenir des tem peratures specifiques de sortie (65 - 80°C).
Un court enroulem ent helicoidal therm iquem ent isole a la sortie du deuxiem e four a ete
em ploye com e tube transitoire.
Les fonctions tem ps-tem perature obtenues pendant le
chauffage et le refroidissem ent des echantillons ont ete em ployees pour corriger le tem ps
de montee de la tem perature. Les valeurs D corrigees dans des conditions de chauffage
par m icro-ondes se sont etendues entre 27.0 a 5.7 s a pH 5.6 entre 65 et 80°C,
respectivem ent. Les valeurs pendant la periode therm ique de retention ont varie de 18 a
39 secondes entre 65 et 75°C a pH 5.6. U ne com paraison avec les valeurs D entre le
system e de flux continu M W et la retention therm ique, ainsi que la cinetique en lots,
dem ontre que la destruction enzym atique s'est produite beaucoup plus rapidem ent dans
V
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sous les conditions de debit continu via le chauffage par micro-ondes que dans les
conditions conventioneiles de chauffage.
VI
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ACKNOW LEDGEM ENTS
I w ould like to extend my truthful thanks to my thesis supervisor, D r. Hosahalli S.
R am asw am y, w ho guided my study and research throughout the course o f my study, who
offered not only academic advice but also personal suggestions. H is constructive
criticism , valuable help and guidance is the indispensable part o f my work. I think I will
benefit from his teaching through my entire life.
I w ould like to thank Dr. Tatiana Koutchma, for her suggestion and help on my
experim ent. Especially, at the very beginning when I was establishing the continuousflow m icrowave set-up, her technical support is indispensable.
I am also grateful to my friends and colleagues in my group. Their friendship and
suggestions are part o f my success: Dr. Reza Zariefard, Pram od Pandey, Dr. Cuiren
Chen. Thanks are extended to the whole staff o f the D epartm ent o f F ood Science and
A griculture Chemistry.
Finally, I w ould like to express my deep gratitude to my wife, X iaom eng, W ang,
for her love, support and incredible caring. H er encouragem ent inspires me to move
forw ard. Also, my special appreciation goes to my parents and my sister for their
understanding and support.
VII
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NOMENCULTURE
A
M ean residual enzym e activity, unit/ml
Aq
Initial enzym e activity, unit/ml
D ,D j ,D 2
Decim al reduction time, D at T l3 D at T2, s
D„
D ean num ber
Ea
A ctivation energy, kJ/m ole
F
Process lethality, s
k, k l, k2
R eaction rate constant, k at T x, k at T2, S '1
m
M ass o f test sample, kg
n
O rder o f reaction
P
Power, W
R
Universal gas constant, kJ/mole; K, H eating rate, °C /s
Rg
Reynolds N um ber
Q
Q uantity o f heat, KJ
t
Time, s
T
Tem perature, 0 C
te
Effective heating time, s
z
Tem perature sensitivity indicator, 0 C
VIII
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Abbreviations
BA A
Bacterial A lpha-A m ylase ( from Bacillus sabtilis)
CDT
Com e - dow n Time
CUT
Come - up Time
HTST
H igh Tem perature Short Time
MW
M icrow ave
PM E
Pectin M ethyl esterase
RF
Radio Frequency
TDT
Therm al D eath Tim e
TTI
Time Tem perature Indicator
G reek Sym bols
p
D ensity o f the fluid
p
Viscosity o f th e fluid
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L IS T
OF TABLES
Table #
Page
2.1
G eneral characteristics o f three thermal processes applied to foods
5
2.2
M ajor applications o f M W heating in food industry
14
2.3
Tim e-Tem perature Integrators had been studied from different origins
31
2.4
Published data o f studies showing m icrow ave effects on different samples at
2450 M H z
'
39
3.1
Therm al resistance o f various food constituents
44
3.2
The com position o f phosphate buffer solutions
46
3.3
Tim e-Tem perature range and intervals em ployed for kinetic studies at different
pH levels
48
3.4
Procedure used for a-am ylase assay
50
3.5
Regression details for D -values com putation at pH 6.9
55
3.6
D -values and z-values o f a-am ylase under w ater-bath therm al heating
condition
62
3.7
4.1
4.2
K inetic param eter estimates (Dgo z) o f a-am y lase from Bacillus subtilis at
different enzyme concentration under isotherm al condition
66
Summary o f operating temperatures, flow rates, heating rates and absorbed
pow er (initial tem perature 20°C )
79
Thermal kinetics param eters (D-, z-values) o f a-am ylase under m icrowave
continuous-flow heating condition
88
4.3
Thermal kinetic param eters (D-, z-values) com parison o f a-am ylase under M W
continuous-flow and conventional batch heating condition
88
4.4
Thermal kinetic param eters (D-, z-values) com parison o f a-am ylase under Batch,
continuous-flow M W and conventional holding condition
92
X
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4.5
K inetic param eter estimates (Dgo z) o f Bacillus subtilis a-amylase at different
enzym e concentration obtained from nonlinear regression analysis on nonisotherm al inactivation data.
XI
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94
LIST OF FIGURES
Figure #
Page
2.1
Electrom agnetic spectrum
10
2.2
M icrow ave heating mechanism
11
3.1
Flow chart o f calculation steps for kinetic data analysis
53
3.2
D -values curves o f a-am ylase at pH 6.9 at different tem peratures w ithout any
correction
55
z-value curves o f a-am ylase before correction
56
3.3
3.4
Tim e-tem perature profile o f sample during conventional heating
58
3.5
D -values o f a-am ylase at pH 6.9 at different tem peratures after correction
59
3.6
z-value o f a-am ylase at pH 6.9 after correction
60
3.7
Therm al inactivation curve o f a-am ylase at pH 5.6 before correction
61
3.8
Therm al inactivation curve o f a-am ylase at pH 5.6 after correction
61
3.9
z-value o f a-am ylase at different pH before correction
64
3.10
z-values o f a-am ylase at different pH after correction
64
3.11
pH dependency o f z-values o f a-am ylase
65
4.1
Configuration o f M W heating setup
74
4.2
H eating characteristics o f the continuous-flow M W setup
76
4.3
M icrow ave heating curves as a function o f tim e at different flow rate
80
4.4
M icrow ave exit tem peratures as a function o f (a) flow rate (b) residence tim e
81
4.5
M icrow ave exit tem peratures as a function o f (a) Reynolds num ber (b) D ean
number
83
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4.5
M icrow ave (a) pow er absorbed and (b) exit tem peratures as a function o f flow
rate at different pH
4.6
Inactivation rate o f a-am ylase at different pH under
m icrow ave condition
4.7
Tim e-tem perature profile o f test samples subjected to continuous-flow
m icrow ave vs. w ater-batch heating condition at exit tem perature 65 °C
4.8
Tem perature sensitivity com parison betw een continuous-flow m icrowave
heating and conventional batch heating at (a) pH 5.6 (b) pH5.0
4.9
Tem perature sensitivity com parison between continuous-flow m icrowave
heating and conventional holding at pH 5.6
continuous-flow
XIII
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TABLE O F CONTENTS
Page
A BSTRA CT
RESU M E
A CK N O W LED G EM EN TS
N O M EN CLA TU RE
TA BLE OF C O NTEN TS
LIST OF TABLES
LIST OF FIG U RES
II
IV
VII
VIII
XIV
XI
XII
CHAPTER 1
IN T R O D U C T IO N
1
CHAPTER 2
L IT E R A T U R E R E V IE W
4
• Thermal processing
• N ew processing methods
4
6
>
>
•
M ic ro w a v e heating
r>
>
>
>
•
Non-thermal processing
New thermal processing
9
Principle o f microwave heating
Development o f m icrowave heating technology
Applications o f m icrowave heating
Advantages and lim itations o f m icrowave heating
Fouling problem
Inactivation/D estruction kinetics
>
>
>
7
8
Theoretical basis o f therm al kinetics
Lag correction
Lethality concept
9
12
13
14
15
17
17
21
21
•
Assessment o f thermal processing
23
•
Tim e-tem perature integrator
25
>
>
Definition o f Tim e-Tem perature Integrator
The need for T T Is as process evaluation tools
XIV
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25
25
>
>
Criteria for a Tim e-Tem perature Integrator
W orking principle o f TTIs
•
®
•
•
•
•
CHAPTER 3
32
40
KINETICS OF ENZYME INACTIVATION UNDER
ISOTHERMAL WATER BATH CONDITION
41
A bstract
Introduction
M aterials and M ethods
41
42
45
> Preparation o f solutions
45
•
•
•
•
>
>
>
>
•
Phosphate buffer solutions
Enzym e solution
Substrate preparation
C olor reagent
Isothermal heat treatm ent
a-am ylase assay
Tim e-tem perature profile
K inetic data analysis
Results and Discussion
46
46
47
47
47
49
50
51
54
> First order inactivation models for a-am ylase
> Com e up time and com e dow n tim e corrections
> Comparison w ith literature values
Conclusions
CHAPTER 4 INACTIVATION OF a-AMYLASE BASED TTI UNDER
NON-ISOTHERMAL CONTINUOUS FLOW M ICRO­
WAVE HEATING CONDITION IN RELATION TO
CONVENTIONAL HEATING
•
28
28
29
30
M icrow ave thermal, non-thermal and enhanced effects
Summary o f the literature review
•
•
•
•
M icrobiological TTIs
Enzymatic TTIs
Chemical T T Is
Physical TTIs
27
27
A bstract
54
57
65
67
68
68
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•
In tro d u c tio n
69
•
M aterials and M ethods
73
>
M icrowave heating system
> K inetic data analysis
73
77
Results and Discussion
79
•
>
>
>
>
>
•
CHAPTER 5
M icrow ave heating characteristics
79
Enzym e inactivation profiles under continuous-flow microwave
heating system
85
Decim al reduction time com parison
87
Enzym e inactivation profiles under continuous-flow therm al
hold
91
Comparison o f literature values
93
Conclusions
95
CONCLUSIONS AND RECOMMENDATIONS
REFERENCES
96
98
XVI
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CH APTER 1
INTRODUCTION
C onsum ers’ demands are strongly influencing the trends in the food market.
A ccording to W elti-Chanes (1997), changes in social consum ption patterns, availability
o f certain products, consum er’s lifestyles, purchasing pow er as well as preferences, and
greater aw areness o f health and safety as related to food are the main reasons for the
increase in dem and o f new food processing methods.
It is w ell know n th at therm al processes are very important to stabilize foods and
assure its m icrobiologic safety. D epending on the severity o f the heat treatm ent and on
the objectives to be accomplished, different thermal processes, such as pasteurization and
sterilization can be defined (Lund, 1975). Optim ization o f thermal processing for safety
and quality has been the main them e for producing better quality foods. Several
techniques including high pressure processing, electrical pulse treatm ent, light pulses,
ultrafiltration, irradiation, addition o f preservatives o r their com bination have been
investigated as alternatives to conventional thermal processing (M ertens and K norr,
1992).
M icrow ave heating is attractive for heating o f foods due to its volum etric origin,
fast tem perature rise, and controllable heat deposition and easy clean up. M icrow ave
ovens have gained w idespread acceptance in home application for re-heating and
preparation o f foods, and m icrow ave processing has recently gained better acceptance as
an effective m ethod for food preservation in several processes such as cooking, drying,
pasteurization and sterilization (Decareau, 1985). It is reported that in both Europe and
1
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U.S. there are many microwave operations in food industry. Presently, successful
m icrow ave processing operations in food industry are cooking, blanching, drying,
tem pering, baking, pasteurization and sterilization (D ecareau and Peterson, 1986;
M udgett, 1989; Giese, 1992).
H owever, industrial m icrowave processing o f food did not develop rapidly due to
lack o f inform ation on product safety and quality (M udgett, 1986). Also, there is a
controversy w hich is whether the non-therm al effect o f m icrow ave heating exist.
Recently, studies have focused on different procedures, approaches, experimental
designs, techniques, and biological systems to distinguish therm al and non-thermal
effects o f m icrowave heating and resolve the controversial question. Several theories
have been advanced to explain how electrom agnetic fields m ight kill microorganism s
w ithout heat as summarized in a review by K norr et al. (1994). On the other hand, some
researchers refute any m olecular effects o f electric fields com pared w ith thermal energy
using classical axiom s o f physics and chemistry.
M any researchers have used different m icroorganism s and food borne enzym es as
indicator to evaluate the lethality o f the m icrowave heating treatm ent. H owever, because
these is no standard indicator being used, so, different approach draw s controversial
conclusion (Tajchakavit, 1997). There are difficulties to precisely com pare efficiency o f
m icrow ave heating to conventional because o f the different approaches em ployed and
lack o f tem perature history data. Enzym atic tim e-tem perature integrators (TTIs) are
inexpensive, quick and accurate method to evaluate accum ulated process values (F), rate
constants and im pact o f critical process param eters on the target safety o r quality
2
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attributes, a-am y lase from Bacillus subtilis was used to evaluate inactivation efficiency
o f m icrow ave and conventional heating.
The follow ing w ere the objectives o f this research:
1. T o determ ine the thermal kinetics o f a-am y lase based TT I from Bacillus
subtilis at different pH, as influenced by conventional heating w ith
consideration o f come-up tim e effectiveness.
2. T o evaluate the characteristics o f a continuous-flow m icrowave heating
system in order to establish the procedure fo r m icrowave kinetic studies.
3. T o determ ine the thermal inactivation kinetics o f T T I under m icrowave
continuous-flow heating system, and com pare them w ith their conventional
therm al counterparts.
The study is a further investigation o f m icrowave pasteurization concept for liquid
foods by introducing T T I as indicator to evaluate the process, also, it provides another
m ethod to clarify the inconclusive issues over the existence o f non-therm al m icrowave
effects.
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CHPTE.R 2
L IT E R A T U R E R E V IE W
An overview o f therm al processing and new processing methods is briefed in the
chapter. H istorical developm ent o f microwave, principles o f m icrow ave heating, its
application in food industry, its advantages and limitations are discussed. Also, a b rief
review o f tim e-tem perature integrator is presented in this chapter.
Thermal processing
Conventional thermal processing generally involves heating o f foods packaged in
herm etically sealed containers for a long enough tim e period at a high enough
tem perature to elim inate the pathogenic microorganism s that endanger the public health
and those
m icroorganism s and enzymes that cause deterioration o f the food during
storage. It is one o f the im portant preservation techniques for producing packaged sh elf
stable food products.
In order to establish a successful heat treatm ent procedure, determ ining the proper
tem perature and length o f time, good knowledge o f m icrobiology and m echanism o f heat
transfer are required. M ost o f the food processing techniques involve heat transfer
operations. H eat processing can be achieved by a variety o f techniques using hot w ater or
steam (cooking, blanching, pasteurization, sterilization, evaporation and extrusion), hot
air (baking, roasting, and drying), hot oil (frying), and irradiated energy (m icrowave,
infrared radiation, and ionizing radiation). D epending on the severity o f the heat
treatm ent and on the objectives to be accomplished, different therm al processes, such as
4
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pasteurization and sterilization, can be defined (Lund, 1975). Pasteurization is a relatively
m ild form o f heat treatment designed to inactivate enzymes and destroy spoilage
vegetative m icroorganism s (bacteria, yeast, and molds) present in low-acid foods (pH <
4.6). On the other hand, if the pH o f the food is high (pH > 4.6) the main objective o f this
therm al treatm ent is to kill pathogenic microorganisms. This process is usually used in
com bination w ith other extrinsic factors (e.g., acidification, cooling, preservatives, and
packaging) to m inim ize microbial grow th and assure product safety during storage and
distribution. Sterilization or “commercial sterilization” requires a severe heat treatm ent
to destroy heat-resistant bacterial spores. The severity o f the heat treatm ent depends on
the nature o f the food (e.g., pH, and w ater activity), the m icroorganism ’s heat resistance,
the initial m icrobial load, the heat transfer characteristics o f the food and heating
medium, and on the storage conditions follow ing the thermal processing (Lund, 1975;
Fellows, 1988). The general characteristics are summarized in Table 2.1.
T able 2.1. G en eral characteristics of three thermal processes applied to foods
Thermal Process
Blanching
Pasteurization
Commercial
Sterilization
Temperature
Comments
Usually < 100 °C Inactivate enzymes,
Usually < 10 minutes remove tissue gases,
wilt tissue, preheat
tissue, other special
treatments follow
(canning, freezing,
dehydration)
Usually < 100 °C Inactivate vegetative
cells of pathogenic or
spoilage organisms,
other special treatment
follow(e.g. refrigeration
fermentation)
Usually > 100 °C Inactivate spores of
pathogenic or spoilage
organisms, usually
used in conjunction
with anaerobic storage
conditions
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Therm al processing methods are extensively used for foods preservation and
preparation. H owever, there has been an increasing public concern tow ard the quality o f
these processed foods w hich are inferior in nutritional quality. As Lund (1975) pointed
out, therm al processes are designed to reduce the activity o f undesired biological
m aterials (e.g. enzymes or microorganisms) contained in or on the food, however,
concom itant w ith that is the simultaneous destruction o f essential nutrients. On one side,
therm al treatm ent leads to desirable results,
such as inactivation o f unhealthy
m icroorganism s, protein coagulation, textural softening, and
formation o f aroma
com ponents. B ut undesirable changes also occur at the same time, such as loss o f
vitam ins and minerals, formation o f therm al reaction com ponents o f biopolym ers, and, o f
course, loss o f fresh appearance, flavor, and texture. This concern has prom oted
researchers to find new food processing techniques to minimize quality degradation in
processed foods.
New food processing methods
D uring the last 15 years, consum er dem ands for higher quality, fresh, and
convenience food have resulted in the developm ent and application o f new food
processing technologies. Basically, there are tw o types o f methods, N ew therm al
processes and Non-thermal processes.
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Non-thermal processes
A m ong non-therm al processes (NTP) are pulsed electric fields, high hydrostatic
pressures o r ultrahigh-pressure, irradiation, ultrasonic radiation, and oscillating magnetic
fields.
A ccording to Palaniappan et al. (1990) and Castro etal. (1993), the application o f
electricity to foods in order to inactivate undesirable m icroorganism s may be achieved by
(1) electroporation, (2) low-voltage alternative currents,
(3) ohm ic heating,
(4)
m icrow ave and dielectric heating, and (5) high electric fields. A m ong these, only high
electric field pulses has the advantage o f m inim izing the generation o f heat inside a food.
The use o f high hydrostatic pressure, high voltage electric pulses and ultrasonic radiation
are procedures for the inactivation o f m icroorganisms in foods that offer alternatives to
heat. These techniques are highly effective in inactivating vegetative cells o f bacteria,
yeast and filam entous fungi, at pressures and at voltage gradients that are com patible with
the retention o f high quality in some foodstuffs. H owever, bacterial spores rem ain more
difficult to control by both these procedures, so that their use for the preservation o f foods
other than relatively short shelf-life or products in w hich spores are not a problem
because they are inhibited by the intrinsic properties o f the food (e.g. low pH o r low
w ater activity) must await further research (Gould, 1995).
The use o f ionizing radiation to preserve foods or to eradicate pathogens from
them, is already w ell established. In addition to its value as a preservation technique, it
offers a very effective route o f the reduction in food poisoning, e.g. via th e irradiation o f
the often Salmonella- and Campylobacter- contam inated food such as poultry and other
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foods o f anim al origin. W hilst the use o f radiation continues to grow worldwide, negative
consum er in m any countries holds back its w ider use.
New thermal processes
There are several neo-thermal technologies such as continuous processing in
rotary retorts, m icrow ave heating, ohmic heating and light pulse technology have been
studied. W ith respect to the im provement o f techniques for the inactivation o f
m icroorganism s in foods, most effort and new application has concerned thermal
processing. A particular aim has been to minimize damage to product quality. This is
being pursued in tw o ways. Firstly by the w ider application o f m ore high tem peratureshort time processing, w ith associated aseptic packaging where relevant.
Secondly, by
delivering heat in new ways, e.g. by microwave or by electrical resistance heating o f
foods, which allow better control o f heat delivery and minim ize the over-cooking that
com m only occurs in m ore conventional thermal processes.
D unn and his team think that only light pulse technology seems to be a prom ising
option in the future and he explained that by using intense flashes o f broad-spectrum
“w hite” light to inactivate microorganisms in foods and packages w hich based on the
treatm ent o f foods w ith short light pulses that allow the spatial localization o f this light
on the food surface to induce lethal effects on microorganisms. R esearchers claimed that
this technology w as capable o f killing microorganisms on food surfaces o r in transparent
m edium s such as w ater, and they pointed out that the related costs could be “very
favorable.” H owever, m ore research is required to determine its future application (D unn
et al., 1995).
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Microwave heating
M icrow ave heating involves conversion o f electrom agnetic energy into heat by
selective absorption and dissipation. M icrow ave pasteurization and sterilization promise
to give very quick heat processing, w hich should lead to small quality changes due to its
volum etric origin, fast tem perature rise, and controllable heat deposition, according the to
H TST principle. A s one o f the new thermal process, microwave heating are currently
found for m ost o f the heat treatm ent operation in both dom estic and industrial
applications.
The desire to develop new food processing techniques has led researchers to study
radio frequency energy in its various forms. M icrow ave heating is a long recognized
heating process w hich has potential to enhance heating rate. In conventional heating one
relies on th e processes o f conduction and convection to transport heat from th e heating
source (a hot surface o f fluid) the bulk o f the product. These m echanism s require
relatively a longer time.
Principles o f microwave heating
M icrow ave are very short w aves o f electrom agnetic energy that travel at the speed
o f light. Figure 2-1 provides the schem atic o f the electrom agnetic spectrum o f
microwaves are located. M icrow ave heating, w hich is one o f the radio frequency heating,
is also dielectric heating but refers to the heating that takes place in a nonconductor due
to polarization effects at frequencies betw een 300 M H z and 300 G H z (D ecareau, 1985).
For example, the only frequency is allow ed for com m ercial foodservice and hom e use is
2450 M Hz, which m eans 2450 million cycles per second.
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X
Gamma]”
R ad io­ M icro­ Infrared VISIBLE SPECTRUM UV
Light Rays Rays
waves waves Light
25
1
1
700
500
1
pm
nm
nm
pm
nm
mm
Wavelength
Figure 2 -t. E lectrom agnetic S p ectru m
to
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M icrow ave in themselves are not heat, it is the material that absorbs m icrowaves
converts the energy to heat. In food system, the heat is generated by interaction between
m icrow aves and tw o other materials, polar molecules and ions. W ater is the m ost
com m on polar molecule and also is a major com ponent o f m ost foods. Because w ater
m olecule has negative and positive end, so in the presence o f a m icrow ave electric field,
it attem pts to line up with the electric field. Since m icrow ave field reversing its polarity at
m illions o f tim es per second, the w ater molecule, constrained by the natural structure o f
the food o f w hich it is a part, begins doing flip-flop m ovem ent m illions tim es p er second
also. In doing so, heat is generated by the m olecular friction. Ionic conduction is another
m icrow ave heating mechanism. Electrically charged ions are influenced by m icrow ave
electric field so they are migrate first in one direction then in the opposite direction as the
electric field is reversed. Figure 2.2 is the schematic picture o f microwave heating.
A
W ater Mo lecu Le
Ch b rin e
Ion
Ionic Interaction
R otation
Alternating
1 Electric
' Field
D ipolar Interaction
Figure 2.2. Microwave heating mechanism
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As Schiffm ann summarized the dielectric properties o f food materials, physical
geom etries o f food, thermal capacity and conductivity o f food, and initial tem perature o f
food are param eters that effecting microwave heating (Schiffmann, 1986). Besides,
m icrow ave penetration depth is generally much deeper that o f conventional heating. The
penetration depths range from 8 cm to 22 cm for 915 M Hz and 3 cm to 8 cm for 2450
M H z depending on the moisture content (Decareau, 1985).
D evelopm ent o f m icrowave heating technology
W hen Louise Pasteur was still fighting w ith the false spontaneous generating,
Jam es C lerk M axw ell predicted m athematically the existence and behavior o f radio
w aves based on M ichael Faraday’s hypothesis. M ore that 10 years later, in 1885,
H einrich H ertz verified M axw ell’s theory by experiment.
A lthough there w ere
applications o f radio frequency heat production, such as Jacques A rsene d’arsonval’s
high-frequency heat therapy, the potential application to food processing appears not to
have been recognized until the early 1940s.
Just prior to W orld W ar II, the microwave oven was invented as a by-product o f
another technology — radar, and it marks a new era o f radio frequency heating
application. As a result o f the mass o f m icrowave technology developed for radar during
W orld W ar II, m icrowave heating or “heating by radar” becam e one o f the enchantm ents
or “darlings” o f the im m ediate post- W orld W ar II years. In 1946, w hen Dr. Percy
Spencer w ith Raytheon com pany was testing a new vacuum tube called m agnetron, he
found that the candy bar in his pocket had melted. He was intrigued and did many other
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related experiments. By 1947, the Raytheon company dem onstrated the first commercial
m icrow ave w hich is 5.5 feet tall and weighting over 750 pounds, named "R ad aran g e"
(D ecareau, 1985). Several applications including blanching o f vegetables, coffee roasting
and cooking, and baking for better vitamin retention reported (D ecareau and Peterson,
1986). The concept o f microwave freeze-drying was discussed by Van Leer. The concept
o f tem pering had been recognized in 1946 (Cathcart, 1946).
Although the first patent describing a m icrowave heating system in conjunction
w ith a conveyor was issued by Spencer in 1952, conveyorized oven designs w ith large
product openings w ere not developed until 1962. The first m ajor application was the
microwave finish drying o f potato chips in late 1960s.
AnvUcation o f m icrowave heating in food industry
F or many years the largest application o f microwave heating has been defrosting
or thawing o f frozen foods, such as blocks o f meat, prior to further processing. Further
application o f m icrow ave heating is for drying in com bination w ith conventional hot-air
drying. Often, m icrowave are prim arily used for moving w ater from the w et interior o f
solid food pieces to the surfaces, relying on the preferential heating o f w ater by
microwaves. A pplications can be found for pasta, vegetable, and various cereal products,
where also puffing by rapid expansion o f the interior o f the food m atrix can be
accomplished using m icrowave energy (Tem pest, 1996). Table 2 .2 shows the major
successful applications o f m icrowave heating process in food industry.
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T ab le 2-.1. M a jo r ap p licatio n s o f M W h eatin g in food in d u stry
A p p lica tio n s
Cooking
G eneral C h a ra cteristics
Modifying flavor and texture
P r o d u c ts
Bacon, Sausage,
Poultry, Sardines,
Meat patties
Blanching
Inactivate spoilage enzymes
Drying
Reducing moisture content
Pasta, Snack foods,
Onions, Fruit juices
Tempering
Raising temp, below freezing
Meat, Fish, Poultry
Vegetables, Fruits
Pasteurization Inactivate vegetative microorganisms Milk, Fruit juice,
and
Inactivate microbial spores
Fresh pasta,
Sterilization
Prepared meals,
Sliced bread
A dvantages and limitation o f microwave processing
M icrow ave processing is one o f the alternatives techniques that offers high
quality retention and reduced processing tim e as com pared w ith conventional processing,
especially for liquid food, such as fruit juices and milk. Its volum etric heating m akes it
possible to reduce the processing time greatly, therefore, im proving the retention o f
therm oliable food constituents. From the processing aspect, since m icrow ave heating
occurs only within the food and the heat is generated only w here it is needed, so energy
efficiency is greatly improved. In addition, it is easy to control the tim e and heating
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condition o f m icrow ave processing due to the development o f the microwave heating
equipm ent. Finally, after the processing, it is easy to clean the equipment.
H ow ever, with all these advantages mentioned above, microwave processing is
not a m ature application in food industry because o f many other reasons. According to
B uffler (1993), the reasons are (1) only 50% efficiency can be expected compared with
gas thus rendering microwave advantageous only under som e circumstances, (2)
unfam iliar heating techniques and (3) poor perception due to operation safety. Also,
O hlsson (1991) argued that for a big volum e o f food m icrowave heating is not
appropriate because o f the uneven heating characteristic, also, he m entioned that
in
m icrow ave pasteurization and sterilization, very high requirem ent on heating uniform ity
m ust be m et in order to fulfil the quality advantages.
D ecareau (1985) pointed out that the difficulty o f developm ent o f m icrowave
processing in food industry is that the communication between m icrow ave engineers and
food m anufacturers had been very bad and besides, the food industry is reluctant to
receive new technology. Therefore, the understanding o f interaction o f m icrowave and
food system need to be deepened, the knowledge o f safety and quality o f m icrowave
processing
need to be clarified, the microwave equipm ent technologies need to be
improved.
Fouling problem
Continuous high-tem perature short-time (H TST) pasteurization o f liquid foods is
the appropriate processing that applied presently. Fouling is a severe problem in the
H TST processing, leading to both an increase in the pressure drop through food
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processing equipm ent and a decrease in the transfer efficiency. Presently, in HTST
processing, th e heat is transferred from the heating pipe to the center o f the pipes,
therefore, a tem perature ingredient exist, where the tem perature close to the pipe is higher
than that o f to th e center. For viscous liquid foods, the constituents o f food are exposed to
the high tem perature inner surface to formed solid deposit due to a series o f chem ical
reactions. T he solid deposits that are formed on the inner surface o f the heating pipes
represent a hazard to quality and sterility since not only the heat transfer efficiency is
reduced but also the presence o f the deposits degenerate the tastes and color o f the
product.
The incidence o f fouling in heat exchangers can represent excessive costs in terms
o f capital invested and the maintenance o f equipment (Boot, 1989). Currently, in food
industry there are tw o ways to remove the deposits, chemical cleaning and mechanical
cleaning, depending upon the nature o f the deposits. For example, a scraped surface heat
exchanger has been developed to remove the deposits by continuously sweeping the inner
surface by scraper blade.
Com paring to conventional heating, microwave heating provides a better
alternative to solve the fouling problem. Because m icrowave heats foods volum etrically,
so the tem perature ingredient from the inner surface to the center o f the heat exchanger is
greatly reduced. Hence, com bine with other technologies (helical coil, for example),
m icrowave possesses a great potential to completely solve the fouling problem.
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Inactivation/Destruction kinetics
Theoretical basis o f thermal kinetics
To accom plish the prim ary objectives o f therm al processing, it is necessary to
obtain quantitative data on the thermal kinetics o f enzymes, microorganism and quality
factors. R eaction kinetic param eters for therm al inactivation/destruction o f enzymes and
m icroorganism s as well as quality attributes are required for basically three reasons (Lenz
and Lund, 1980): (1) for establishing a therm al process, (2) for minimizing loss o f a
quality factors and (3) for shelf life testing. The kinetic param eters available in the
literature as described in H ayakaw a et al. (1981) can be categorized into the following
tw o groups: (1) those based on uni-step o r multi-step reaction kinetics em ploying
application o f some characteristic formulae and (2) those based on empirical analysis o f
tim e-tem perature curves o f the inactivation. The latter has been w idely used for kinetic
data analysis for therm al processing. The mechanism o f inactivation/destruction o f
various attributes including enzymes and microorganism s can be based on the
generalized n
-order kinetic model (Labuza, 1980)
Generally, for many food com ponents, enzym es and m icroorganisms, the first
order kinetics model adequately describes the destruction (Stumbo, 1973). However,
according to Labuza (1982a,b), nutrient losses due to thermal processing can generally
characterized to follow a zero- or first -o rd e r kinetics as shown below:
- { d d d t) = kC n
(2.1)
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w here C = concentration o f the desired quality attribute, t =
constant,
n = the order o f reaction. Assum ing a
time, k = the reaction
rate
first-order reaction rate, the decimal
reduction tim e D can be found to be reciprocally related to k (D=2.303/k). W ith the
exception o f photochemical reactions and some physical reactions, the rate constant o f a
reaction is strongly dependent on the temperature. The relationship betw een rate constant
and tem perature is usually modeled either by the A rrhenius equation:
k = K 0e~Ea,RT-------- (2 .2 )
where k = reaction rate constant at T, K0 = frequency factor, E a = activation energy, R =
gas constant, and T = absolute tem perature or the TDT concept:
D -
Z>010(r°~r)/z------- (2.3)
w here D = decimal reduction time at T, D 0 = D -value at the reference T (usually
121.1°C), and z = tem perature range required to change D by a factor o f 10.
Considering a first -o rd e r reaction rate, integrating Eqn. (2.1) between limits C t
ant tim e t and C2 at time t2 and converting the natural logarithm to base 10 results in:
logC, = logC, - - W ( f 2 2.O0O
—
(2.4)
Rearranging Eqn. (2.4) for tw o tem peratures Tj and T2 with their corresponding
reaction rate constants, kj and k2, results in:
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The therm al death time w hich is the alternate concept o f describing the
tem perature dependency o f the reaction rates in food that is based on empirical
consideration proposed by B igelow (Lund, 1975)
The D-value or decimal reduction tim e represents the tim e required to reduce
concentration by 90%. This can be obtained from the sem i-logarithm plot o f logarithm ic
concentration vs. tim e as the time take for the straight line portion to traverse one log
cycle. The use o f the D -value concept makes it easier to visualize the reduction o f any
particular food component at that time. The D-value is reciprocally related to k as
follows:
The tem perature dependency o f k and D are, however, at variance w hen
considering the TD T and Arrhenius concepts. The TDT approach is based on the
assum ption that the thermal death time o f m icroorganisms, enzym es, or nutrients follows
a sem i-logarithm ic relationship and relates proportionally w ith tem perature as follows:
w here D } and D 2 are D -values at Tj and T2 respectively. Since the D -values in based on
a logarithm ic destruction, the complete inactivation/destruction o f enzym e activity o f the
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m icrobial population is theoretically not feasible. An adequacy o f heat treatm ent
therefore em ployed a probability approach for process calculation.
From E qn. (2.5) and Eqn. (2.7), the relationship betw een E a and z can be obtained
as follows:
2303R TJ\
It should be noted that both TDT and Arrhenius concepts contradict each other
since the reaction rate constant (k) is a reciprocal o f the absolute tem perature for the
A rrhenius concept w ith E a as the slope index o f a semi-logarithmic plot. W hereas the D value is a direct proportional to tem perature in the TDT concept with z as the slope index
o f a sem i-logarithm ic plot. However, Lund (1975) suggested that the tw o concepts are
reconcilable a t small tem perature ranges. According to Ram aswam y et al. (1989), both
concepts have been proven to be suitable for studying inactivation/destruction kinetics.
H owever, they dem onstrated that erroneous results could be associated w ith conversion
o f param eters from one concept to another depending on associated
reference
tem perature and tem perature range employed. The authors recom m ended the use o f the
low er and upper limit o f the experimental tem perature range instead o f the approach
suggested by Lund (1975) where T is assum ed to be proportional to 1/T for small
tem perature ranges. The TD T concept is m ore widely applied in therm al process
calculation (Ram asw am y et a l, 1989).
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L a s correction
To
determ ine kinetic param eters o f inactivation/destruction o f many food
com ponents, th e food com ponents in appropriate m edia are subject to heat treatm ent at
different tem perature. However, there is always a lag period associated prior to reaching
the tem peratures o f the surrounding heating or cooling media. It is, therefore, essential to
properly correct the times associated during the lag periods especially when the heating
tim e is fairly short. In some processes, a shorter lag period could be obtained by using
steam as the heating medium which provides a better heat transfer. This, however, does
not necessarily elim inate the lag corrections.
Lethality concept
Lethality (F-value) is a measure o f the heat treatment or sterilization processes. To
compare the relative sterilizing capacities o f heat processes, a unit o f lethality needs to be
established. For convenience, this is defined as an equivalent heating o f 1 minute at a
reference tem perature, which is usually taken to be 250 °F for the sterilization processes.
Thus the F-value w ould represent a certain multiple or fraction o f the D -value depending
on the type o f the m icroorganism ; therefore, a relationship is established. For the food
com ponent passes through changes in tem peratures and the lethal effects o f tem peratures
are integrated over the heating time based on the tim e-tem perature profile giving a
process lethality (F):
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t
F = JlO
( T - r ref)
dt
*
(2.9)
0
For real processes w here the food passes through a tim e-tem perature profile, it
should be possible to use this concept to integrate the lethal effects through the various
tim e-tem perature combinations. The com bined lethality so obtained for a process is
called process lethality and is also represented by the symbol: F0 . Furtherm ore, with
reference to th e processing situation, the lethality can be expressed as related to a specific
location (norm ally thermal center) or any other arbitrarily chosen location or a sum o f
lethality at all points inside a container. From m icrobiological safety point o f view, the
assurance o f a minimal lethality at the thermal center is o f utm ost im portance, while from
a quality standpoint it is desirable to m inim ize the overall destruction throughout the
container.
M ost o f the early literature relevant to PM E inactivation discarded the heating
lag correction by assum ing instantaneous increase in tem perature (Rouse and Atkins,
1952; Atkins and Rouse, 1953; Ulgen and O zilgen 1991). There are several reports
showing application o f the time correction to the lag periods (Eagerm an and Rouse, 1976;
N ath and Ranganna, 1977a,b,c; V ersteeg et al, 1980; Ram asw am y and Ranganna, 1981;
M arshall et a l, 1985; W icker and Tem elli, 1988; Tajachakavit and Ram aswam y, 1997).
Graphical and numerical integration based on tim e-tem perature history o f the sample.
The procedure is similar to the one described in H ayagaw a et al. (1981) who suggested
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an iterative com puterized procedure for estimation o f kinetic param eters by correcting
isotherm al heating times for heating and cooling lags.
Assessment of thermal processing
As discussed previously, thermal processing, including both conventional and
new therm al processing techniques, is one o f the most applied, traditional forms o f food
preservation. H owever, it is well known, it affects the quality o f food products
significantly. Therefore, minimization o f the detrimental effects o f heating on nutrients
w hile m aintaining the lethality target have been pursuing. Currently, the main objective
o f the optim ization o f therm al processing conditions is the maxim ization
o f the final
nutritional and/or sensorial product quality. The nutritional quality, such as vitam in
content, is im portant in some specific products (e.g., baby foods) and in term s o f public
well being, but the consum er’s perception goes to food sensory attributes, such as texture,
color, and flavor (Isabel et a l, 1999).
H eat penetration studies and m icrobiological assays are the tw o classical methods
used to determine adequate thermal processes for a given product and to evaluate the
im pact o f a heat treatm ent from a safety point o f view. In therm al processing it is possible
to find out an appropriate tim e-tem perature com bination w ith desired lethal effect, but
leading to different quality losses. An accurate, safe, and reliable process design depends
on the ability to assess the treatm ent delivered in term s o f the impact on both quality and
safety factors by studying microbial and enzym atic kinetics. Also, it is necessary to
obtain quantitative data on the thermal degradation o f m icroorganism s, enzym es and
quality factors. T he destruction o f m icroorganism s by m eans other than has been the
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objective o f num erous studies since the science o f bacteriology w as developed by Pasteur
as a result o f his w ork on wine (Decareau, 1985). It is well known, microorganisms are
m ost resistant to heat at their optimum pH o f growth, which is generally about 7.0 and the
pH is low ered or raised from this optimum value, there is a consequent increase in heat
sensitivity. A dvantage is taken o f this fact in the heat processing o f high-acid foods,
w here considerably less heat is applied to achieve sterilization com pared to foods at or
near neutrality (Jay, 1996). Previously, a variety o f m icroorganisms w ere applied to heat
treatm ent as indicator to evaluate the lethality o f the treatment. H owever, the tim e after
therm al processing needed to calculate the process impact by readout o f a indicator
depends on the nature o f the m onitoring system, as for microbiological assays could be
w aiting for several days to get the results.
Experim ental data in microorganism inactivation studies are generally analyzed
by m eans o f tw o successive linear regression. In the first, the logarithm o f the
concentration o f the therm olabile factor remaining after heat treatm ent is plotted against
time, giving D-value. In the second regression the logarithm o f D -value is plotted against
treatm ent tem perature, giving the z-value. This methodology generally provides high
confidence ranges, due to the small num ber o f degrees o f freedom. B ecause the
evaluation methods have limitations with regard to their applicability in continuous
systems an in new
heating technologies,
recently,
enzym atic T im e-Tem perature
Integrators, that allow fast, easy, and correct quantification o f the therm al process im pact
in term s o f food safety w ithout the need for detailed information o f the actual tem perature
history o f the product, has gain grow ing attention.
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Time-Temperature Integrator (TTI)
Definition o f Time-Tenwerature Integrator
A TTI is defined as a small, inexpensive device that shows a time- and
tem perature-dependent, easily measurable and irreversible change that can be correlated
to the changes o f a target attribute o f a food undergoing the same variable tem perature
exposure (Taoukis and Labuza, 1989a,b; Weng <?/ al., 1991a, b). The target attribute can
be a safety o r other quality attribute o f interest such as microorganism (spore)
inactivation, or loss o f a specific vitamin, texture, or color.
The major advantage o f TTIs is the ability to quantify the integrated timetemperature im pact on a target attribute without information on the actual tim etem perature history o f the product. All TTIs are by definition post factum indicators o f
the im pact o f the thermal process, because with TTIs, the lethality calculation is simply
based on the change in status o f the TTI after thermal treatm ent, as compared with its
initial status.
Therefore, TTIs can be used as alternative tools when the physical
mathematical m ethod or the in situ m ethods are infeasible.
The need for TTIs as process evaluation tools
Thermal processing, including blanching, pasteurization and sterilization, has
been and still is one o f the most widely used physical m ethods o f food preservation. The
primary purpose is to destroy food-spoilage microorganisms and/or inactivate undesired
enzym es to obtain a com m ercially sterile food with an acceptable sh elf life. Also, at the
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sam e time maxim um quality retention should be pursued. Therefore, in the context o f
food preservation, the quantitative measurement o f the impact o f a thermal process in
term s o f food safety is o f utmost im portance in process design, optim ization, evaluation.
Tw o methods are commonly used for process lethality evaluation. They are in situ
m ethod and physical-m athem atical method. In the in situ m ethod the level o f the food
safety attribute o f interest is evaluated before and after therm al processing to provide
direct and accurate inform ation on the process impact. However, in practice, the
m easurem ent o f microbial counts, texture, vitamin content, organoleptic quality, etc., is
either laborious, tim e-consum ing or expensive, and in som e cases even impossible
because o f the detection limit o f the analytical techniques at hand and/or sampling
requirem ents. In the physical-mathematical method, based on the tem perature history o f
the product combined with knowledge on the heat inactivation kinetics o f the safety
attribute, the impact o f the thermal treatm ent on the param eter o f interest is calculated.
This means that from a heat penetration curve, assum ing a target z-value and identifying
a reference temperature, to calculate lethality. For the case o f a z-value o f 10 °C and
reference tem perature o f 121.1 °C the symbol o f a Fo-value is com m only used. However,
the need for tim e-tem perature data can seriously limit the applicability o f this method.
Direct registration o f the tim e-tem perature profile o f the product is not appropriate under
some processing conditions, for example, for agitated, forced convection
heated
(particulated) foods, it is difficult to m onitor their tem perature history.
Because the in situ and the physical-m athem atical m ethods have certain
lim itations when they are applied to these new technologies, so, it is quiet necessary to
develop Tim e-Tem perature Integrator (TTIs) as an alternative process evaluation tools.
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Criteria for a Time-Temperature Integrator
For successful application in food processing systems, a TTI should need the
follow ing criteria: (1) for convenience, the TTI has to be inexpensive, quickly and easily
prepared, easy to recover, and give an accurate and user-friendly read-out; (2) the TTI
should be incorporated into the food without disturbing heat transfer within the food, and
it should experience the same tim e-tem perature profile as the param eter under
investigation; (3) the TT I should quantify the im pact o f the process on a target attribute.
Because the aim o f using a TTI is to calculate the processing value F relying, solely on
the TTI response, it is necessary that the TTI response kinetics obey a rate equation that
allows separation o f the variables (Hendrickx et al., 1995). Also, it can be easily shown
that the tem perature sensitivity o f the rate constants (z-value) o f the TTI and the target
attribute should be equal to assure equality in process-value (M aesm ans, 1993).
W orking principle o f TTIs
Any system, whether biological (m icrobiological or enzym atic), chemical or
physical, can be used to mimic changes o f intrinsic food quality attributes provided that
the tem perature sensitivities o f the rate constants (z-values) o f the TTI and the target
attribute are equal in the relevant temperature range, and the reaction rates encountered
during heating o f the TTI will induce a detectable response to the tem perature history.
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M icrobiological TTIs
The use o f microbiological systems to m onitor the efficacy o f the sterilization
process (e.g. the use o f Bacillus spp. And Clostridium sporogenes spores) is the most
frequent application o f TTIs In the food and pharmaceutical industries. Because any heat
treatm ent m ust meet the strict requirement o f guaranteeing food safety, the z-value o f a
spore utilized to m onitor safety must closely resemble the z-value reported for spores o f
proteolytic strains o f Clostridium botulinum (z =10 °C), the reference microorganism
used for m onitoring the safety o f the in-pack sterilization o f low -acid foods. The
advantage o f a microbiological system to monitor the safety o f thermal processes in that
the m easuring device and target attribute are sensitive to heat in the sam e tem perature
range. The major disadvantage o f any microbiological monitoring system is the length o f
the assay. The long incubation time between process and read-out o f the system does not
allow for rapid intervention upon any kind o f system atic failure o f process deviation.
Q uantification o f a microbiological TTI requires skill, and the analytical precision o f
currently available techniques is rather low Additional problem s arise from the inherent
variability o f living organisms. Thus, before use as a marker to determ ine the killing
pow er o f a given heat treatment, spore should be thoroughly calibrated to determ ine their
heat resistance. Contam ination risks associated with microbiological m onitoring systems
can be reduced by w orking with permeable or isolated systems rather that dispersed ones.
Enzym atic TTIs
The potential o f protein-based systems, in particular enzym e-based system s, is
receiving considerable interest. The relative easiness o f read-out and handling o f these
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system s gives them a significant advantage over microbiological TTIs. In enzyme-based
m onitoring system s, often the amount o f enzym atic activity remaining after the heat
treatm ent is assayed to determine the im pact o f the tem perature history, although other
properties, such as the heat o f enzym e deterioration, can be determined instead.
A ttem pts to select heat-resistant enzymes and to control enzym es heatinactivation kinetics have been reported. To a limited extent, by changing the conditions
o f an enzym es (e.g. through enzym e im mobilization or protein engineering) and/or its
environm ent (e.g. by ‘solvent engineering’; changing the ion concentration, pH, m oisture
content, additives, etc.), the thermal sensitivity o f the enzyme can be altered to match the
kinetic behavior o f a target quality param eter. In the context o f the developm ent o f an
enzym e-based TTIs, much work has been reported on a-am y lase o f Bacillus spp. The
feasibility o f using Bacillus licheniformis a -am y lase covalently im mobilized on glass
beads or Bacillus amyloliquefaciens a -a m y la se in the presence o f polyolic alcohols o r
carbohydrates as TTIs based on Bacillus subtillis a —am ylase to m onitor the safety o f
pasteurization processes have been developed.
Chemical TTIs
Chemical system s are based on a purely chem ical response tow ards time an
temperature. Detection o f the concentration change o f a chemical com pound added to the
food product, as a measure for the impact o f a thermal process, was advocated more than
30 years ago
to
overcom e
inherent
disadvantages
associated
with
quantitative
microbiology. Flexibility o f handling and high analytical precision in the detection o f
chem ical reactions m ake chem ical TTIs prom ising tools for the evaluation o f therm al
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processes. A crucial deficiency, however, is that no chemical
reactions have thus far
been identified, in the open literature on heat treatm ents o f foods, that feature the Ea
required for m onitoring food safety in the sterilization tem perature range, and only a few
are available that can be used to follow the deterioration o f other quality attributes.
Physical TTIs
The tem perature-sensing mechanism o f existing physical TTIs for the evaluation
o f therm al processes is based on diffusion. The disadvantage o f this system is that it is
activated by steam, and hence can not be used to m onitor other types o f heating media;
nor can it be embedded in a solid particle (Van Loey et al., 1995, 1996). Table 2-3 shows
the tim e-tem perature integrators had been studied and their z-value.
M ost o f the systems described above still have to be subm itted to validation tests
to ascertain their effective functioning and accuracy as T T Is under tim e-tem perature
variable conditions, which is usually the case in, for example, continuous processing in
rotary retorts, aseptic processing, volum etric (ohm ic and m icrow ave) heating and hybrid
processes. The rapid development o f new, accurate TTIs is urged to prevent modern
heating technique from eluding their marketplace.
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Table 2.3. Tim e-tem perature integrators had been studied from different origins
O rigin
N am e
z-value
Bacillus stearothermophilus in alginate
z=8.5 °C
Clostridium sporogen es in alginate
z=12.5-12.7 °C
Clostridium sporogen es in a turkey cube
z=8.5 °C
Bacillus stearothermophilus in
z=11.7°C
M icrobiological
polyacrylamide gel
Bacillus anthracis in perspex
z=60°C
Bacillus stearothermophilus in plastic
z=7.8-10 °C
Bacillus stearothermophilus in a glass bulb
z=10 °C
Immobilized peroxidase in decanol
z=11.6 °C
Immobilized peroxidase in dodecane
z=10.1 °C
Bacillus subtilis a-amylase in tris
z=8.4-12.8°C
Enzymatic
buffer (5-30mg/ml)
Bacillus subtilis a-amylase (200mg/ml)
z=8.6 °C
Bacillus subtilis a-amylase (200mg/m!)+
z=6.2 °C
trehalose (500 mg/ml)
Chemical
Thiamine breakdown
z=26°C
Hydrolysis of dissaccharides
z=18 °C
Methylmethionine sulfonium (pH4-6)
breakdown
z=20-22.8 °C
Thermologs
z=9.4-9.6 °C
Physical
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Microwave thermal, Non-thermal and enhanced effects
M icrow ave used for domestic and industrial heating application are part o f the
electrom agnetic spectrum with a specific frequency o f 915 or 2450 M Hz. T he waves
have the capacity to penetrate the food and create heat by friction o f dipole m olecules o f
water, w hich w ill try to orient and align with the field. This is the m acroscopic therm al
effect o f increasing tem perature within material. Traditionally, non-therm al effects under
the application o f electro-m agnetic radiation refers to lethal effects w ithout involving a
significant rise in tem perature as in the case o f ionizing radiation. O ne o f the effects o f
such quantum energy is breakage o f chemical bonds. Roughly, one electron volt o f
energy is required to break from a molecule a covalent bond to produce one ion pair and
this is referred to as a direct non-thermal effect. According to Tong, microwave nontherm al (o r non-therm al) effects are defined here as the existence o f additional lethal or
destruction effects that cannot be explained by heat alone (Tong, 1996).
Since the beginning o f the use o f m icrowave in chem istry, biology the argum ent
betw een non-therm al and thermal effects o f m icrowaves was assumed. First o f all it
should be clearly defined w hat characterizes thermal, non-therm al, enhanced o r specific
microwave effects. Non-therm al effects o f radio frequencies (RF) w ere dem onstrated in
the early w ork o f Flem ing (1944). Olsen et al. (1966) was probably the first person who
postulated the non-therm al effects o f microwave heating. The review o f Stuerga and
G aillard (1996) provide some definition used in biological studies based on irradiation or
pow er flux density in W /m2 or specific absorption rate in W /kg and does not involve any
assumptions relating to mechanisms o f interaction. Three dom ains o f pow er density w ith
com parison to the thermal capacity o f biological m etabolism w ere defined. The threshold
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selected for standard definition o f biological metabolism corresponds to 4 W /kg o f living
m atter; hence therm al effects have been defined for irradiation pow er density greater than
4 W/kg, w hen organism, can net dissipate the energy supplied by the irradiation; nontherm al effects correspond to density between 0.4 an 4 W/kg. In such conditions, the
therm o-regulation system is able to com pensate for the effect o f irradiation.
A ccording to Risman (1996), any non-therm al effect must not be explicable by
m acroscopic tem peratures, ti m e-tem perature histories or gradients. This means that any
effects, w hich can be explained by applying, verified theories to experimental data and
m acroscopic tem perature are not non-thermal. The cases w here microwave heating gives
a particular tim e-tem perature profile and gradients, which can not be achieved by other
means, are only m icrowave specific. The effect o f higher tem perature achieved by
m icrow ave heating w as explained by Gedye et al., (1988) According to Gedye, it is
possible that m icrowave reactions could produce different products than from reactions
achieved by using conventional reflux techniques. Since microwave heating significantly
increases the reaction tem perature, it is possible that the m icrowave reaction tem perature
could exceed the tem perature required o f a new reaction that was not possible at the
low er reflux temperature. Also, these results are im portant because they confirm that
microwave heating does not alter the reaction but simply provides a much faster and
m ore efficient (high tem perature) method o f carrying out organic reactions. These effects
w ere defined as enhanced m icrowave effects.
Decareau has argued that it is difficult to com pare results o f the many researchers
who have carried out studies on the effect o f m icrowave heating on the m icrobiological
population o f food in foodservice situations. N ot only are details lacking, but most o f the
JJ
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studies w ere carried out with a less than com plete knowledge o f m icrowave heating
phenom ena (D ecareau, 1992). Tong (1996) summarizes that studies on non-therm al
effect can be conducted under either steady-state or unsteady-state conditions. For the
steady-state techniques, samples are originally stored at a tem perature low enough so that
no reaction(s) is expected to occur within a reasonable length o f time. The samples are
quickly brought to a high tem perature and m aintained at that tem perature for a period o f
time by a heating mechanism. Samples, at various time intervals, are w ithdrawn from the
high tem perature environment, cooled immediately and analyzed for the com positions o f
interest by the appropriate instrument and techniques. It is often desirable to have a
com e-up time. By definition, in order to prove an non-therm al effect, samples heated by
both m icrowave and conventional m eans must have identical tim e-tem perature history at
every internal location. Tajchakavit (1997) argued that it is crucial to accommodate the
effective portions o f the lag periods during heating and cooling in order to obtain more
accurate kinetic data. In her study, she corrects the heating and cooling tim es during
come-up and com e-down periods (lag), and applies it to the cum ulative lethality based on
the tim e-tem perature history o f the sample. She suggests that in order to explain the
differences between microwave and therm al heating modes, additional investigations
should be carried out with samples o f various sizes subjected to progressively increasing
tem peratures in a batch mode. Also, she w as using “enhanced thermal effects o f
microwave
heating”
rather
than
“ non-therm al
effects” .
The
enhancem ent
was
characterized using a concept o f mass norm alized m icrow ave heating tim e to effective
therm al time ratio and a microwave enhancem ent ratio (M ER) w hich was defined as the
m icrowave to thermal
inactivation/destruction
ratio.
These
w ere
quantified
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and
m icrow ave heating conditions which provide enhancem ent o f therm al effects were
elucidated.
In connection with studies supporting thermal effects o f microwave heating
results o f G oldblith and Wang (1967) showed no difference in inactivation o f E.coli and
B.subtilis exposed to the same tim e-tem perature conditions o f microwave and
conventional heating. However, microwave treatments w ere made in transient-state and
tem perature gradient as well as cooling conditions w ere not discussed. Lechow ich et al.
(1969) studied the exposure o f S. faecalis and S. cerevisiae to m icrow ave at 2450M H z
and conductive heating, and concluded that the inactivation by m icrow ave could be
explained solely in term s o f heat generated during the exposures. K inetics o f destruction
w as not m ade in the study. Vela and Wu (1979) used a different approach by exposing
various bacteria, actinomycetes, fungi and becteriophages to m icrow aves in the presence
and absence o f water. They found that microorganisms w ere inactivated only in the
presence o f w ater and killed by thermal effect. There was no tem perature increase w hen
lyophilized cells were irradiated. The destruction o f E.coli, S. aureus, P. fluorescens and
spores o f B. cereus by m icrowave irradiation at 3 pow er levels w ere studied by Fujikaw a
et al. (1992) assuming uniform tem perature distributions these authors didn’t find the
destruction profiles by m icrowave exposure. No com parison between m icrow ave and
conventional hating tem perature profiles was included in the study. W elt and Tong
(1993) introduced an apparatus to evaluate possible non-therm al effects o f m icrowaves
on biological and chem ical systems and explained the limits and requirem ents to
com parative studies between kinetics under m icrowave conventional heating. They
com pared inactivation o f C. sporagenes and o f thiam in under equivalent time-
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tem perature treatm ents by perfectly stirred batch treatm ent by conventional and
m icrow ave heating and did not observed atheram l effect. Diaz and M aritinelli (1991)
perform ed experim ents to see if the lethality o f microorganisms is dud to microwave
radiation or heat generated by microwave radiation with 4 types o f m icroorganism s
treated, as indicated by the authors, to the same exposures o f tim e and tem perature. They
concluded that killing effect was due to the heat generated by m icrow ave energy based on
the percentage o f the survivors. Heddleson et al. (1994) showed that the relationship
betw een tim e o f heating, temperatures, and microbial destruction achieved by m icrowave
heating followed conventional reaction kinetics and this illustrated its therm al origin.
In connection w ith additional enhancement o f microbial and enzym es destruction
by m icrow ave energy the following studies indicted and supported the phenom ena o f
non-therm al and specific effects o f microwave. First, the study o f Culkin et a l, (1975)
should be mentioned who exposed single portions o f soups inoculated with E. coli and S.
typhimurmm to 915 M H z, microwaves and found that for any exposure tim e, the closer
the sampled organism s were to the top the low er w ere their level o f survival. They
suggested that the heat generated during microwave exposure alone is inadequate to fully
account o f the nature o f the lethal effects o f microwaves. D reyfuss and C hipley (1980)
characterized some o f the effects o f sub-lethal m icrowave heating on cells S. aureus.
They determ ined higher enzym atic activities in microwaveO-treated cells that can not be
explained solely by therm al effects. However the results cannot be com pared due to the
lack o f therm al control. M udgett (1986) calculated the lethality o f E.coli strain in the
continuous system and com pared it with experim entally m easured values. Experim ental
m icrobial lethality w as som ewhat greater that the predicted by num erical integration. The
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author opinioned that it could have resulted from sensitivity o f the kinetic effects form
the selective absorption o f m icrowave energy by the test organism based on high
intracellular conductivity.
A com parative study o f Khalil and Villota (1988) showed consistently higher
lethality w hen exposed to microwave irradiation and lower D -values (D212 =157 min for
m icrowave and D 212 = 171 min for conventional heating in distilled water) for B.
stearothermophilus spores. Khalil and Vilotta (1988) conducted a study on injury an
recovery o f S. aureus using m icrowave and conventional heating at sub-lethal
tem perature (50 °C) was carried out in which kerosene was chosen as a cooling medium
to keep the sam ple tem perature constant. They concluded that m icrowave-heated cells
suffered greater injury as well as greater membrane damages. K erm asha et al. (1993a)
analyzed inactivation o f w heat germ lipase and soybean lipoxygenase at various
tem peratures sing conventional and m icrowave batch heating, and found higher enzyme
destruction rates under microwave heating conditions. Odani et al. (1995) used
microwave irradiation to kill E. coll, S. aureus and B. cereiis in frozen shrimp,
refrigerated pilaf, and saline. M icrow ave irradiation was shown to result in the release o f
proteins from E. coll as detected by gel electrophoresis o f cell-free supernatants using
sensitive silver staining. They suggested that the m echanism s o f killing bacteria depend
not only on temperature, but also other effects o f m icrowave irradiation. Comparing
m icrowave and conventional m ethods o f inactivation o f B. subtilts, W u an Gao (1996)
showed that the D 100 o f m icrowave was 0.65 but for conventional heating is was 5.5 and
dem onstrated non-thermal effects o f such energy on m icroorganism s. In a review o f
contribution to the study o f m icrowave action, Joalland (1996) considered the action o f
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m icrow aves u p o n bacteria by taking into account the different cellular com ponents such
as genetic m aterial, enzymatic activity, mitochondrias, membranes and cytoplasma, and
concluded th a t beside thermal effects there could exist some non-therm al effects. Riva et
al. (1993) reported different z-values for Enterohacter cloacae and Streptococcus
faecalis in batch conventional heating (4.9 °C and 5.8 °C) and microwave heating (3.8 °C
and 5.2 °C). The results were explained rather to a different heating kinetics and nonuniform local tem perature distributions during m icrowave heating that existence o f
specific non-therm al effects. The result o f study by Altas and Ozilgen (1992) o f the
injury o f E. coli and degradation o f riboflavin during pasteurization with microwave in a
tubular flow reactor also indicated the effect o f the flow behavior and other experimental
conditions on the death mechanism in the m icrowave field. They suggested that microbial
death m ight be caused through a damage to a different sub-cellular part under each
experim ental conditions.
There are a number o f studies made recently using different approaches for
estim ation o f m icrowave heating effects. Kozempel et al. (1998) developed a pilot scale
non-therm al flow process using microwave energy to inactivate Pediococcus sp.
N RRLB-2354. a cooling tube within the process line to maintain the tem perature below
40 °C removed the heat generated by the application o f microwave energy to the system.
The significant reduction in microbial count was reported. Shin and Pyun (1997)
published the results o f com parative study o f the inactivation o f L. plantarum using
m icrow ave and conventional heating at 50 °C for 30 min and indicated significant nontherm al effects under continuous and pulsed m icrow ave heating. Table 2 .4 shows the
published data supporting or opposing w hether m icrow ave heating has additional effects.
38
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T ab le 2 .4 . P u b lish ed d ata o f studies show ing m icrow ave effects on d iffere n t
sam ples a t 2450M hz
Test sam ple
Additional effects
Reference
C. sporogen es
Yes
Grecz e t al. 1964
Fusarium solai, Fusarium phaseoli
Yes
Olsen e t al. 1966
E.coli, B.subtilis
No
Goldblith and Wang 1967
S. faecalis, S. cerevisiae
No
Lechowich e t al. 1969
E.coli
No
Vela and Wu 1979
S. aureus
Yes
Dreyfuss and Chipley 1980
B. stearothermophilus
Yes
Khali an Villota 1988
E.coli
No
Fujikawa e t al. 1992
Cl. S porogen es
No
Welt e t al. 1994
E.coli, S. aureus
Yes
Odani et al 1995
Bacillus Subtilis
Yes
Wu and Gao 1996
S. cerevisiae, L. plantarum
Yes
Tajchakavit et al 1997
Peroxidase
Yes
Henderson et al. 1975
Peroxidase, PPO
Yes
Lorenz 1976
Lipoxygenase, Trypsin inhibitor
Yes
Esaka et al. 1987
Lipoxygenase
Yes
Kermasha e t al. 1993a
Lipase
Yes
. Kermasha e t al. 1993b
PME
Yes
Tajchakavit et al 1997
Thiamine
No
Goldblith eta l. 1968
Thiamine
No
van Zante and Johnson 1970
Thiamine
No
Welt and Tong 1993
Microorganisms
Enzymes
Nutrients
From the Table 2.4, it indicates that results from enzym es are uniform ly
supporting the additional effects o f microwave heating w hile the results from nutrients
39
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are uniform ly not showing the additional effects o f microwave heating. Evidence from
m icroorganism s are controversial partly due to the disadvantage o f procedure o f
m icrobial m ethod, e.g. contamination, length o f inoculation etc.
Summary of the literature review
O ne factor that should have a favorable influence on the adoption rate o f
m icrow ave processing is the cost o f energy and the increasing use o f electrical energy.
U se o f prim ary energy sources by the food and beverage industry decrease 15.2% from
1974 to 1984, while the use o f electrical energy increased 22.4%. This trend is expected
to continue in the food processing industry, and greater use o f m icrow ave energy will
follow (D ecareau, 1985). Although microwave heating o f food as one o f the m inim ally
processing methods
is well documented, mechanism o f microwave pasteurization o f
liquid foods need to be thoroughly understood and clarified, especially the kinetic o f
enzym e inactivation and microorganisms destruction. In order to apply this technique into
industrial application and finally verify the controversial argum ent o f non-therm al
m icrow ave
effects,
further experiments by
using
standard
TT I
for m icrow ave
pasteurization kinetics study is required.
40
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CH A PTER 3
INACTIVATION KINETICS OF a-AM YLASE BASED TTI UNDER
IS O T H E R M A L W A T E R B A T H H E A T IN G C O N D IT IO N
ABSTRACT
Thermal inactivation kinetics o f a-am ylase based enzym atic tim e-tem perature
integrators (TTIs) from Bacillus subtilis (BAA) was studied by subjecting small test
samples to isotherm al heating conditions in a well stirred tem perature controlled bath at
several tem peratures in the range from 50 to 95°C and at pH in the range from 5.0 to 6.9.
Transient tim e-tem perature profile o f test samples during the come-up and com e-down
period (usually less than 90 s) w ere m easured and corrected to take into consideration
only their effective portion. From the evaluated residual activity o f TT I follow ing heating
for various time intervals, and using first order rate o f inactivation kinetics, the associated
rate constant was computed as decimal reduction time (D value). U sing TD T concept, the
tem perature sensitivity o f the D values were obtained as z value. The tim e corrected D (at
a reference tem perature o f 70 °C) and z-value were: 72.5 min and 30.3 °C at pH 6.9, 5.5
min and 33.6 °C at pH 6.5, 2.3 min and 32.7 °C at pH 6.0, 1.1 min and 29.5 °C at pH 5.6
and 0.4 min and 21.1 °C at pH 5.0, respectively. The results indicated that by varying the
pH o f test solution, the sensitivity o f TTI to thermal inactivation can be varied and T T Is
appropriate for evaluating pasteurization and
cooking
conditions
can easily
formulated.
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be
INTRODUCTION
A tim e-tem perature integrator (TTI) is a means by w hich a tim e-tem perature
dependent change can easily be measured and correlated to changes in a target attribute
o f a food undergoing the same variable tem perature exposure (Taoukis and Labuza,
1989a,b; W eng et al., 1991a, b). The target attribute can be any safety or other quality
attribute o f interest such as m icroorganism (spore) inactivation, or loss o f a specific
vitamin, texture, or color. The major advantage o f TTIs is the ability to quantify the
integrated tim e-tem perature im pact on a target attribute w ithout inform ation on the actual
tim e-tem perature history o f the product. All TTIs are by definition post factum indicators
o f the im pact o f the thermal process. T T Is can be used as an alternative indicator when
the physical mathematical m ethod or the in situ m ethods are infeasible.
In the food industry, m icrowave heating is categorized as one o f the minim ally
processing methods and the electrom agnetic heating that directly heats the whole volume
o f the foods is a m ethod that may overcom e these lim itations caused by the low heat
diffusivity o f the foods (Ohlsson, 1994). M icrow ave heating treatm ent have been
ascertained to inactivate enzymes, elim inate m icrobial grow th while retaining quality
attributes o f the food products. M udgett and Schw artzberg (1982) gave a detailed review
on m icrowave pasteurization and sterilization and their study show s that there has been a
clear advantage o f m icrowave pasteurization o f pulpy fruits and vegetable juices in glass
or polypropylene bottles in term s o f im proved flavor and low er operating costs compared
with conventional processes (M udgett and Schwartzberg, 1982).
42
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To evaluate the effectiveness o f M W heating systems, the integrated tim etem perature effect m ust be measured based on the analysis o f specific therm al history o f
the heat tran sfer mode and destruction reactions kinetics. A lthough m any researchers
have used different m icroorganisms and food borne enzym es as indicators to evaluate the
lethal effects o f the microwave heating treatm ent (Tajchakavit and Ram aswam y, 1997;
Tajchakavit,
et al.,
1998; Koutchm a and
Ram aswam y,
1999), there
are some
disadvantages o f both microbial and food borne enzym atic indicators w hich are not
overcom e, and also, the dispute o f non-thermai m icrowave effect is still continuing in the
academ ic circle. The disadvantage o f using m icroorganism as an indicator is that it is
very tim e consum ing and laborious. Also, contam ination o f the m icroorganism during
test run is another concern. The disadvantage o f using food borne enzyme is that the
specific enzym e is ju st appropriate for one food system and can not be used for other kind
o f food.
It is w ell docum ented that the environm ental factors, such as pH, can affect the
heat resistance o f microorganisms o r quality factors o f the foods systems. Xezones and
H utchings (1965) studied the inactivation o f Clostridium botulinum in various groups o f
food with different pH values. They have concluded that design, operation, and costs o f
sterilization w ere significantly affected by pH. Cam eron et al. (1980) indicated that
acidification to interm ediate pH values could be used to achieve better utilization o f
sterilization for certain groups o f foods products. Rodrigo and M artinez (1988) studied
the effect o f an interm ediate pH level o f 5.2 on the heat resistance o f Clostridium
sporogenes spores in artichoke extract and observed D -value low er that those obtained in
phosphate buffer, the phenom enon being m ore evident as treatm ent tem perature
43
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increased.
Table 3.1 shows different food constituent has different thermal resistance
(Lund, 1977).
Table 3.1. Thermal resistance o f various food constituents
C onstituent
z-value (° F)
D 121 (min)
Vitamins
45-55
100-1000
Color, texture, flavor
45-80
5-500
Enzym es
12-100
1-10
V egetative cells
8-12
0.002-0.02
Spores
12-22
0.1-5.0
The pH value can affect the D -value o f m icroorganism s in various ways. On the
one hand, acid pH may reduce the heat resistance o f the spores, an effect directly
reflected in the severity o f the sterilization process since the lethality required for
m icrobiological safety depends, am ong other things, on the D -value at a given
tem perature. On the other hand, the pH o f the food product may inhibit or dim inish
m icrobial grow th, especially o f spores sensitized to pH after heat treatm ent, and
consequently reduce the apparent heat resistance o f the spore (M artinez, 1999).
In order to achieve microbiological safety and high quality in therm ally
processed foods, it is necessary to have correct kinetic data for predictive m odels w hich
makes it possible to use tim e-tem perature integrators as a widely applicable tools to
evaluate the im pact o f heat treatm ent on m icroorganism s and quality factors in a variety
44
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o f processing situation. Recent developm ents o f process evaluation methodologies
(H endrickx, et al.,
1995)
showed that standardized enzymatic tim e-tem perature
integrators (T T Is) can be successfully used fo r fast and correct quantification o f thermal
processes. T he relative easiness o f read-out and handling o f these system s gives them a
significant advantage over microbiological TTIs. In enzym e-based m onitoring systems,
often the am ount o f enzym atic activity rem aining after the heat treatm ent is assayed to
determ ine th e im pact o f the tem perature history, although other properties, such as the
heat o f enzym e deterioration, can be determ ined instead. Prom ising results have been
reported (V an Loey, et a l, 1995) for the TTIs based on a-am ylase from Bacillus suhtilis
w hich was chosen as T T Is to study the effectiveness o f m icrowave heating system.
The objective o f this study was to evaluate the kinetic param eters (D- and zvalues) o f a-am y lase based TT I from B A A at different pH under isothermal heating
conditions in the pasteurization tem perature range (50-95°C) by taking into account the
effective portion o f com e-up and com e-dow n time o f the heating. The purpose w as to
generate an appropriate inactivation kinetics database for the TT I for subsequent use in
the evaluation o f continuous flow conventional and m icrow ave heating systems under
non-isotherm al heating conditions.
M A T E R IA L S A N D M E T H O D S
Preparation o f solutions
All test solutions w ere prepared according to standard specifications (Christian
and Purdy, 1962)
45
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Phosphate buffer:
1.97 g N a 2H P 0 4 .2 H 2 0 (Sigma Chemical Co., St. Louis, M O) and 1.23 g K H 2 PO 4
(Sigm a Chem ical Co., St. Louis, M O) w ere dissolved in 800 ml distilled water, to which
65 ml 0.9% N aO H (Sigma Chemical Co., St. Louis, M O) solution w as added and the pH
w as adjusted to 6.9 with N aO H (Sigm a Chemical Co., St. Louis, M O). The solution was
finally diluted to 1000 ml w ith distilled w ater and kept at room tem perature until use. To
get buffers o f other pH which are used in this study, the com position w as shown in Table
3.2.
Table 3.2. The composition of Phosphate b u ffer Solutions
KH2P 0 4
N a2H P 0 4
M easured pH
(g)
(g)
5.168
0.0858
5.0
4.855
0.2014
5.5
3.68
0.805
6.0
2.17
1.13
6.5
0.942
1.57
7.0
Enzym e solution:
Commercial a-am y lase (Validase B A A 1200L, V alley R esearch, Inc. South
Bend, IN) was used to prepare the enzym e solution. The com m ercial enzym e w as kept in
a refrigerator ( 4 °C) until use. The solution w as diluted to 10*4 w ith the appropriate
buffer prepared as detailed above.
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Substrate preparation-.
0.3 g starch (soluble starch, Sigma Chemical Co., St. Louis, M O ) w as dissolved in
the buffer and made up to 100ml. In order to dissolve the starch completely, the buffer
w as heated m ildly on a heater with continuous stirring. A fresh substrate solution was
used for each experiment.
C olor reagent:
10 g 3,5-dinitrosalicylic acid (Sigm a Chemical Co., St. Louis, M O) was dissolved
in 300 ml w arm distilled w ater and 400 ml 1 N N aO H (Sigm a Chem ical Co., St. Louis,
M O), the solution was heated mildly with stirring w hile adding 300 g potassium -sodium
tartrate (Sigm a Chemical Co., St. Louis, MO). The solution w as then diluted to 1000 ml
w ith distilled w ater and kept at room temperature until use.
Isothermal heat tre a tm e n t
2 ml aliquotes o f the prepared (diluted) a-am y lase solution w ere taken in 4 ml
glass tubes and immersed in a well-stirred w ater bath (Fisher Scientific, Ltd., M ontreal,
Q C) at a selected tem perature for certain period o f tim e as isotherm al heating treatm ent.
D etails o f the time tem perature com binations employed are show n in T able 3.3.
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Table 3.3. TIm e-Temperature range and interval employed for kinetic studies at
different pH levels
Temperature
°C
pH 5.0
pH 6.0
pH 5.6
pH 6.5
pH 6.9
95
10 min @
1.0 min
90
10 min @
1.0 min
5 min @
0.5 min
10 min @
1.0 min
5 min @
0.5 min
5 min @
0.5 min
10 min @
1.0 min
2 min @
0.25 min
5 min @
0.5 min
5 min @
0.5 min
25 min @
5.0 min
25 min @
5.0 min
85
80
75
70
2 min @
0.25 min
2 min @
0.25 min
5 min @
0.5 min
5 min @
0.5 min
65
2 min @
0.25 min
5 min @
0.5 min
5 min @
0.5 min
10 min @
1.0 min
60
5 min @
0.5 min
5 min @
0.5 min
10 min @
1.0 min
55
5 min @
0.5 min
10 min @
1.0 min
50
5 min @
0.5 min
10min @
1.0 min
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A fter th e heating, the tubes w ere removed quickly from the w ater-bath and
subm erged into an ice-w ater bath immediately to cool the solution down. The residual
enzym e activities w ere measured according to a-am ylase assay described below. The pH
o f the buffers in w hich the commercial a-am y lase w ere dispersed w ere 5.0, 5.6, 6.0, 6.5,
6.9 and tem peratures em ployed for the isothermal heating treatm ent varied from 50°C ~
95°C.
a-am ylase assay (Bergmeyer, 1974)
The principle o f the enzym e essay is that a-am ylase hydrolyses the substrate
starch to soluble reducing groups such as maltose and glucose, and maltose reacts with
3,5-dinitro-salicylic acid to nitro-am ino-salicylic acid. The concentration o f the resulting
nitro-am ino-salicylic acid is measured using a spectro-photom etric technique. The
procedure followed is shown in Table 3-4.
The activity is expressed as 0.1 mg starch hydrolysed in unit tim e and is
expressed as:
V olum e activity (absorption units/m in) = 4950* AE
W here AE is the difference in absorption betw een the sam ple and the blank.
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T able 3.4. P ro ced u re used for a -a m y la se assay
Pipette the following into 12 ml centrifuge tubes (at room
temperature)
Test
solution
Blank
Buffer
1.0 ml.
1.0 ml.
Test sample (Enzyme solution)
(before and after isothermal heating treatment)
0.05 ml.
Substrate solution
1.0 ml.
1.0 ml.
2.0 ml.
2.0 ml.
M ix thoroughly and incubate for 10 min.
Add Color reagent
Add Enzyme solution to the Blank
—
0.05 ml
M ix thoroughly, place the tubes in a boiling w ater bath
(TH ERM O M IX 1480) immediately and heat for 5 min.
Cool in cold w ater and after 20 min read the absorption
from spectrophotom eter (BECKM AN, DU-65)
T im e -te m p e ra tu re profile
The tim e-tem perature profiles o f test samples subjected to the isothermal heating
treatm ent were evaluated using a needle type 0.381 mm diam eter copper-constantan
therm ocouple (Omega Engineering Inc , Stamford, CT) attached to a data-logger. The
therm ocouple is inserted into the test sample in the tube and tim e-tem perature data were
recorded using data-logger (Dash-8, M etra-Byte Corp., Taunton, MA) to get the timetem perature data during come-up tim e (CU T), hold tim e as well as com e-down (CDT)
times o f the samples. The hold time data represent full isothermal exposure o f test sample
to the set temperature. A certain fraction o f the com e up and com e down periods also
contribute to enzyme inactivation and must be included in the kinetic analysis. The
procedure followed to accom m odate the effective portion o f com e-up and com e-down
periods is detailed later.
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Kinetic d a ta an aly sis
B ased on previous studies, inactivation o f a-am y lase w as assum ed to follow a
first order reaction kinetics w hich means a logarithm ic order o f destruction, w hich can be
expressed as:
w here A is the residual a-am ylase activity; A0 is the initial a-am y lase activity; k is the
reaction rate constant (s _1) at a specific temperature; t is the inactivation tim e (s); the k
value is obtained as the negative reciprocal slope by regression o f loge (A / Ag) vs. time;
the D -value, decim al reduction time, is defined as the heating time required to result in a
90 percentage inactivation o f the initial activity at a given tem perature. It reduces the
residual activity to one tenth (one decimal reduction) o f the initial activity. D value is
m ore frequently em ployed in therm al kinetics studies and is related to the rate constant, k
can be expressed as:
The D value can also be obtained as the negative reciprocal slope o f the lo g 10 (A/
Aq) vs. tim e curve. On a semi-logarithmic plot, it can be obtained easily as th e time
interval between w hich the lo g i0 (Al
vs. tim e curve passes through one logarithm ic
cycle.
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The z-value, w hich is the tem perature sensitivity o f D- value, can be determ ined
as the negative reciprocal slope o f log D vs. Tem perature regression w hich m eans the
tem perature required to achieve a ten-fold change in D-value. It can be expressed as:
x.
<r » - r '>________ (3.3)
log A - log d 2
w here Tj and T2 are the tem peratures corresponding to D j and D 2
In m ost food processing situation, the lethality (F) accum ulated at the cold spot is
calculated by integration o f the lethal effects o f the tem perature profile during the come
up, hold and com e-down time. It can be expressed as:
te = F = £ l 0 iT~Tr‘fVzd t -------(3.4)
w here te is th e effective tim e and Tref is the reference or the bath tem perature.
In therm al inactivation studies involving non-isotherm al heating conditions, the
contribution o f the CUT and CDT should be added to the heating tim e by calculating the
effective portion o f the CUT and CDT using a sim ilar concept. C alculation o f the
effective tim e or lethality requires data on z-value which needs to be obtained from a
regression o f log D -value vs. temperature. F or this purpose, first estim ates o f the D-value
associated at the various tem peratures (T) are obtained using uncorrected heating tim es
assum ing isothermal heating conditions. These values are used to calculate the z-value by
regression o f log D vs. tem perature as the negative reciprocal slope. This z value is then
used to calculate the effective tim e under each condition using th e above equation (3.3)
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and from these th e corrected D-values are calculated. These D -values are then used to get
a new z-value w hich is then used again to get m ore precise value o f effective tim es and
used
recalculate the D -values and another z-value. This procedure is repeated many
tim es until the chance o f z-value is less than 0.5%. A schematic flow chart o f calculation
steps is shown in Figure 3.1 (Ramaswamy, 1993; Tajchakavit, et al., 1997).
START
Regression ofLog(AAo x 100) vs. t
D=-l/sIope
Regression of LogDvs. T
z=-1/dope
recalculate D, z-values
END
F ig u re 3.1. Flow c h a r t of calculation steps for kinetic d a ta analysis
According to Ross et al. (1979), the accuracy o f the accum ulated lethality (F o r te)
calculated for a therm al process using num erical integration technique is depended on the
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num ber o f data points included in the calculation. Therefore, it is better to have as many
tim e-tem perature data points as possible for a better calculation.
RESULTS AND DISCUSSION
First O r d e r Inactivation Model for a-am y lase
Figure 3.2 shows the typical residual activity curves for a-am y lase at pH 6.9
following heat treatm ent for various time intervals at selected tem peratures. The linearity
o f the curves indicate the adequacy o f using the first order plots. The D values obtained
as the negative reciprocal slope o f the regression o f log (A/Ao) vs. time data are shown in
Table 3.5 along w ith the associated R2 values which are quite high. As expected the
curves w ere steeper at higher tem peratures yielding low er D values. T o relate the D
values to tem perature, a similar semi-logarithmic approach is used relating log 10 D
values vs. temperature. The z-value is obtained as the negative reciprocal slope o f log D
vs. tem perature regression. The z value plot for a-am ylase inactivation at pH 6.9 is
shown in Figure 3.3. Again the curve shows a reasonable fit w ith an associated R2 o f 0.93
and a com puted z value o f 31.1°C at pH 6.9. The relatively high m agnitude o f D- and zvalues o f the TTI under isothermal heating in buffer solution at pH 6.9 dem onstrates a
fairly high resistance o f the enzym e to therm al destruction.
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0
5
10
15
20
25
30
35
40
45
50
55
60
♦ 85C ■80c"Sn^<5T1*70C ®65C
Figure 3.2. D-values curves of a-amylase at pH 6.9 at different temperatures
without any correction
Table 3.5. Regression details for D-value computations at pH 6.9
Tem perature °C
65
70
75
80
85
z-value
D value, min
104.2
72.5
66
29.8
25.5
3 i.r c
R2
0.93
0.91
0.94
0.90
0.96
0.93
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2.5
a>
3
CO
>fi
Q
R = 0.9347
o'
z=31.1 oC
O!
o
0.5
60
65
75
70
80
85
90
Temperature, C
Figure 3.3. z-value curves of a-amylase at pH 6.9 before correction
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Come-Up Tim e and Come-Down Time Corrections
As discussed earlier, even w hile heating small (2 ml) test samples in well agitated
w ater baths, a certain time lag is inevitable before the sample can reach the target
temperature. Likew ise a small delay is accom panied during cooling. The test sample w ill
not be at the desired tem perature during these come-up and com e-dow n periods. W hile
the lethal contribution during the come-up period could be relatively small, the same
during cooling could be significant since the test sample is at the highest tem perature
prior to cooling. Ignoring com e-up and com e-dow n time contributions to lethality could
result in serious errors in com puted kinetic param eters especially at high tem peratures
w here a short tim e can induce appreciable inactivation. Hence these need to be corrected
in order to get m eaning kinetic parameters.
Similar concepts have been traditionally followed in most therm al processing
applications. The time interval from adm itting the heating medium (steam o r water) until
the actual processing condition is established in the retort is called the come-up tim e
(CU T), which is characteristic o f all batch-type retorts. Thermal process is usually tim ed
from when the retort actually reaches the operating condition. The lethal effects at the
product center contributed during the CUT are small. However, the product tem perature
is elevated during the CUT, thereby shortening the time required for it to reach lethal
levels (Ramaswamy, 1993). Come-up tim e effectiveness has been traditionally taken to
be 42% o f CUT as originally suggested by Ball (1923) for thermal process calculations.
Spinak and Wiley (1982) found CUT effectiveness vary from 35% to 77% in retort
pouches calculation, and Ram asw am y and Tung (1986) found the effectiveness o f 42% to
be very conservative for application to thin-profiie geom etry due to fast heating.
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F igure 3.4 shows a typical com e-up period tim e-tem perature profile o f sample
subject to conventional w ater bath heating condition, with w ater bath maintained at 74°C.
All com e-up and com e-down curves w ere corrected using the procedure described earlier
taking the effective tim e o f the w hole heating process including com e-up, hold and com e­
dow n period. The kinetic param eters w ere recalculated using the iterative procedure
described earlier. The corrected D and z value curves for pH 6.9 are presented in Figures
3.5 and 3.6. In this case, since the CUT is only small portion o f the heating time, so the
corrections w ith respect to D values and z value are small.
80
70
60
50
40
30
20
50
30
60
80
90
Time, sec
F ig u re 3.4. Time-temperature profile of sam p le d u rin g co n v en tio n al heating
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Log10(A/Ao)
0 - .............................
-
0.1
-
-
0.2
-
-0.3
D85 =24.1 min
085
-°-4
-°-5 '
075=64.2 min
-0-6 '
-0.7 -------------------------------------------------------------------------------------------------0
5
10
15
20
25
30
35
40
45
50
55
60
♦ 8 5 C ■ 8 0 C ’& % d 11*70C ® 65C
F ig u re 3-5. D -values cu rv es of a-amylase at pH 6.9 at d iffere n t temperatures after
correction
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2.5
©
J3
CB
>
Q
o
0.5
60
65
75
70
80
85
90
Tem perature, C
Figure 3.6. z-value curves of a-amylase at pH 6.9 after correction
W hen the heating condition has a significant CUT in relation to th e total heating
time, the contribution o f com e-up tim e would becom e more significant and in such cases
the corrections becom e m ore apparent. Figures 3.7 and 3.8 show the inactivation curves
at pH 5.6, and unlike at pH 6.9, the D -values before CUT correction are much different
from D -values after CUT correction. Table 3.6 shows the calculated D and z-values at
different tem peratures and different pH before and after CUT correction.
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0.2
!
-
I
S
0.2
150
100
200
—
-0.4
-
0.6
-
1.2
-1.4
T im e, s e c
!♦ 75 0 7 0 A 65 * 60 !
Figure 3.7. Thermal inactivation curve of a-am ylase at pH 5.6 before correction
o
20
-
25
0.2
-0.4
<
- 0.6
-
0.8
■1
1.2
1.4
Time, sec
■♦60 0 6 5 A 70 X 75 j
Figure 3.8. Thermal inactivation curve of a-am ylase at pH 5.6 after correction
61
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 3.6, D-values and z-values of a-amylase under water-bath thermal heating condition
Temperature
C
at pH 6.5
uncorrected corrected
50
55
60
65
70
75
80
85
z-value
10.22
6.6
4.7
3.9
2.4
33.7
9.9
5.53
4.38
3.67
2.2
33.6
D-values min
at pH 6.0
uncorrected corrected
5.8
3.2
2.6
2.1
1.2
36.5
at pH 5.6
uncorrected corrected
4.3
3.7
5.1
3.6
3.3
3.2
2.3
2.4
1.5
1.2
1.9
0.83
33.1
29.5
1.96
1.1
at pH 5.0
uncorrected
5.1
2.9
1.8
1.6
0.6
corrected
4.2
2.3
1.7
1.1
0.4
1.05
32.7
62
23.6
21.1
For monitoring o f the evolution o f a certain food safety or quality param eters, TTI
must have z-value equal to that o f the target param eter and conveniently low or high Dvalue in the considered tem perature range to give m easurable response. The pH
dependency o f the decimal reduction time o f a-am ylase was studied in a w ater bath at the
range o f 5.6 to 6.9. From the figure 3.9 shown below, the z-values before correction are
23.6°C, 3 3 .1°C, 36.5°C, 33.7°C, 31 f ’C at pH 5.0, 5.6, 6.0,6.5, 6.9, respectively. The time
corrected z-values o f a-am ylase are shown in Figure 3-10. As shown in figure 3.10, after
correction, the z- values are: 21.1 °C, 29.5 °C, 32.7 °C, 33.6 °C and 30.3 °C at pH 5.0, 5.6,
6.0, 6.5, 6.9, respectively.
Figure 3.11 shows the z-value o f a-am ylase, as related to the pH. As can be seen,
the z value varies appreciably depending on the pH level em ployed, thus the TTI from a am ylase can be used as an indicator for a variety o f food systems. It is well known the
changes in the pH o f the reaction media may affect enzym e activity. The changes are due
to protonation or deprotonation o f ionizing groups in the enzym e, in the substrate, or in
the enzym e-substrate complex. Shifting the pH away from neutral may w eaken the forces
stabilizing protein conformation, leading to an enhanced rate o f enzym e denaturation at
the temperature o f the assay.
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2.5
c
£s
pH6.0
Q
-z=36£--
o
pH 6.5
z = 3 3 .7
0 .5
60
100
90
-0 .5
T e m p e r a tu r e
C
Figure 3.9. z-value of a-amylase at different pH before correction
2 .5
pH 6 .9
2 --3 0 .3 C
c
E!
Q
pH 6.5
o
0)
o
45
60
65
- 0 .5
75
80
pH 5 .6
85
90
z = 2 9 .5
Temperature, C
Figure 3.10. z-values of a-am ylase at different pH after correction
64
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100
35
30
X
w.
./.
J
® 25
3
(0
>
n 20
15
10
4
4 .5
5
5.5
j
6
6 .5
7
7 .5
!
pH
Figure 3.11. pH dependency of z-values of a-amylase
C o m p ariso n with literature values
Although thermal inactivation kinetics o f a-am ylase from Bacillus spp. had been
widely studied, a-am y lase from Bacillus subiilis w ere not extensively investigated. Also,
the source employed and the buffer used in various studies has been generally variable.
Most studies were done by enzym e im mobilization in stead o f solvent engineering which
employed in this study. Van Loey et al. (1996) reported that the z-value o f heat
inactivation o f a-am y lase from Bacillus subiilis is dependent o f enzym e concentration,
whereas the decimal reduction tim e at 80°C increases as enzyme concentration increases,
indicating that dissolved a-am ylase from Bacillus subiilis is more therm ostable at higher
to
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enzym e concentration. Thermal inactivation o f a-am ylase from Bacillus subtilis at
concentration o f 5, 10, 20, 30 mg/ml could clearly be described by first-order kinetics.
K inetic param eters
estimated using nonlinear regression
analysis
on
isothermal
inactivation data were reported as Table 3.7.
Table 3.7. Kinetic parameter estimates (D80 z) of a-am ylase from Bacillus subtilis at
different enzyme concentration under isothermal condition (Van Loey et al., 1996)
a -a m y la s e from Bacillus
subtilis (m g/m l)
5
10
20
30
z ( °C )
Dso(mirs)
9.79 ± 0.22
10.10 ±0.25
9.46 ± 0.28
9.58 ±0.21
9.08 ± 0.32
12.88 ± 0.35
17.43 ± 0.42
33.41 ± 0.95
Also, a z-value o f 8 .9 1°C and a D l0()- value o f 0.17 min were obtained when
concentration o f a-am ylase from Bacillus subtilis was 200 mg/ml. Additionally, Bacillus
lichenifortnis a-am ylase (type X ll, Sigma) and Bacillus amylo/k/iiefaciens a-am ylase
(type II-A, sigma) were under the same thermal inactivation, and it was found that all
these three a-am ylases at different environmental condition could be adequately
described by a first-order decay. The dependence o f the decimal reduction tim e on the
initial enzym e concentration was found (Van Loey et al ., 1999)
Since inactivation o f enzym es depends on several factors including the source o f
enzyme, nature o f substrate, experimental approach and heating conditions etc, it is not
surprising to see differences in the published kinetic data, in fact, this is principal reason
for the detailed evaluation o f inactivation kinetics carried out in this study. Accurate data
66
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w ere required under the testing conditions employed in this study for use in evaluating
their inactivation behavior under continuous flow systems. The z-values obtained in this
study are higher than that o f z-values in the literature, nevertheless, the D-values in this
study, for example, DXlj -values are much lower than that o f Dxo -values in the literature.
C O N C L U S IO N S
Thermal inactivation kinetics o f a-am ylase based TTI from Bacillus subtilis at
different pH (5.0 to 6.9) were studied in the pasteurization tem perature range (50 to 95
°C). The kinetics o f enzyme inactivation could be determ ined by the first-order reaction
rate and the result showed that the inactivation rates increased while the tem perature o f
the heat treatment increased
By considering the effectiveness o f CUT, the D-values and z-value o f TTI from
a-am ylase were calculated at different pH The z-values o f a-am y lase before correction
are 23.6 °C, 33.DC, 36.5"C, 33.7°C, 31.1 °C at pH 5.0, 5.6, 6.0,6.5, 6.9 respectively, after
correction, the z- value are. 21. 1°C, 29.5°C, 32.7”C, 33.6°C and 30.3°C at pH 5.0, 5.6,
6.0, 6,5, 6.9 respectively.
The result also shows a pH dependency o f a-am ylase, therefore, by adjusting pH
o f the solution, a-am ylase can be applied to various food system with different pH
67
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CHAPTER 4
K IN E T IC S O F E N Z Y M E IN A C T IV A T IO N U N D E R N O N -IS O T H E R M A L
C O N T IN U O U S -F L O W M IC R O W A V E H E A T IN G AND T H E R M A L H O L D IN G
C O N D IT IO N S
ABSTRACT
Inactivation kinetics o f an a-am ylase enzym atic tim e-tem perature integrator
(TTI) from Bacillus subtilis (BAA) under continuous-flow microwave and conventional
hold conditions w ere evaluated and compared. The TTI dispersed in a buffer solution (pH
5.0 o r 5.6) initially at 20°C was continuously circulated through two helical coils
connected in series for heating. The tw o coils w ere positioned in tw o domestic
m icrow ave ovens (2450 M H z and 1000 W nominal capacity each) and connected by a
short tubing. The sam ple flow rates w ere adjusted to result in specific exit tem perature
(65-80°C). A short fully insulated helical coil at the exit o f the second oven was used as a
holding tube. Test samples could be drawn either at the exit o f the second M W oven or
im m ediately after the holding tube. After establishing steady state heating conditions,
small volum e o f test sam ple was collected at the intended exit (after M W o r after the
holding tube) directly in to a beaker surrounded by crushed ice to rapidly cool the sample.
U nder the range o f experim ental conditions, the extent o f enzyme inactivation following
the heat treatm ents w ere evaluated. The inactivation data w ere then com bined w ith
experim entally m easured tim e-tem perature data to com pute the associated decimal
reduction times. Tim e-tem perature profiles obtained during heating and cooling o f the
samples w ere used to correct come-up times. The corrected D -values under m icrowave
heating condition ranged from 16.0 s to 4.9 s at pH 5.0 between 65 and
75°C and
68
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27.0 to
5.7 s at pH 5.6 between 65 and 80°C, respectively. D-values during the thermal hold
period varied from 39 s to I B s between 65 and 75°C at pH 5.6. Com paring with the Dvalues betw een continuous-flow M W and therm al holding, as well as from the batch
kinetics from chapter 3, enzym e destruction occurred much faster under continuous-flow
m icrow ave heating condition than under conventional thermal heating condition. Hence,
there w as evidence that microbial lethality under microwave heating conditions can not
be fully accom m odated by conventional models em ploying thermal kinetics data.
IN T R O D U C T IO N
Conventional pasteurization o f liquid foods (milk, juices and other beverages) is
carried out in continuous flow high-tem perature short-time (HTST) heating systems using
heat exchangers (tubular/plate) followed by a brief period o f holding followed by
subsequent cooling, again in heat exchangers, and packaging, usually under aseptic
conditions. A com m on problem encountered in these continuous H TST pasteurization
processes is the contact surface fouling caused by the exposure o f fluids to high surface
temperature. R ecent innovations such as scraped surface heat exchangers help to
m inim ize fouling problem s by continuously sweeping the hot surfaces with scraper
blades.
M icrow ave heating could provide an alternative for continuous pasteurization o f
som e liquid food products. The greater penetration depth and faster heating rates
associated with m icrowave heating have been recognized as potential factors to im prove
the retention o f therm olabile constituents in liquid foods such as m ilk and fruit juices
69
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(M udgett,
1986). Also, a potential advantage o f continuous-flow sterilization or
pasteurization o f fluid food over conventional sterilization m ethods for canned foods is
that product quality can be improved due to higher nutrient retention. ■
A num ber o f studies have reported successful m icrowave pasteurization o f milk
(H am id et al., 1969; Jaynes. 1975; Chiu et al., 1984; M erin and Rosenthal. 1984;
K nutson et a l, 1988; Kudra et a l, 1991). M icrow ave pasteurization o f enzymes (e.g.
PM E) and m icroorganism s (e.g. L. plantarum) in fruit juices w ere conducted by Copson
(1954), N ikdel and M ackellar (1992), Nikdel et al. (1993), Tajchakavit and Ram aswam y
(1998). Tajchakavit and Ram aswam y applied m icrowave energy for inactivation o f PM E
in orange ju ice and destruction o f spoilage microorganisms, S. cerevisiae and L.
plantarum w ere used as indicators to study the kinetics under continuous-flow
m icrow ave heating condition. They found out that the inactivation/destruction followed
typical first-order reaction kinetics showing linear destruction rate on a logarithm ic plot
o f residual activity/survivors vs. residence times. A fter accom m odating the effective
portion o f come-up time and com e-dow n time, it was reported that within the range o f
tem peratures and sam ples size em ployed in these studies, m icrow ave heating proved to
inactivate the enzyme and destroy the m icroorganism s by an o rder o f m agnitude faster
that conventional thermal heating (Tajchakavit, 1997). However, the two selected strains
w ere very sensitive to microwave heating, rapid destruction w ere achieved at tem perature
as low as 60°C for S. cerevisiae and 65°C for L. plantarum. A nother problem is that the
tem perature range is from 52.5 ~ 60°C for S. cerevisiae and 57.5 ~ 65°C for L. plantarum
w hich
w ere
very
narrow.
Therefore,
a
tim e-tem perature
indicator
w ith
70
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high
therm ostability w hich allow developing enzymatic monitoring system s w orkable in a
broad therm al im pact range is required.
Several studies have been carried out to evaluate the effect o f m icrowave heating
on biological and chemical systems using different approaches, experimental designs and
techniques, som e dem onstrating additive non-thermal effects to be associated w ith them.
V arious strains o f m icroorganisms and enzymes have been em ployed in these studies. It
is generally believed that the microwave effects on biological system s are mostly caused
by the heat generated by the friction o f dipole m olecules under the influence o f
oscillating electrical field; however, any possible non-therm al effects will always overlay
the thermal effect. It is difficult to precisely evaluate the effectiveness o f m icrowave
heating vs. conventional heating from the literature because o f the techniques em ployed
or a lack o f detail in the m ethods or materials, especially in relation to the tem perature
monitoring. Lack o f tem perature measurement, uneven heating due to m icrowave field
distributions and inability to control tem peratures o f m icrow ave-heated samples during
heating have been generally cited as reasons for the difficulty in resolving the therm al vs.
non-therm al effects controversy. Evaluation o f non-therm al effects on biological systems
(which can not be explained by particular tim e-tem perature history) have been the topic
o f interest in several recent studies. Several studies (Culkin et a l, 1975; D reyfuss and
Chipley, 1980; M udgett, 1986;
Khalil and Villota, 1988; K erm arsha et a l, 1993a,b;
Tajchakavit and Ram aswam y, 1995,1997; Tajchakavit et a l, 1997; Koutchm a, 1997;
Kozempel et a l, 1997,1998) have observed non-therm al or enhanced thermal effects o f
microwaves. Others (G oldblith and W ang, 1967; Lechow ich et a l, 1969; Vela and Wu,
71
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1979; Fujikaw a e t a l , 1992; Fujikawa, 1994; W elt et a l, 1994; Tong, 1996) have refuted
the non-therm al effects o f microwave.
Several theories have been developed to explain how electrom agnetic energy
m ight kill m icroorganism s w ithout heat. O ne explanation w as selective absorption o f
m icrow ave energy w ithin the organism resulting in its inactivation w hile the surrounding
tem perature could still be low (Palaniappan et a l, 1990). Theoretical analysis o f the
controversial question o f non-therm al effects o f m icrowave heating in chem istry w as
recently m ade by Stuerga and Gaillard (1996). U sing classical axiom s o f physics and
chem istry they concluded that there is no doubt that an electric field can have different
m olecular effects com pared w ith thermal energy. M icrow ave specific effects may occur
in cases w here m icrow ave heating gives particular tim e-tem perature distributions, w hich
can not be achieved by other means. This m eans that in the absence o f non-therm al
effects, m icrobial destruction under m icrowave heating results from established tim etem perature relationships o f therm o-bacteriology. The use o f this approach is rather rare
in studies concerning m icrowave effects and m ost results refer to evaluation o f lethality
effects w ithout considering therm al history o f the product (Riva et a l, 1993).
The objectives o f this study were (1) to evaluate the kinetic param eters (D -values
and z-values) for inactivation o f a-am ylase based T T I in different pH solutions during
continuous-flow
non-isotherm al
m icrowave
heating
conditions
by
taking
into
consideration o f com e-up tim e and (2) to com pare the results w ith those obtained from
the conventional batch therm al treatm ent as well as continuous therm al holding tim e
kinetics.
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M A T E R IA L S AND M E T H O D S
M icro w av e h e a tin g system
T he m icrowave heating system used for subjecting the enzyme solution to
continuous-flow treatm ents are detailed in Tajchakavit and R am sw am y (1995) and Le
Bail et al. (2000). A schematic diagram o f the system is shown in Figure 4.1. Briefly, it
consisted o f tw o 1000 W, 2450M Hz microwave ovens (SHARP Carousel, Model. R4 1 1AWC; G oldstar W aveplus II) with two helical glass coil heat exchangers (one in
each) centrally located inside each oven cavity. The enzym e solution was circulated
through the helical coils made from Pyrex glass tubing [inside diameter, 0.5 cm with
volum e o f coil 78.6 ml (first oven) and 80.4ml (second oven)]. These tw o ovens w ere
connected by a short plastic tubing. The flow o f the test solution in the system w as
adjusted using a calibrated variable-speed m etering pump (Cole-Parm er Instrum ent
Company, M asterflex® ). The direction o f the fluid flow in the tube was upw ard in order
to have a better control o f flow rate. Inlet, outlet and middle tem peratures were
continuously gathered by using thin w ire copper-constantan therm ocouples centrally
inserted in the tubes and attached to the data-logger (H P-34970A D V M +H P- 34.901A
m ultiplexer). In order to get better m ixing condition and reduce the tem perature gradient
across the radius o f the tube, two static mixers w ere installed at the outlet o f the first and
the second microwave oven.
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J4
Holding tube
cooler
sample
pump
F ig u re 4.1. C o n fig u ratio n o f M W h eatin g setu p
74
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The average residence time o f the test liquid in the m icrowave heat exchanger
w as obtained by dividing total volume o f test sample inside the oven (which w as
subjected to m icrowave heating) by the steady state volum etric flow rate o f the liquid
through the system. After the second oven, the sample exiting from the microwave oven
w as run through an isothermal holding tube made o f Pyrex glass tubing (volum e 39.6ml,
inside diam eter, 0.9 cm). The tubing was insulated to prevent heat loss. The length o f
tubing and flow rate w ere pre-adjusted based on preliminary runs to obtain desired exit
tem peratures. H eat treated test samples w ere withdrawn both at the exit o f the second
m icrow ave oven and at the exit o f the holding tube during steady state heating periods
w ith exit tem peratures in the range from 65 to 80°C. Each exit tem perature w as achieved
by pre-adjusting and changing the flow rate.
A t the flow rate used, the fluid flow profile w as expected to be essentially
laminar. H owever, the use o f helical coils creates secondary flow w hich can result in
thorough mixing o f the fluid as it passes through the system. In order to assess the flow
characteristics o f the system, several param eters associated w ith the heating system w ere
evaluated. The heating param eters and the form ulas used for its calculation are detailed in
F igure 4.2.
75
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1111
l l l l l : temperature rise vs. residence
time, s
[1
'.e a -n k
.:,r
t
AT= ! exit —. i
t = Fiow rate (V) /voiume
*'
-AT/1
* \-
Absorbed power, W/g
3
:4
Formula
Heating parameters
,
" etnbsrSiUi-e «s Rey*elo
Temperature vs. Dean number
Re - Ovp/jpt ,
Dc ^ R g^ r J I ^
F ig u re 4.2. H eatin g c h ara cteristics o f th e continuous-flow M W setu p
76
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K in etic D a ta A nalysis
The destruction o f microorganisms is generally modeled based on first order rate
reaction kinetics:
d C /d t = -kC
(4-1)
in w hich dC/dt is the tim e rate o f change concentration C, k is the reaction rate constant.
The thermal resistance o f microorganisms is also tradionally characterized by food
m icrobiologists by means o f the D and z values:
D = 2.303 Ik
ill—
log Dx / D 2
(4-2)
(4-3)
D value represents the heating tim e at a given tem perature to cause one decimal reduction
in surviving m icrobial population. The z value represents the tem perature range between
w hich the D values change by a factor o f 10. W hen the therm al resistance o f a
m icroorganism is known, it is possible to calculate the equivalent lethality (F) or tim e
(tref) necessary for therm al treatm ent by integration o f the tim e-tem perature history using
the equation (4-4):
'
i 7 - j 10
T(Q-Tr
z
dt
(4-4)
w here T (t) is the transient tem perature at time t and T R a reference tem perature. This
approach has been traditionally used in the thermal process calculations.
77
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Sim ilar concept can be applied for determining kinetics param eters during
continuous-flow heating systems; however, non-isotherm al heating conditions are
involved in these cases. The D values will have to be com puted using equation (4-5):
D - t eff /lo g ( C J C )
(4-5)
where teff is an effective tim e (same as F in Eqn.4-4) w ith T r as exit tem perature,
obtained using either a com puter model predicted or experim entally determ ined timetem perature profile; C0 and C are initial and final concentration o f microbial cells. The
use o f this approach is rather rare in studies concerning m icrowave effects. There have
been only
few studies describing kinetics during microwave heating and m ost results
refer to evaluation o f lethality effects w ithout considering therm al history o f the product.
B ut as in thermal destruction, microwave destruction kinetics o f food constituents such as
quality attributes, enzymes and m icroorganisms are required for establishing microwave
processing.
The procedure for gathering kinetic param eters during continuous-flow heating
have been detailed in (Tajchakavit and Ram aswam y, 1997). Briefly, the D-values at the
exit tem peratures can be first calculated from the regression o f log residual numbers o f
survivors vs. uncorrected heating time (residence time), and then z value is obtained as
the negative reciprocal slope o f log D vs. temperature. U sing the calculated z value, the
heating tim es can be corrected using Eqn. (4-3), and D -values and subsequently the z, can
be corrected. This step can be repeated as many times as necessary until the convergence
o f z-value.
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RESULTS AND D ISC U S S IO N
M icro w av e h e a tin g ch aracteristics
Typical evolution o f tem perature as function o f time at different mean fluid flow
rates during continuous-flow microwave heating are shown in Figures 4.3. It indicates
typical lag periods prior to achieving steady state. As K udra et al. (1991) explained, the
non-linearity in the tim e-tem perature profile during the early phase o f heating is
contributed by the coil and the environm ent w ithin the oven cavity. In the m icrowave
heating set-up used, it took about a minute after turning the m icrowave on to reach the
target tem perature in the range 65 to 95°C, w hile equilibrated steady state exit
tem peratures w ere achieved after about 2 min.
The m ean exit tem perature at the end o f first and second oven as a function o f
flow rate and residence time are shown in Figures 4.4 (a) and (b). As expected higher
residence tim es (low er flow rates) resulted in higher exit tem peratures. The associated
d ata on Reynolds and D ean numbers, mean heating rate, pow er absorbed and pow er
absorption efficiency are summarized in Table 4.1.
Table 4.1. Summary of operating temperatures, flow rates, heating rates and
absorbed p o w er (initial temperature 20°C )
Exit
temperature,
°C
95
88
81
75
70
66
Flow rate,
Residence
time, s
Heating rate,
ml/s
°C/s
Power
absorbed, W
Efficiency,
%
Re/De
numbers
4.17
5.00
5.50
6.67
7.50
8.33
64.8
54.0
49.1
40.5
36.0
32.4
1.09
1.18
1.14
1.23
1.25
1.27
1228.2
1327.3
1287.3
1395.7
1413.1
1441.3
61
66
64
69
71
72
600/178
720/214
792/236
960/286
1080/322
1200/357
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90
4 .2 m l/s
5 .4 m l/s
6 .5 ml/s.
7 .6 m l/s
70
50
30
20
40
50
60
70
80
90
Time, sec
F ig u re 4.3. Microwave heating curves as a function of time at different flow
ra te
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100
90
CL
40
Flow rate, ml/s
A 1 oven ® 2 oven
Figure 4.4a. Microwave exit temperatures as a function of flow rate
100
90
80
70
CL
60
50
40
30
50
70
R esid en ce time, s
A 1 oven SS2 oven
Figure 4.4b. Microwave exit temperatures as a function of residence time
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The heating rates varied from 1.09 to 1.27 °C/s depending on the flow rate and
exit tem peratures. The microwave pow er absorption efficiency varied from 61 % to 72%,
with low er levels associate with higher exit tem peratures and low er flow rates. This can
easily be explained by the higher loss o f heat to the m icrowave oven environm ent at the
higher tem peratures. The associated Reynolds numbers w ere considerably low er than the
2100 indicating the flow to be essentially laminar. H ow ever, the use o f helical coil
creates secondary flow w hich results in thorough mixing o f the fluid as it passes through
. the system. It has been argued that, at high D ean numbers, the secondary mixing o f fluids
w ithin a helical coil provides a ‘ perfectly mixed or plug flow profile’ for the flowing
liquid (Dravid et al., 1971). The Dean numbers associated w ith the flow varied from 178357 in the present studies. The exit tem peratures achieved as a function o f Reynolds and
D ean numbers are shown in Figures 4.5a,b.
The m icrowave heating patterns also depended on the pH o f the test solutions.
The mean exit tem peratures (a) and pow er absorbed (b) as a function o f flow rate at
different pH are shown in Figures 4.6. The power absorbed and hence the exit
tem peratures w ere higher at lower pH. The differences in the heating patterns at different
pH w ere ascribed to the differences in the concentrations o f various chem icals used for
the preparation o f the buffers.
82
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90
70
60
Reynolds number
A 1 oven ®2 ovens
Figure 4.5a. Microwave exit temperatures as a function of R eynolds number
90
80
<a 70 3 60
S
50
§40-
20
60
Dean number
A 1 oven B 2 ovens
Figure 4.5b. Microwave exit temperatures as a function o f Dean number
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100
90
o
e3
X 1
CO
X
80
—
m
Q.
E
♦
70
x
UJ
60
50 44
5
10
6
Flow rate, ml/s
♦ pH 5.6
mpH 6.0
A pH 6.5 XpH 6.9
Figure 4.6a. Microwave exit temperature as a function o f flow rate at
different pH
5
1400
6.0
7.0
10.0
Flow rate, mL/s
-pH5.6 —
pH6. 0
-pH6.5 '
-pH6.9
Figure 4.6b. Microwave power absorbed as a function o f flow rate at
different pH
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Enzyme inactivation profiles under continuous flow microwave h eatin g
Typical inactivation curves obtained in th e continuous-flow m icrowave system
for steady state exit tem peratures in the range 65 and 97°C are shown in F igure 4.7.
U nder the
sam e
microwave
continuous-flow
heating
condition,
the
m icrowave
inactivation o f a-am y lase at different pH are characteristically different. For example, at
pH 6.9, alm ost no inactivation was observed in th e range o f 70 to 90°C. Increasing exit
tem perature to 97°C achieved only about 7 % inactivation. H owever, as the pH lowered,
the extent o f inactivation significantly increased. This form o f presentation has been the
conventional approach used by researchers for presenting data for com parative purposes.
Temperature, C
60
65
70
75
80
85
90
95
100
1
0.1
pHS.O —A— pH5.6
pH6.0
pH6.5 —H— pH6.9
Figure 4.7. Inactivation ra te of a-am y lase at different pH u n d e r continuous-flow
microwave condition
85
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W hat is not apparent from the conventional residual activity plotted against
tem perature (Figure 4.7) is the fact that the flow rates em ployed for achieving the
different tem peratures (at each pH) w ere different. Hence, for the sam e exit temperature,
the residence tim es under different heating conditions w ere also different. Obviously, the
residence tim e at each pH steadily increases as the exit tem perature is elevated, thus
giving a com pounded destruction effect due to higher tem perature and longer residence
tim e com bination. The steeper drop in residual activity shown in figure 4.7 is the result o f
such com bination effects. Quantitative com parisons under these heating conditions can
be made using com puted D values [D = heating time /(log reduction in residual activity)].
T he heating time, however, should be the effective time com puted as before by
integrating the kinetics into the heat penetration data. Come-up tim e-tem perature profiles
under the heating conditions are needed for this purpose. Since the com e-dow n w as
alm ost instantaneous (w ith small volum es o f heated test solution collected directly in to
ice-chilled conical flasks), the lethality accum ulated during com e-dow n period was
neglected. The experimentally determ ined tim e-tem perature profile for m icrowave and
conventional heating conditions are shown in F igure 4.8. R elative to conventional
heating, the heating profile under m icrowave has been reported to be fairly linear
(R2=0.97) w hich is also apparent from the tem perature continuously m easured at the m id­
point o f the system (betw een the tw o m icrow ave ovens). The linear profile betw een the
initial and final tem peratures over the com e-up period was subdivided to 100 elem ents
for accurate computation o f accum ulated lethality during the com e-up period (Equation
4-4). As outlined before, a z-value w as initially com puted from uncorrected residence
tim e and later refined using the effective time, tef.
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60
50
40
20
30
40
Time, s
MW
Figure 4.8. Time-temperature profile of test samples subjected to
continuous-flow microwave vs. water-bath heating condition at an exit temperature
65°C.
Decimal Reduction Time Comparisons
The D -values estim ated (uncorrected and corrected) from the experim ental
survivor data at different tem peratures and pH is shown in Table 4.2. D values from the
conventional batch system (C hapter 3) and m icrow ave heating are com pared in Table 4.3
w hich provides some interesting com parisons betw een the enzym e inactivation in the tw o
heating systems.
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Table 4.2. Thermal kinetic parameters (D and z-values) of a-amylase under
m icrowave continuous-flow heating conditions
Temperature
65
70
75
80
85
z-value
D-values, s
at pH 6.0
at pHS.O
at pH5.6
uncorrected corrected uncorrected corrected uncorrected corrected
N/A
N/A
16
37.1
27
21.4
105
89
15.3
7.6
27.4
16.2
76
61.2
11.7
9.4
4.9
18.2
49
43
10.5
5.7
5.1
N/A
29
22.1
N/A
N/A
N/A
N/A
26.8
25.3
31.8
23.3
27.5
19.6
Table 4.3. Thermal kinetics parameters (D-, z-values) co m p ariso n of a-amylase
under conventional batch heating and MW continuous-flow heating conditions
Temperature,
65
70
75
80
85
z-value
at pH6.0
microwave thermal
N/A
192
89
138
61.2
120
43
66
22.1
N/A
25.3
32.7
D-values, s
at pH5.6
at pH5.0
microwave thermal microwave thermal
16
27.0
198
102
114
66
15.3
7.6
66
4.9
24
11.7
50
N/A
N/A
5.7
N/A
N/A
N/A
N/A
19.6
21.1
23.3
29.5
The D -values obtained under conventional batch heating conditions (com m only
em ployed in therm al death studies by subjecting small test samples to various times
under isothermal heating conditions in a w ater bath) varied from 1.1 min at pH 5.0 and
1.9 min at pH 5.6 to 9.9 min at pH 6.5 at 65°C. The values at 70°C varied from 0.4 min at
pH 5.0 and 1.1 min at pH 5.6 to 5.5 min at pH 6.5. The D -values observed with the
continuous-flow m icrowave heating ranged from 16 s at pH 5.0 and 27 s pH 5.6 at 65°C
to 7.6 s and 15.3 s at 70°C. Thus the D-values associated with m icrow ave heating w ere
considerably lower than those obtained for conventional batch heating. O verall, the D -
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considerably low er than those obtained for conventional batch heating. Overall, the Dvalues obtained under conventional batch heating w ere 4 to 8 tim es higher than those
obtained un d er m icrow ave heating.
2.5
2
Conventional batch heating
"'NB
2=29.5 C
1.5
1
MW continuous-flow heating
2=23.3 C
0.5
0
40
80
60
100
Temperature, C
Figure 4.9a. Temperature sensitivity comparison between continuous-flow
microwave heating and conventional batch heating at pH 5.6
2.5
iventional batch heating
z=21.2 C
1.5
OJ
0.5
MW continuous-flow heating
2=19.6 C
90
100
Temperature, C
F ig u re 4.9b. Temperature sensitivity co m p ariso n between continuous-flow
m icrow ave heating and con v en tio n al batch heating a t p H 5.0
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In a recent study, Ram aswam y et al. (2001) reported that the D-values for E. coli
obtained under conventional batch heating conditions vary from 173 s at 55°C to 2 s at
65°C. In com parison, the D-values w ith continuous-flow m icrow ave heating were
reported to range from 13s at 55°C to 0.78s at 65°C, considerably low er than those
obtained for conventional batch heating. In earlier studies w ith similar set-up,
Tajchakavit et al., (1998) evaluated the destruction kinetics o f S. cerevisiae and L.
plantarum in apple juice under continuous flow m icrow ave heating conditions (52.565°C) and com pared with conventional w ater bath treatm ents. The tim e-corrected Dvalues show n for both strains under conventional heating w ere reported to be 4-12 tim es
higher than those observed in microwave heating. Thus m icrobial destruction occurred
much faster under microwave heating than under therm al heating suggesting some
enhanced effects associated w ith microwave heating. R iva et a l, (1991) attributed the
differences found in destruction kinetics betw een conventional and microwave to
different heating kinetics and non-uniform local tem perature distributions during
m icrow ave heating than existence o f non-therm al effects. A ktas and Ozilgen (1992)
evaluated the injury o f E. coli during pasteurization with m icrowaves in a tubular flow
reactor and indicated the destruction effect to be influence by th e flow behavior and other
experim ental condition. They also suggested that microbial death m ight be caused
through a damage to a different sub-cellular part under each experim ental condition.
Sastry and Palaniappan (1991) that the effects o f m icrowave are clearly th e field in w hich
the knowledge gap is still vast and much further studies are needed to understand the
details is still valid. Although no non-therm al effects are im plicated in this study, it may
be possible that the microbial destruction under m icrowave heating could be different
90
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from the conventionally assumed first order which m ight explain the enhanced effects.
K erm asha et a l (1993a,b), while reporting non-therm al effects due to m icrowave
heating, gave the following explanation. Proteins,
as com plex m acro-m olecules,
generally have num erous polar and/or charged moities (i.e., COO-, and NH4+) which can
be affected by the electrical component o f the microwave field (D ecareau, 1985).
A lthough th e m icrow ave energy may be insufficient to disrupt covalent bonds, the noncovalent bonds such as hydrophobic, electrostatic and hydrogen bonds, may well be
disrupted. T hus the direct microwave effect could be m ore pronounced, im m ediate and
specific than th e random kinetic energy mechanism associated w ith conventional heating.
Enzyme inactivation profiles under continuous-flow th e rm a l hold
As indicated in Figure 4.1, the enzym e test solutions after m icrow ave heating
w ere passed through a small holding tube.
Only
a short residence time w as
accom m odated in this section since the test solution entering the holding tube is at its
highest tem perature. There was only a small drop in tem perature (m axim um 2°C) in the
holding tube since it w as well insulated. The drop in tem perature w as likewise
accom m odated as in the case o f come-up tim e for the m icrowave heating, and from the
logarithm ic reduction in the residual activity estim ated w ithin the hold tube section, the
associated D values w ere computed. Table 4.4 and Figure 4.10 com pare the D values
betw een the tw o continuous-flow systems - m icrow ave heating and therm al holding at
pH 5.6.
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Table 4.4. Kinetics param eters (D-, z-values) comparison of a-amylase under batch,
continuous-flow thermal holding and MW heating conditions
Temperature
(C)
65
70
75
80
z-value
Thermal
Continuous
flow
pH 5.6
(s)
Thermal
Batch
heating
Microwave
Continuousflow
39
30
18
N/A
31.9
114
66
49.8
N/A
29.5
27.0
15.3
11.7
5.7
23.3
The D value obtained under continuous flow thermal holding (39, 30 and 18 s at
65, 70 and 75°C, respectively at pH 5.6) w hich were low er than sim ilar data under
therm al batch (114, 66 and 49.8 s at 65, 70 and 75°C, respectively at pH 5.6) heating
conditions. This difference was m ainly attributed to the batch vs. continuous mode
heating conditions.
W hile it is possible to
carry out predom inantly isotherm al
experim ents under batch heating conditions em ploying small sam ple volum es, the
continuous flow system s are necessarily non-iso thermal, especially in the absence o f a
holding time. The uncertain residence time and tem perature distribution in the
continuous-flow systems, and the conservative averages em ployed generally yield lower
D values.
The D values associated with microwave heating (27, 15.3 and 11.7 s at 65, 70
and 75°C) were, however, considerably lower than the corresponding values found under
conventional batch and even under continuous-flow therm al
Ram asw am y et a l
holding conditions.
(2001) also reported D values obtained for continuous-flow
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Raraasw am y et al. (2001) also reported D values obtained for continuous-flow
m icrowave heating to range from 13 s at 55°C to 0.78s at 65°C w hich w ere considerably
low er in com parison w ith both continuous flow thermal heating (73s and 3s at 55°C and
65°C) and batch heating (173s and 2s at 55°C and 65°C). This indicates that microwave
continuous-flow heating condition is much more efficient in inactivating enzym e than
conventional therm al treatment.
1.6
Thermal holding period
7=31 g p._______
1.4
1.2
MW heating period
z= 2 3 .3 C
0.8
0.6
0.4
0.2
60
Temperature, C
Figure 4.10. Temperature sensitivity comparison between continuous-flow
microwave heating and conventional thermal holding at p H 5.6
Comparison with literature values
M any
m icrobiological
systems
had
been
studied
under
continuous-flow
microwave heating system, for example, E.coli, Saccharomyces cerevisiae, Lactobacillus
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plantamm, etc. however, a-am ylase from Bacillus subtilis w ere not extensively
investigated. V an Loey et al. (1996) applied Bacillus stbtilis a-am y lase (200 mg/ml)
under non-isotherm al heating condition and g o t z-value o f 8.64 ± 0.25 °C and D 100 -v alue
at 0.14 ± 0.02 min. Therm al inactivation o f a-am y lase from Bacillus subtilis at
concentration o f 5, 10, 20, 30 mg/ml could clearly be described by first-order kinetics.
The decimal reduction tim e at 80°C increases as enzym e concentration increases,
indicating that dissolved a-am ylase from Bacillus subtilis is m ore therm ostable at higher
enzym e concentration. K inetic param eters estim ated using nonlinear regression analysis
on non-isotherm al inactivation data w ere reported (Table 4.5).
Table 4.5. Kinetic parameter estimates (Dgo? z) of Bacillus subtilis a-amylase at
d iffe re n t enzyme concentration obtained from nonlinear regression analysis on nonisothermal inactivation data (Van Loey et al., 1996)
Bacillus subtilis a-am ylase ( m g /m l)
Z(°C)
5
10
20
30
12.78 ± 0 .6 4
10.27 ± 0 .3 8
8.39 ± 0 .3 8
9.82 ± 0 .3 1
D 8 q (min)
10.89
13.82
18.51
26.49
± 0 .4 2
± 0 .4 0
± 0 .7 0
± 0 .6 1
Bacillus licheniformis a-am ylase and Bacillus amyloliqmfaciens a-am ylase w ere
also tested under the non-iostherm al treatm ent. For Bacillus licheniformis a-am ylase, the
DgQ-values were: 16.8 ± 1.0 min, 34.7 ±1.8 min, 115.8 ± 34.4 min w ith concentration o f
lOmg/raJ, 30mg/ml and 200mg/ml, respectively; F or Bacillus amyloliquefaciens a-
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CONCLUSIONS
T he application o f microwave energy for destruction o f enzym atic timetem perature indicator in continuous-flow condition was studied. Kinetics o f continuousflow m icrow ave heating system and continuous-flow therm al holding period w ere
evaluated. C ontribution o f lethality during come-up time was corrected to get D-value
and z-value. A ccording to the experiment, inactivation o f a-am y lase based TTI from
B A A in the continuous-flow microwave heating conditions is more effective than the
conventional batch heating condition and the continuous-flow thermal holding condition.
The result indicate further that the possibility o f exist o f non-therm al or enhanced thermal
m icrow ave effects.
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CHAPTER 5
G E N E R A L C O N C L U S IO N S AND R E C O M M E N D A T IO N S
Therm al inactivation kinetics o f a-am ylase based enzym atic tim e-tem perature
integrators (T T Is) from Bacillus subtilis was studied by subjecting small test samples to
isothermal heating conditions in a well stirred tem perature controlled bath at several
tem peratures in the range from 50 to 95°C and at pH in the range from 5.0 to 6.9.
Transient tim e-tem perature test data were gathered and corrected for the come-up and
com e-dow n lag periods. The time corrected D (at a reference tem perature o f 70 °C) and
z-value were: 72.5 min and 30.3 °C at pH 6.9, 5.5 min and 33.6 °C at pH 6.5, 2.3 min and
32.7 °C at pH 6,0, 1.1 min and 29.5 °C at pH 5.6 and 0.4 min and 21.1 °C at pH 5.0,
respectively. The results indicated that by varying the pH o f test solution, the sensitivity
o f TTI to therm al inactivation can be varied and TTIs appropriate for evaluating
pasteurization and cooking conditions can easily be formulated.
Inactivation kinetics o f the a-am ylase TTI was also evaluated under continuousflow microwave and conventional hold conditions. The TTI dispersed in a buffer solution
(pH 5.0 or 5.6) initially at 20°C was continuously circulated through two helical coils
connected in series for heating. The sample flow rates were adjusted to result in specific
exit tem perature (65-80°C). A short fully insulated helical coil at the exit o f the second
oven was used as a holding tube. Under a range o f experimental conditions, the extent o f
enzym e inactivation follow ing the heat treatm ents w ere evaluated. The corrected Dvalues under microwave heating condition ranged from 16.0 s to 4.9 s at pH 5.0 between
65 and 75°C and 27.0 to 5.7 s at pH 5.6 between 65 and 80°C, respectively. D-values
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during the thermal hold period varied from 39 s to 18 s between 65 and 75°C at pH 5.6.
Com paring with the D-values between continuous-flow M W and thermal holding, as well
as from the batch kinetics, it was found that enzyme destruction occurred much faster
under continuous-flow microwave heating condition than under conventional thermal
heating condition. Hence, there was evidence that microbial lethality under microwave
heating conditions can not be fully accom m odated by conventional m odels em ploying
thermal kinetics data
The future use o f TTIs will enhance the further introduction o f new processing
technologies for the production o f microbiologically safe, high-quality convenience
foods, where existing approaches are not applicable The use o f TTIs provides a better
approach for establishing thermal processing conditions, leading to possible reductions in
production costs and an improved safety-quality balance. Minimal therm al processing
conditions and their efficient evaluation lead to a less intensive im pact on food products,
and consequently to advantages in term s o f improved nutritional value and sensory
characteristics. TTIs can also be used in m onitoring critical control points (e.g.
determ ination o f the in-park coldest point, or evaluation o f the coldest zone in a retort) as
part o f a hazard analysis and critical control points (H A CCP) program, both for
conventional therm al processing as well as for new technologies to produce heatpreserved foods.
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