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Kinetics of destruction of potato polyphenol oxidase (PPO) during vacuum microwave blanching Miladinović, Zoran 2006

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K I N E T I C S of D E S T R U C T I O N of P O T A T O P O L Y P H E N O L O X I D A S E (PPO) d u r i n g V A C U U M M I C R O W A V E  BLANCHING  by  Zoran Miladinovic  B . S c . ( H u m a n Nutrition), T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 9 6 B . E d . ( S e c o n d a r y ) , T h e University of British C o l u m b i a , 1 9 9 8  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES  (Food Science)  T H E UNIVERSITY O F BRITISH C O L U M B I A February 2006 © Zoran Miladinovic, 2006  ABSTRACT V a c u u m m i c r o w a v e (VM) blanching d e c r e a s e s e n z y m a t i c food browning at low t e m p e r a t u r e s (between ~40-55°C) w h e n c o m p a r e d to c o n v e n t i o n a l p r o c e s s i n g methods.  T h e impact a n d reaction of m i c r o w a v e blanching on p o l y p h e n o l o x i d a s e  ( P P O ) of the R u s s e t potato w a s investigated a n d c o m p a r e d with the effects of conventional convective heating.  In order to c l o s e l y e x a m i n e P P O kinetics in a  m i c r o w a v e field and to establish a P P O a s s a y for u s e in further investigations, potato p u r e e plus polyvinylpolypyrrolidone ( P V P P ) w a s centrifuged a n d filtered to p r o d u c e a treatment ready s u s p e n s i o n . W h o l e potatoes w e r e cut into F r e n c h fry strips a n d b l a n c h e d using a pilot-sized V M dehydrator. T h i s w a s c o m p a r e d to a s t e a m kettle blanch to determine the blanch effects on p h y s i c a l form.  Finally, two  different m i c r o w a v e treatments w e r e utilized along with a heated water bath in order to c o m p a r e the microwave heated potato s u s p e n s i o n s a m p l e s with conventional heating.  M i c r o w a v e treatments u s e d a 7 0 0 W , 2 4 5 0 M H z v a c u u m m i c r o w a v e o v e n  a n d a n E t h o s S y n t h ( E S - M R ) m i c r o w a v e reactor set at 2 4 5 0 M H z , w h i c h d r o p p e d the m i c r o w a v e power level from 700 W to below 100 W after reaching the d e s i r e d temperature.  E x p e r i m e n t s in all three treatments w e r e c o n d u c t e d under controlled  temperatures between 40°C a n d 70°C.  T h e d e c i m a l reduction v a l u e s (D-value) of  P P O w e r e r e d u c e d by heating in v a c u u m m i c r o w a v e treatments a s c o m p a r e d to heating in a water bath at temperatures  b e t w e e n 40°C a n d 57°C.  At these  temperatures, the D-values, z - v a l u e s a n d activation e n e r g i e s ( E ) for the P P O m o d e l a  reaction w e r e significantly lower (p < 0.05) for the V M a n d the E S than the heated water  bath.  The  results  s u g g e s t the  e x i s t e n c e of  an  alternative  inactivation  m e c h a n i s m for P P O w h e n heated in a m i c r o w a v e field c o m p a r e d to a water bath at low p r o c e s s i n g temperatures.  TABLE OF CONTENTS  ABSTRACT  II  TABLE OF CONTENTS  IV  LIST O F T A B L E S  VI  LIST O F F I G U R E S  VII  LIST O F S Y M B O L S A N D A B B R E V I A T I O N S  VIII  ACKNOWLEDGEMENTS  X  1.  INTRODUCTION  1  2.  L I T E R A T U R E REVIEW  3  2.1  General Introduction / Central C o n c e p t of Thesis  2.2 F o o d Spoilage / Deterioration and Browning 2.2.1 Introduction 2.2.2 Enzymatic Browning 2.2.2.1 Introduction 2.2.2.2 P P O a s Indicator of Enzymatic Browning 2.2.2.3 P P O Characteristics 2.3 Physical Processing / Microwaves 2.3.1 Introduction 2.3.2 Enzymes and Radiation / Heat 2.3.3 Enzymes in Solution 2.3.4 Novel Microwave Interactions 2.3.5 E M Radiation and the E M Spectrum 2.3.6 Microwave Heating 2.3.6.1 Introduction 2.3.6.2 Factors Affecting Microwave Heating 2.3.7 Microwave Applications 2.3.7.1 Introduction 2.3.7.2 Effects of Microwaves on F o o d and Enzymes 2.3.7.3 Blanching and Microwave Blanching  3 6 6 6 6 8 9 13 13 13 15 15 17 18 18 19 20 20 21 23  iv  2.4 The Potato 2.4.1 Measuring Potato P P O Activity 2.4.2 Potato Blanching  24 24 24  3  26  HYPOTHESIS AND OBJECTIVES  3.1  Hypothesis  26  3.2  Objectives  26  4. M A T E R I A L S A N D M E T H O D S 4.1 Materials 4.2 Sample Preparation 4.3 Overview of Methods 4.3.1 Heat Treatments 4.3.2 Enzymatic Activity Determination 4.3.3 Kinetic Studies 4.3.4. Statistical Analysis  27 27 27 28 33 35 35 36  5.  37  5.1  R E S U L T S A N D DISCUSSION P P O Enzyme A s s a y and P P O Characteristics  37  5.2 Heat Treatments with Experimental Microwave Apparatus  46  5.3 Industrial Sized Vacuum Microwave  53  6. C O N C L U S I O N S  58  7. R E F E R E N C E S  61  LIST O F T A B L E S  T a b l e 5.1 T a b l e 5.2 T a b l e 5.3  T a b l e 5.4  P P O a s s a y s of the P V P P treated crude h o m o g e n a t e T a b l e s u m m a r i z i n g reaction rate w h e n the substrate concentration is varied T e m p e r a t u r e d e p e n d e n c e of the rate constant a n d activation e n e r g y for water bath, v a c u u m m i c r o w a v e a n d E t h o s S y n t h m i c r o w a v e reactor at various temperatures T h e blanching of fries using various treatments  40 41  48 54  vi  LIST O F  FIGURES  Figure 2 . 1 : Flowchart to s h o w the logical progression of this thesis 5 Figure 2.2: P P O catalyzed reaction that a l s o includes c o p p e r a s a co-factor 7 Figure 4 . 1 : S c h e m a t i c diagram of a v a c u u m m i c r o w a v e a p p a r a t u s 29 Figure 4.2: Microwave oven 29 Figure 4 . 3 : T h e pump 30 Figure 4.4: Internal M e c h a n i s m of the M i c r o w a v e a p p a r a t u s for v a c u u m m i c r o w a v e p r o c e s s i n g of liquids 30 Figure 4 . 5 : S c h e m a t i c diagram of the E t h o s Synth m i c r o w a v e s y s t e m 31 Figure 4 . 6 : Front view of m i c r o w a v e reactor 32 Figure 4 . 7 : T o p view of m i c r o w a v e reactor 32 Figure 4 . 8 : V a c u u m microwave drying ( V M D ) m a c h i n e 33 T a b l e 5.1: P P O a s s a y s of the P V P P treated crude h o m o g e n a t e 40 T a b l e 5.2: T a b l e s u m m a r i z i n g reaction rate w h e n the substrate concentration is varied 41 Figure 5.1: S a m p l e graphical representation of the crude potato h o m o g e n a t e P P O assay 43 Figure 5.2: S a m p l e graphical representation of the P V P P treated crude potato homogenate P P O assay 43 Figure 5.3: S a m p l e graphical representation of the P V P P treated c r u d e potato homogenate P P O assay 45 F i g u r e 5.4: S h o w n here is the velocity of the P P O c a t a l y z e d reaction v e r s u s varying substrate (catechol) concentrations 46 T a b l e 5.3. T e m p e r a t u r e d e p e n d e n c e of the rate constant a n d activation e n e r g y for water bath, v a c u u m microwave a n d E t h o s S y n t h m i c r o w a v e reactor at various temperatures 48 Figure 5.5: E t h o s Synth R e a c t o r at 60°C 49 Figure 5.6: M i c r o w a v e at 4 0 ° C 49 Figure 5.7: Hot water bath at 60°C 50 Figure 5.8: T h e D-value curve of the E S reactor, the V M a n d the hot water bath. T h e z v a l u e is e q u a l to the negative reciprocal s l o p e 51 Figure 5.9: A r r h e n i u s relationship for the E S reactor, the V M a n d the hot water bath. T h e s l o p e is u s e d to calculate the activation e n e r g i e s for e a c h treatment 52 T a b l e 5.4: T h e blanching of potato fries using various treatments 54 Figure 5.10: M i c r o w a v e potato fries 55 Figure 5 . 1 1 : G r a p h i c a l representation of the m i c r o w a v e (w/o v a c u u m ) b l a n c h data from T a b l e 4.1 56 Figure 5.12: G r a p h i c a l representation of the m i c r o w a v e (w/ v a c u u m ) b l a n c h data from T a b l e 4.1 57 Figure 5.13: G r a p h i c a l representation of the Hot water blanch data from T a b l e 4 . 1 . T h e moisture content c h a n g e varies slightly b e t w e e n runs but d o e s not s h o w any consistent trend 57  VII  LIST OF S Y M B O L S AND ABBREVIATIONS Symbols °C  Degrees Celsius  °F  D e g r e e s Fahrenheit  D-value  T i m e required to c h a n g e the concentration of reactants or products by 9 0 %  E  Activation energy  a  K  Kelvin  Log  Logarithm  M  Mole/liter  mM  Millimole/liter  fjL  Microliter  cm  Centimeter  nm  Nanometer  MHz  Megahertz  s  seconds  h  Hour  W  Watts  z  T e m p e r a t u r e c h a n g e required to c h a n g e the d e c i m a l reduction time by a factor of 10  Abbreviations  ANOVA  A n a l y s i s of V a r i a n c e  EM  Electro-magnetic  ES-MR  E t h o s Synth M i c r o w a v e R e a c t o r  PER  Protein Efficiency Ratio  PPO  Polyphenol oxidase  PVPP  Polyvinylpolypyrrolidone  VM  V a c u u m microwave  VMB  V a c u u m microwave blanching  VMD  V a c u u m microwave drying  ACKNOWLEDGEMENTS  I would like to take this opportunity to thank the m a n y p e o p l e w h o a s s i s t e d a n d supported m e during this long term e n d e a v o u r . First of all, I want to e x p r e s s my deepest  gratitude  and  thanks  to  Dr.  Tim  e n c o u r a g e m e n t , a d v i c e a n d continued support.  Durance,  my  supervisor,  for  his  I would a l s o like to thank the other  m e m b e r s of the supervisory committee; Dr. David Kitts a n d Dr. Bruce T o d d for their constructive r e c o m m e n d a t i o n s a n d a s s i s t a n c e .  In addition, I would like to extend my s i n c e r e appreciation to Mr. S h e r m a n Y e e a n d M s V a l e r i e S k u r a for their technical a s s i s t a n c e a n d invaluable a d v i c e during the c o u r s e of this thesis.  A s well, a huge thank y o u to P a r a s t o o Y a g h m a e e a n d others in the lab for a n s w e r i n g q u e s t i o n s w h e n I didn't k n o w what o n e plus o n e w a s a n y m o r e a n d to d i s c u s s topics related to this thesis.  A s p e c i a l thank you to my family a s well a s to Katrin a n d S l o b o d a n for their help with the computer.  1. INTRODUCTION C o n v e n t i o n a l methods of drying f o o d s s u c h a s air drying h a v e b e e n u s e d for hundreds  of  years.  However,  certain  preservation  methods  undesirable characteristics, o n e being e n z y m a t i c browning. effective method u s e d to prevent e n z y m a t i c browning. c o m b i n e d with a v a c u u m environment  produce  some  B l a n c h i n g is usually a n  R a d i a n e n e r g y (microwaves)  forms the b a s i s of V a c u u m  Microwave  B l a n c h i n g ( V M B ) which will be d i s c u s s e d with respect to polyphenol o x i d a s e ( P P O ) a n d b l a n c h i n g . T h e objectives of the p r o p o s e d r e s e a r c h include: determine if a n o n thermal denaturing effect on polyphenol o x i d a s e m a y o c c u r in the V M B p r o c e s s a n d to provide a blanching application ( V M B ) .  T h e s e objectives, hopefully, will be  a c h i e v e d with the experimental a p p a r a t u s that our laboratory h a s c r e a t e d that allows the testing of the effects of V a c u u m M i c r o w a v e Drying ( V M D ) on the e n z y m e P P O . A c c o r d i n g to s o m e studies, there m a y be a reduction in both time a n d temperature for the inactivation of P P O using a m i c r o w a v e field treatment a s c o m p a r e d to a water bath treatment. where  In a normal m i c r o w a v e field, the temperature rises to a point  P P O begins to denature;  however,  in a v a c u u m  m i c r o w a v e field,  the  temperature might not reach the denaturing point found in conventional heating d u e to the reduction in the boiling point of water at reduced p r e s s u r e .  Non-thermal  effects, therefore, may b e c o m e dominant during V M D P P O inactivation.  Some  e v i d e n c e for the non-thermal effects of m i c r o w a v e s on e n z y m e s w a s reported for the e n z y m e pectin methyl e s t e r a s e in o r a n g e juice (Tajchakavit et a l . , 1995).  The  1  p o l y p h e n o l o x i d a s e a s s a y results for s a m p l e s b l a n c h e d in different w a y s will provide a quantitative a n a l y s i s of how m i c r o w a v e e n e r g y in a v a c u u m environment interact with polyphenol o x i d a s e .  might  T h e significance of this r e s e a r c h lies in the fact  that V M B provides a relatively new method of p r o c e s s i n g f o o d s by blanching t h e m . A s relatively few studies have e x a m i n e d m i c r o w a v e blanching, this study m a y enrich future r e s e a r c h . U n l e s s eaten fresh, food preservation is vital to maintain food integrity a n d quality.  O n e method of food preservation is blanching, which is a p r o c e s s u s e d to  destroy undesirable e n z y m a t i c activity  in v e g e t a b l e s a n d fruits prior to further  p r o c e s s i n g a n d conventionally includes a hot water or s t e a m m e d i u m .  A major  p u r p o s e in blanching fresh foods is to prevent e n z y m a t i c browning during storage w h i c h mainly o c c u r s w h e n the e n z y m e P P O is e x p o s e d to 0 p o l y p h e n o l s ( P P ) , which are abundant in plant materials.  2  in the p r e s e n c e of  M i c r o w a v e s c a n a l s o be  u s e d to blanch food materials and destroy P P O . Specifically, the m e t h o d of heating (microwave v s . hot water) a n d its effects o n P P O w e r e investigated in this study. A b s o r p t i o n spectrometry h a s b e e n s u c c e s s f u l l y u s e d in s e v e r a l studies to determine the rate of e n z y m a t i c browning by m e a s u r i n g the P P O activity of the m a c e r a t e d fruits at an a b s o r b a n c e of 4 2 0 nm ( P i z z o c a r o et a l . , 1 9 9 3 ; A l m e i d a a n d Nogueira,  1995).  Furthermore,  the  heat  resistance  of  enzymes  can  be  c h a r a c t e r i z e d by D-values a n d z - v a l u e s of different p r o c e s s e s w h i c h then c a n be c o m p a r e d to s e e the effectiveness ( d e c r e a s e in e n z y m a t i c browning) of  different  heating m e t h o d s . T h e a b s o r b a n c e at 4 2 0 nm of heat treated s a m p l e s in this study w a s u s e d to m e a s u r e e n z y m a t i c browning reaction rate; extent of formation of the  2  c o l o u r e d products, q u i n o n e s , a n d the m e a s u r e m e n t of colour c h a n g e s d u e to the p r e s e n c e of soluble pre-melanoidins. A c c o r d i n g to M a y e r a n d Harel (1979), colour formation is likely d u e to both the formation of low m o l e c u l a r weight c o m p o u n d s , i.e. q u i n o n e s , a n d to the p r e s e n c e of m e l a n o i d s with high m o l e c u l a r weights. m e a s u r e d c h a n g e in concentration w a s c o n s e q u e n t l y u s e d to obtain  The  parameters  s u c h a s D-values, z - v a l u e s and activation e n e r g i e s  2. L I T E R A T U R E REVIEW  2.1  General Introduction / Central Concept of Thesis  F o o d p r o c e s s i n g is a critical field in F o o d S c i e n c e , being e s s e n t i a l for practical production a n d distribution of m a n y f o o d s . T e c h n o l o g y plays a n important role in the food s y s t e m of today s i n c e the world's population is increasing at a rapid rate a n d it is therefore, impossible to feed e v e r y b o d y with n o n - p r o c e s s e d f o o d s . the s a m e time, however, people d o not want f o o d s to be altered e x c e s s i v e l y .  At For  e x a m p l e , w h e n c h e m i c a l s are a d d e d to preserve food or more importantly, w h e n the taste  is c h a n g e d with a physical p r o c e s s , f o o d s  may  be  less acceptable  to  consumers. In this thesis, therefore, a p h y s i c a l p r o c e s s , blanching, w h i c h conventionally o c c u r s in a s t e a m or hot water m e d i u m , w a s investigated.  A blanching m e d i u m  u s e d in this study involved a v a c u u m m i c r o w a v e environment w h i c h represents a n e w technology in food p r o c e s s i n g that will hopefully be c o n s i d e r e d in the future study of F o o d S c i e n c e .  3  B l a n c h i n g is a n established p r o c e s s , widely u s e d in the food industry, for instance, to prevent e n z y m a t i c browning.  T h e major e n z y m e a s s o c i a t e d with  e n z y m a t i c browning is polyphenol o x i d a s e ( P P O ) .  T h e a m o u n t of this e n z y m e in a  food c a n be m e a s u r e d by m e a n s of a n a s s a y . T h e potato (white b a k e r ' s potato) w a s c h o s e n from s e v e r a l candidate v e g e t a b l e s a s the food that w o u l d m e a s u r e c h a n g e s in P P O d u e to blanch treatments.  be u s e d to  Finally, conventional p r o c e s s e s  w e r e c o m p a r e d to this new technological p r o c e s s , V a c u u m M i c r o w a v e B l a n c h i n g (VMB).  T h e flowchart s h o w s a graphical representation of the c o n c e p t u a l idea  behind this thesis. (Figure 2.1).  4  Browning h a s a negative influence on v e g e t a b l e quality  Prevent e n z y m a t i c browning by processing  S o m e p r o c e s s e s not a s effective a s others  Therefore, c h o o s e a p r o c e s s to m a x i m i z e food's quality by m e a s u r i n g a variable, in this c a s e , the amount of e n z y m e d e s t r o y e d  Microwave and vacuum  Process  Beneficial s i n c e m i c r o w a v e heating (radiant) is faster than conventional heating (conduction). A l s o , lower p r o c e s s i n g temperatures m a y be obtained in v a c u u m m i c r o w a v e heating Figure 2 . 1 : Flowchart to s h o w the logical progression of this thesis.  2.2  Food Spoilage / Deterioration and Browning  2.2.1 Introduction F o o d in today's c o n s u m e r c o n s c i o u s society is constantly under  scrutiny  regarding its nutritional value or lack of it. Daily w e hear of reports c o n d e m n i n g certain f o o d s a s being unhealthy or e v e n inedible.  Today's consumer considers  fresh food to be preferable for various p e r c e i v e d r e a s o n s , s u c h a s it being healthier or having a high nutritional value. H o w e v e r , it s e e m s increasingly difficult within our temporary North A m e r i c a n culture to eat fresh food consistently a n d on a regular basis.  Therefore,  the  need  a r i s e s to  prevent,  or  delay  food  spoilage  and  deterioration, w h i c h c a n be c a u s e d by various factors, s u c h a s oxidation, microo r g a n i s m growth, n o n - e n z y m a t i c a n d e n z y m a t i c browning.  T h e f o c u s of this thesis  will be e n z y m a t i c browning a n d its prevention, in particular, using the potato a s the m o d e l food.  2.2.2 Enzymatic Browning  2.2.2.1 Introduction G e n e r a l l y , the browning of f o o d s is undesirable a n d r e d u c e s not only the a p p e a r a n c e but the nutritional value of that particular food.  In s o m e c a s e s , s u c h a s  coffee a n d t e a , the brown a p p e a r a n c e is d e s i r e d a n d a required a s s e t . H o w e v e r , in  6  v e g e t a b l e s and fruits, browning  and/or brown  spots reduce consumer appeal,  thereby d e c r e a s i n g the monetary value of the food. Specifically, browning of foods, c a t a l y z e d by e n z y m e s , is referred to a s e n z y m a t i c browning.  F o r a significant  portion of the thesis, the d i s c u s s i o n will include e n z y m a t i c browning involving the e n z y m e , polyphenol o x i d a s e ( P P O ) .  For the s c h e m a t i c e n z y m e reaction d i a g r a m ,  p l e a s e s e e Figure 2.2.  0 \  J  +  2H 0 2  Figure 2.2: P P O c a t a l y z e d reaction that also includes c o p p e r (cuprous) a s a c o factor (Whitaker, 1994) P o l y p h e n o l o x i d a s e ( P P O ) has a d i c h o t o m o u s role in the plant a n d a n i m a l kingdoms.  P P O is present in all plants, with particularly higher concentrations, for  e x a m p l e , in potato tubers, m u s h r o o m s , b a n a n a s a n d a p p l e s .  P P O plays a critical  role in h u m a n functions, for e x a m p l e , in eye, hair a n d skin pigmentation a n d is specifically critical in the formation of the e x o s k e l e t o n s of i n s e c t s . P P O is instrumental in the curing of tea and coffee. coffee in this respect.  Furthermore,  T o b a c c o is similar to tea a n d  T h e colour of prunes a n d raisins is attributed to P P O a l s o .  T h e main d i s a d v a n t a g e of P P O is d u e to its detrimental browning effects on bruised a n d broken plant t i s s u e s .  7  2.2.2.2 P P O as Indicator of Enzymatic Browning E n z y m a t i c browning c a u s e d by P P O h a s b e e n studied extensively in fruits a n d v e g e t a b l e s and is r e s p o n s i b l e for the e n z y m a t i c browning horticultural  products, following  (Martinez & Whitaker, 1995).  bruising,  cutting or other  of t h e s e  damage  to  the  fresh cell  In g e n e r a l , browning results from both e n z y m a t i c and  n o n - e n z y m a t i c oxidation of phenolic c o m p o u n d s .  Browning usually impairs the  s e n s o r y properties of products b e c a u s e of the a s s o c i a t e d c h a n g e s in colour, flavour a n d texture softening (Martinez & Whitaker, 1995).  U n f a v o u r a b l e e n z y m a t i c browning o c c u r s in m a n y plants a n d v e g e t a b l e s a n d is of a great c o n c e r n to F o o d T e c h n o l o g i s t s a n d p r o c e s s o r s . T h e discolouration of browning, however, is not a c h e m i c a l quality defect (Jeon et al., 1996), but is less a p p e a l i n g to c o n s u m e r s a n d therefore, r e d u c e s the market v a l u e of the fruits a n d vegetables.  P P O is the major c a u s e of e n z y m a t i c browning in higher plants ( T h y g e s e n et al., 1995).  P P O c a t a l y z e s the c o n v e r s i o n of m o n o p h e n o l s to o-diphenols a n d o-  d i h y d r o x y p h e n o l s to o-quinones ( T h y g e s e n et al., 1995).  B l a c k or brown  pigment  d e p o s i t s result w h e n the quinine products polymerize a n d react with a m i n o acid groups of cellular proteins. A s a result, P P O activity c a u s e s c o n s i d e r a b l e e c o n o m i c a n d nutritional loss in the c o m m e r c i a l production of fruits a n d v e g e t a b l e s .  Browning c a n be prevented  by various m e t h o d s : e x c l u s i o n of m o l e c u l a r  o x y g e n , methylation of the p h e n o l s with o-methylase, addition of reducing a g e n t s  8  such  as  ascorbate,  bisulfite,  thiols,  which  prevent  the  accumulation  and  polymerization of o - b e n z o q u i n o n e , utilizing metal c o m p l e x i n g a g e n t s s u c h a s s o d i u m fluoride a n d a z i d e which inactivate the e n z y m e by reacting with the e s s e n t i a l copper, heat treatment which thermally destructs the e n z y m e , lowering the p H to b e l o w p H 4.5 a n d using competitive inhibitors s u c h a s s o d i u m b e n z o a t e .  T h e reducing agent  L - a s c o r b i c acid is a n e x a m p l e of a s u b s t a n c e which prevents browning by reducing the o - b e n z o q u i n o n e back to o-diphenol a s rapidly a s it is f o r m e d . browning o c c u r s a s long a s a s c o r b i c acid is present.  T h e r e f o r e , no  A s well, a s c o r b i c acid h a s a  direct effect o n polyphenol o x i d a s e , e s p e c i a l l y in the p r e s e n c e of micromolar c o p p e r ions.  2.2.2.3 P P O Characteristics P o l y p h e n o l o x i d a s e ( P P O ) ( E C 1.14.18.1) is ubiquitous in the plant kingdom (Martinez a n d Whitaker, 1995).  A wide range of m o l e c u l a r weight P P O s exist in  different plant t i s s u e s a n d e v e n in the s a m e plant, which c a n h a v e multiple forms of this e n z y m e ( R i c h a r d s o n a n d H y s l o p , 1994).  V e g e t a b l e s , s u c h a s potatoes are  s u s c e p t i b l e to discoloration during handling a n d p r o c e s s i n g a n d u n d e r g o e n z y m a t i c browning a s a result of cellular disruption a n d a n e x c e s s of o x y g e n in the p r e s e n c e of P P O (Mui et al., 2002). tyrosinases. tyrosine  P P O ' s that exist in the a n i m a l k i n g d o m are the  In fact, the unit definition for P P O in e n z y m e m a n u a l s usually states L-  a s a substrate for  the  standard  P P O assay.  PPOs  have  both  a  m o n o p h e n o l a s e a n d a d i p h e n o l a s e (i.e., catechol) activity, of w h i c h , the latter will be  9  the f o c u s in this study.  A l s o to clarify, P P O h a s b e e n called p h e n o l a s e , c a t e c h o l  o x i d a s e , c a t e c h o l a s e , c r e s o l a s e a n d t y r o s i n a s e (Mui et a l . , 2002). A s p e c i a l quality of P P O is that it c a n c a t a l y z e two s e p a r a t e a n d varied types of reactions, both involving phenolic c o m p o u n d s .  T h e first reaction involves the  hydroxylation of m o n o p h e n o l s by P P O to give o-diphenols.  T h i s reaction is a l s o  often referred to a s c r e s o l a s e activity, s i n c e p-cresol is often u s e d a s the substrate (Whitaker, 1994).  T h e s e c o n d reaction (Figure 1.2) will be the o n e utilized in this  thesis w h i c h involves the p r o c e s s of oxidation. m e a s u r e P P O activity.  Both p r o c e d u r e s c a n be u s e d to  A by-product of both t h e s e p r o c e d u r e s is the formation of  melanin b e c a u s e of the O2 uptake of oxidation of the p-diphenol a n d of s u b s e q u e n t n o n e n z y m e - c a t a l y z e d reactions. It is important  to note that all p o l y p h e n o l o x i d a s e s h a v e activity o n  o-  d i p h e n o l s . It h a s b e e n reported, for e x a m p l e , that polyphenol o x i d a s e s from b a n a n a and  tea  leaves  have  activity  on  o-diphenols  exclusively  but  not  activity  on  hydroxylate m o n o p h e n o l s (Whitaker, 1994).  P o l y p h e n o l o x i d a s e s from potato a n d  apple, however, reflect both types of activity.  C a t e c h o l is the substrate most often  u s e d in the a s s a y of P P O activity o n o-diphenols, therefore, referred to s o m e t i m e s a s c a t e c h o l a s e activity. independently  E v e n though c a t e c h o l a s e activity of P P O c a n be m e a s u r e d  of c r e s o l a s e activity  by the  u s e of  o-diphenols  as  substrates,  c o m p l i c a t i o n s c a n arise from the rapid inactivation of the e n z y m e during reaction a n d inhibition of activity at high substrate concentrations. T h i s inactivation of the e n z y m e , however, is not d u e to the instability of the e n z y m e to the p H , or the temperature u s e d , or the inactivation c a u s e d by c a t e c h o l .  10  Instead, this inactivation is c a u s e d by the reaction of an intermediate product, os e m i b e n z o q u i n o n e free radical.  B e c a u s e of this free-radical oxidation, c o p p e r is  thereby r e l e a s e d a n d the active site s u b s e q u e n t l y d e s t r o y e d (Whitaker, 1994). This p r o c e s s is called reaction inactivation.  M e t h o d s using manometric, polarographic,  chronometric a n d spectrophotometric a n a l y s i s c a n be u s e d to follow the activity of p o l y p h e n o l o x i d a s e on o-diphenols s u c h a s c a t e c h o l .  It c a n be e x p e c t e d that  different results are derived d e p e n d i n g upon the method u s e d .  F o r e x a m p l e , the  m a n o m e t r i c method results in the lowest P P O activity b e c a u s e of the difficulty of obtaining true initial rates.  A s well, the nonlinear r e s p o n s e a n d flattening of the  curve of e n z y m a t i c activity are d u e to limitations p l a c e d on the rate of incorporation of O2 into the solution by s h a k i n g .  U s i n g chronometric a n d spectrophotometric  m e t h o d s resulted in the s a m e o u t c o m e a s low e n z y m e c o n c e n t r a t i o n s (Whitaker, 1994). T h e polarographic method, w h i c h m e a s u r e s 0 with e n z y m e concentration.  2  uptake, g i v e s a linear result  A surprising result, however, w a s that the rate of 0  uptake w a s larger than that found by the m a n o m e t r i c m e t h o d .  2  It w o u l d a p p e a r that  t h e s e two a s s a y s are the preferred m e t h o d s b e c a u s e of the similar initial rates obtained by both the polarographic a n d spectrophotometric m e t h o d s . Polyphenol  o x i d a s e s that  originate  from  different  sources  also  differ  substantially in specific substrate requirements, particularly for potato a n d p e a c h .  In  addition, the activities on m o n o p h e n o l s are substantially lower than o n o-diphenols w h i c h is not u n u s u a l for polyphenol o x i d a s e s . It h a s b e e n e s t a b l i s h e d that a n u m b e r of  polyphenol  oxidases  catalyze  both  o-hydroxylation  of  monophenols  and  d e h y d r o g e n a t i o n of o-diphenols (Whitaker, 1994). T h e ratio of t h e s e two activities,  11  however, differs d e p e n d i n g on the method of preparation. T h i s p r e s e n t e d a c o n c e r n with m u s h r o o m polyphenol o x i d a s e until it w a s clearly e s t a b l i s h e d that this e n z y m e exists in s e v e r a l multiple molecular forms w h i c h d o not affect the s a m e activities on p-cresol a n d c a t e c h o l (Whitaker, 1994). B a s e d on the e n z y m e isolated from various s o u r c e s , it h a s b e e n s h o w n that P P O h a s a p H optimum that is b e t w e e n 5.0 - 7.0, with a s h a r p d e c r e a s e in activity below 4 . 5 ( M a y e r a n d Harel, 1979).  P P O is located in the chloroplast thylakoid  m e m b r a n e s of plant t i s s u e s (Martinez a n d Whitaker, 1995).  T h e e n z y m e , which  c a t a l y z e s the reaction in which p h e n o l s (i.e. catechol) are oxidized to q u i n o n e s , requires two c o p p e r metal ions (cuprous state) for activity a s well a s m o l e c u l a r oxygen.  T h e two main P P O inhibitors are b e n z o i c acid, w h i c h c o m p e t e s with the  phenolic  substrate,  diethyldithiocarbamate  which  interacts  specifically with  the  c o p p e r cofactor in the e n z y m e . T h e e n z y m e itself is an o x y g e n a s e ; c r u d e extracts of m u s h r o o m P P O s h o w multiplicity (different forms of the e n z y m e ) a n d the M W of the predominant form h a s b e e n often reported to be between 116,000 a n d 128,000 Daltons.  T h e r e are four tetramers, e a c h approximately 28 k D a to 32 k D a .  There  h a s b e e n little s u c c e s s in purifying a n d characterizing the entire structure a n d a m i n o acid s e q u e n c e of m a n y P P O s . m u s h r o o m , Neurospora little  difference  in  crassa coding  H o w e v e r , the a m i n o acid s e q u e n c e s of P P O from a n d Streptomyces  sequence,  glaucesens,  demonstrating  the  w e r e found to have close  evolutionary  relationship b e t w e e n the e n z y m e s from different s o u r c e s ( M a y e r a n d H a r e l , 1979). T h e extinction coefficient of P P O is 24.9 at a n a b s o r b a n c e of 2 8 0 n m . using o x y g e n to  catalyze the  dehydrogenation  Besides  of c a t e c h o l s to q u i n o n e s ,  the  12  q u i n o n e s rapidly polymerize to form insoluble brown c o m p o u n d s k n o w n a s m e l a n i n s (Martinez a n d Whitaker, 1995).  2.3  Physical Processing / Microwaves  2.3.1  Introduction W h e n d i s c u s s i n g physical p r o c e s s i n g , it is important to include c h e m i c a l l y  processed foods.  It a p p e a r s that the public wants less material a d d e d to their food;  ideally, the fewer ingredients the better a n d also, ingredients that are known a n d familiar to the c o n s u m e r are preferred. M i c r o w a v e B l a n c h i n g ( V M B ) , which  Therefore, a method s u c h a s V a c u u m  utilizes a v a c u u m environment  along with  m i c r o w a v e s , c a n be s e e n a s a p o s s i b l e alternative for the addition of c h e m i c a l P P O inhibitors.  T h e v a c u u m provides a lower p r o c e s s temperature environment  than  conventional blanching a n d the m i c r o w a v e s provide the energy to support heating.  2.3.2  Enzymes and Radiation / Heat Browning reactions in fruits a n d v e g e t a b l e s are a s e r i o u s problem for the food  industry (e.g. m u s h r o o m processing). browning  T h e principal e n z y m e r e s p o n s i b l e for the  reaction is polyphenol o x i d a s e ( P P O ) .  A microwave  applicator  was  d e s i g n e d a n d u s e d to study m u s h r o o m P P O inactivation ( R o d r i g u e z - L o p e z et al., 1999).  T h e effects of m i c r o w a v e s a n d conventional heating on the kinetics of the  m o n o p h e n o l a s e and d i p h e n o l a s e activities of P P O w e r e studied. C o n v e n t i o n a l a n d microwave  treatments  produced  different  enzyme  intermediates  with  different  13  stability  a n d kinetic properties.  C o n s i d e r a b l e time w a s s a v e d with  microwave  inactivation of the e n z y m e c o m p a r e d with the time n e e d e d w h e n conventional hotwater treatment profitability.  w a s u s e d , possibly resulting  in e n h a n c e d quality a n d  greater  A s well, the short e x p o s u r e time required for s a m p l e s irradiated with  m i c r o w a v e s is very important for maintaining the quality of m u s h r o o m s ( R o d r i g u e z L o p e z et al.,1999). T h e fast m i c r o w a v e treatment typically results in a n i n c r e a s e in antioxidant content a n d a c o n s i d e r a b l e d e c r e a s e in browning.  M i c r o w a v e energy may be a viable alternative to hot water blanching for the a v o i d a n c e of browning.  T h e most restrictive factor for the application of m i c r o w a v e  heating t e c h n i q u e s , however, is the temperature gradients g e n e r a t e d within s o m e s a m p l e s during m i c r o w a v e heating ( R o d r i g u e z - L o p e z et a l . , 1999).  Rodriguez-  L o p e z et a l . , (1999) elucidated in their study the heating inactivation kinetics of m u s h r o o m P P O using 2 4 5 0 M H z m i c r o w a v e radiation. M i c r o w a v e e n e r g y irradiation of P P O at 2 4 5 0 M H z w a s found to c a u s e a significant loss in the d i p h e n o l a s e activity of m u s h r o o m P P O ( S a n c h e z - H e r n a n d e z et a l . , 1999).  T h e r m a l inactivation w a s  irreversible in all c a s e s , which a p p e a r s to s u g g e s t that this loss of activity w a s d u e to a c h a n g e in the overall conformation of the e n z y m e . W h e n s l o w blanching m e t h o d s w e r e u s e d , the e n z y m e remained active for a longer time in the p r e s e n c e of its phenol substrates.  T h e fast m i c r o w a v e treatment, however, resulted in a n i n c r e a s e  in antioxidant content a n d a c o n s i d e r a b l e d e c r e a s e in browning. T h i s t e c h n i q u e a l s o permitted a rapid temperature i n c r e a s e c o m p a r e d to more conventional blanching  14  techniques,  which  has demonstrated  a  large gradient  of  permanent  enzyme  inactivation.  2.3.3  Enzymes in Solution Irreversible inactivation resulted w h e n the effect of m i c r o w a v e (f=10.4 G H z )  irradiation  on a thermostable e n z y m e w a s tested.  T h e activity  of e n z y m a t i c  solutions w a s c o m p a r e d to that of a s a m p l e heated in a water bath at the s a m e temperature.  E n z y m e concentration, m i c r o w a v e power level, a n d e x p o s u r e time  w e r e factors that affected the residual activity of the e x p o s e d s a m p l e s .  When  concentrations w e r e a b o v e 50 ug/ml. m i c r o w a v e effects d i s a p p e a r e d (La C a r a et al., 1999).  T h e s e results w e r e not consistent following water bath heating  using  identical temperatures a n d duration times. T h e results from microwave heating raw milk from c o w s a n d g o a t s in a continuous flow unit up to temperatures ranging from 73.1 to 9 6 . 7 d e g r e e s C , indicate that this continuous m i c r o w a v e p r o c e s s m a y be an efficient method for the pasteurization of milk (Villamiel et a l . , 1996).  T h e o u t c o m e of the heat treatments  w a s c a l c u l a t e d by m e a s u r i n g protein denaturation, lactose isomerization a n d the total bacterial count.  2.3.4  Novel Microwave Interactions Hernandez-Infante et al., (1998) found that microwaving d e s t r o y e d trypsin  inhibitors just a s w a s o b s e r v e d in b e a n s c o o k e d using the conventional m e t h o d . But Protein Efficiency Ratio ( P E R ) for raw s e e d s with low content of anti-nutrients (faba  15  b e a n s , p e a s , c h i c k p e a s a n d lentils), w e r e not affected. In c o m p a r i s o n to m i c r o w a v e heated dry s o y b e a n s , m i c r o w a v e - h e a t e d s o a k e d s o y b e a n s had a higher a m o u n t of d e s t r o y e d trypsin inhibitors a s well a s a higher P E R . B e c a u s e m i c r o w a v e heating of the c o m m o n b e a n s failed to d e m o l i s h hemagglutinins a n d trypsin inhibitors, their digestibility a n d P E R v a l u e s w e r e poor (Hernandez-Infante et al., 1998).  Microwave  heating, therefore, presents a n a d e q u a t e method for destroying hemagglutinins a n d trypsin inhibitors without c o m p r o m i s i n g protein quality of most l e g u m e s e e d s . only e x c e p t i o n to this would  be c o m m o n  b e a n s that p r e s e r v e d  The  anti-nutritional  substances. V u k o v a et al., (2004) reported that m i c r o w a v e e x p o s u r e influences e n z y m e complexes.  Their studies investigating m i c r o w a v e fields p r o d u c e d s o m e stabilizing  a n d prolonged effects on the a c e t y l c h o l i n e s t e r a s e ( A C h E ) activity in skeletal m u s c l e fractions from frog skeletal m u s c l e s . GHz)  of  different  field  intensity  conformation w e r e e x a m i n e d . with  a  low-intensity  T h e effects of continuous m i c r o w a v e s (2.45  on  acetylcholinesterase  activity  and  protein  Differences w e r e found b e t w e e n s a m p l e s irradiated  microwave  m i c r o w a v e ( V u k o v a et al., 2004).  field  and  samples  exposed  to  high-intensity  A n augmentation of r a n d o m coils, a m o r p h o u s  structures a n d (3-sheets w e r e revealed in the s a m p l e s irradiated with a low-intensity m i c r o w a v e field, w h e r e a s the c h a n g e s w e r e less noticeable in the s a m p l e s e x p o s e d to high-intensity m i c r o w a v e s .  E x p o s i n g frog skeletal m u s c l e fraction to m i c r o w a v e s  (2.45 G H z ) results in intensity-dependent, non-thermal a n d prolonged modification of a c e t y l c h o l i n e s t e r a s e activity.  16  B e c k et al., (2002) u s e d the m i c r o w a v e o v e n to thaw fresh f r o z e n p l a s m a u s e d for transfusions.  T h i s proved to be a more superior, gentler a n d quicker  method  using  compared  to  the  water  bath  and  water  bag  system.  An  u n d e r s t a n d a b l e c o n c e r n w a s whether m i c r o w a v e s might h a v e a negative impact on the virus-inactivated p l a s m a by e n h a n c i n g the activation p r o c e d u r e or impairing the proteins w h i c h had b e e n initiated by the inactivation p r o c e s s , therefore hemostatic activity.  reducing  T h e r e were, however, no significant differences found in the  hemostatic parameters which could be attributed to the thawing m e t h o d ( B e c k et a l . , 2002). In addition, the m i c r o w a v e o v e n is suited for thawing of not only fresh frozen p l a s m a but virus-inactivated p l a s m a a s well w h i c h b e c a u s e of the shorter thawing time, is a n a d d e d b o n u s particularly in a m a s s i v e transfusion situation.  2.3.5  E M Radiation and the E M Spectrum M i c r o w a v e energy is o n e form of electromagnetic e n e r g y w h i c h c a n be  v i e w e d a s transmitted a s w a v e s , a n d c a n penetrate food a n d is eventually converted to heat.  M i c r o w a v e s , like all electromagnetic radiation, h a v e a n electric c o m p o n e n t  a s well a s a magnetic c o m p o n e n t .  T h e m i c r o w a v e w a v e is c h a r a c t e r i z e d by  w a v e l e n g t h s b e t w e e n 1 m m to 1m (Fellows, 1987). M i c r o w a v e s are p r o d u c e d at specified frequency b a n d s , for instance, 2 4 5 0 M H z in h o u s e h o l d m i c r o w a v e s a n d s o m e t i m e s 896 M H z in E u r o p e a n d 9 1 5 M H z in the U S A (Fellows, 1987). T h e depth of penetration into a food is inversely related to frequency.  In g e n e r a l , lower-frequency  d e e p l y than higher frequency m i c r o w a v e s .  m i c r o w a v e s , therefore,  penetrate  more  Other factors, s u c h a s dielectrics a l s o  17  influence m i c r o w a v e penetration.  T h e depth of penetration  of m i c r o w a v e s is  determined by the loss factor of the food a n d the wavelength of f r e q u e n c y of the m i c r o w a v e s a s indicated in the equation below:  x=  (1) 2ne*  x [m]: depth of penetration A [m]: w a v e l e n g t h in s p a c e e : dielectric constant £ : loss factor  G r e a t e r penetration a n d more uniform heating is, therefore, obtained using longer w a v e l e n g t h s (896 M H z a n d 9 1 5 M H z ) , with f o o d s that h a v e lower loss factors, or with smaller p i e c e s of food.  H o w e v e r , d e e p penetration into food is not  n e c e s s a r i l y the main requirement a n d the w a v e l e n g t h of m i c r o w a v e s is c h o s e n to suit the required application (Fellows, 1987).  2.3.6 Microwave Heating  2.3.6.1 Introduction In conventional or surface heating, the p r o c e s s time is limited by the rate of heat flow to the body of the material from the surface a s determined by its specific heat, thermal conductivity, density and/or viscosity. S u r f a c e heating is not only slow, but a l s o non-uniform with the s u r f a c e s , e d g e s a n d corners being m u c h hotter than  18  the inside of the material.  C o n s e q u e n t l y , the quality of conventionally  heated  materials is variable and frequently inferior to the desired result. Imperfect heating c a u s e s product rejections, w a s t e d e n e r g y a n d e x t e n d e d p r o c e s s times that require large production a r e a s d e v o t e d to o v e n s . L a r g e p i e c e s of e q u i p m e n t are s l o w to respond to n e e d e d temperature c h a n g e s , take a long time to warm  up a n d have high heat c a p a c i t i e s a n d  radiant  losses.  Their sluggish  p e r f o r m a n c e m a k e s them slow to r e s p o n d to c h a n g e in production  requirements  m a k i n g their control difficult, subjective a n d e x p e n s i v e . Conversely,  with  microwaves,  heating  the  volume  of  a  material  at  substantially the s a m e rate is p o s s i b l e . T h i s is known a s volumetric heating. E n e r g y is transferred through the material electro-magnetically, not a s a thermal heat flux. Therefore, the rate of heating is not limiting a n d the uniformity of heat distribution is greatly improved.  Heating times c a n be r e d u c e d to less than o n e percent of that  required, using conventional t e c h n i q u e s . S i n c e the beginning of the u s e of m i c r o w a v e s in chemistry, athermal effects h a v e b e e n s u g g e s t e d (Stuerga et al., 1996).  M a n y publications in chemistry claim  specific or athermal effects  heating to explain results  of m i c r o w a v e  obtained  (Stuerga e t a l . , 1996).  2.3.6.2 Factors Affecting Microwave Heating T h e m o l e c u l a r structure of water c o n s i s t s of an o x y g e n a t o m with a partial negative c h a r g e s e p a r a t e d from hydrogen a t o m s with partial positive c h a r g e s . T h i s  19  forms a n electric dipole. W h e n a rapidly oscillating electric field is applied to a f o o d , d i p o l e s in the water are reoriented with e a c h c h a n g e in the field direction. n u m b e r of dipoles a n d the c h a n g e s induced by the electric field determine dielectric  constant  The the  £ of a food. T h i s is the ratio of the c a p a c i t a n c e of the food to the  c a p a c i t a n c e of air (or in s o m e c a s e s a v a c u u m ) .  T h e various distortions  and  deformations to the molecular structure, c a u s e d by re-alignment of the dipoles, dissipate the applied energy a s heat. T h e r e is a delay of a fraction of a m i c r o s e c o n d before the dipoles respond to c h a n g e s in the electric field, w h i c h is t e r m e d relaxation  time.  the  T h i s is influenced by the viscosity of the food a n d is, therefore,  d e p e n d e n t on temperature.  F o r e x a m p l e , w h e n water c h a n g e s its state to ice, the  dielectric constant falls a n d continues to d e c r e a s e a s the ice is further c o o l e d . C o n s e q u e n t l y , ice is more transparent to m i c r o w a v e s than water a n d frozen f o o d s that h a v e a significant amount of moisture a b s o r b energy m o r e intensely a s they thaw (Fellows, 1987).  2.3.7 Microwave Applications  2.3.7.1 Introduction Variability  in  colour  and  lack  of  colour  stability  are  major  e x p e r i e n c e d with p r o c e s s e d fruit products (de A n c o s et al., 1999).  problems  Undesirable  s e n s o r y a n d b i o c h e m i c a l c h a n g e s during handling, p r o c e s s i n g a n d storage of fruit products result from e n z y m a t i c browning a n d from n o n - e n z y m a t i c reactions (Maillard m e c h a n i s m s ) . D e v e l o p m e n t of browning or discoloration, off-flavours a n d nutritional  20  damage  were  attributed  to  the  action  of  enzymes  polyphenol  oxidase  and  p e r o x i d a s e . T h e u s e of m i c r o w a v e energy to inactivate e n z y m e s prior to p r o c e s s i n g fruits a n d v e g e t a b l e s is not a c o m m o n practice. T h e potential a d v a n t a g e s of the u s e of m i c r o w a v e energy w h e n c o m p a r e d with conventional heat-blanching are: (1) volumetric heating resulting in a r e d u c e d temperature gradient; (2) inactivation of enzyme  complexes and  (3)  a v o i d a n c e of the  leaching  of  pigments, carbohydrates a n d other water-soluble c o m p o n e n t s .  vitamins,  flavours,  A s well, e x t e n s i v e  studies h a v e s h o w n e q u a l or better retention of s o m e vitamins ( B ^ B , B , C , a n d 2  6  folic acid) after m i c r o w a v e heating c o m p a r e d with conventional heating ( W a t a n a b e et a l . , 1998).  H u m a n s h a v e u s e d the p r o c e s s of drying f o o d s for a g e s .  Today,  however, w e h a v e the potential of drying f o o d s more efficiently, safely a n d quickly. O n e dehydration technology u s e d today c o m b i n e s m i c r o w a v e heat transfer with v a c u u m drying (Durance, 2000).  T h i s dehydration method u s e s m e c h a n i c a l and  electromagnetic d e v i c e s permitting the drying of f o o d s with less heat a n d o x y g e n d a m a g e than by air-drying ( D u r a n c e , 2000).  2.3.7.2 Effects of Microwaves on Food and Enzymes After x-raying the structural a n d d y n a m i c a l effects of m i c r o w a v e fields on tetragonal single crystals of hen egg-white l y s o z y m e , W e i s s e n b o r n et a l . , (2005) found  distinct  results between  m i c r o w a v e levels.  using  high  microwave  power  levels a n d  lower  W h e r e a s high m i c r o w a v e power levels led to i n c r e a s e d , but  r e c o v e r a b l e lattice defects b e c a u s e of the evaporation of crystal water, localized  21  reproducible  changes  in  m i c r o w a v e p o w e r levels.  the  mean-square  displacements  occurred  at  lower  In s o m e c a s e s the B factors e v e n d e c r e a s e d w h e n  m i c r o w a v e power w a s i n c r e a s e d .  In this c a s e there w a s no e v i d e n c e of large  microwave-driven d i s p l a c e m e n t s of structural subunits in the protein w h i c h might be e x p e c t e d if m i c r o w a v e s w e r e to be a b s o r b e d by protein vibrations.  T h e effects of  microwaves  meagre  on  protein  dynamics  and  structure,  therefore,  are  when  m i c r o w a v e s link non-thermally to globular proteins at functioning hydration levels ( W e i s s e n b o r n et al., 2005). T h e relationship between protein denaturation  and textural c h a n g e s w e r e  investigated by S a h i n et al., (2001), by c o o k i n g a n u n c o o k e d trout  (Onchorhyncus  mykiss) in a m i c r o w a v e o v e n using 2 0 , 4 0 a n d 6 0 % power levels for 10, 2 0 , 30, 4 0 s. A s m i c r o w a v e power i n c r e a s e d , texture degradation w a s r e d u c e d b e c a u s e of the proteolytic e n z y m e s .  W h e n time and/or m i c r o w a v e power i n c r e a s e d , proteolytic  activity d e c r e a s e d , indicating an i n c r e a s e in e n z y m e inactivation. results w e r e  o b s e r v e d : 1) 6 0 % power  conditions,  2)  proteolytic  correlation  of (r-0.973)  a n d 20 s w e r e  enzymes were  effective  w a s established between  the  Four conclusive optimum  especially on the variation  cooking  m y o s i n , 3) of texture  a  and  proteolytic activity, a n d 4) most of the fatty a c i d s r e m a i n e d in tact during  the  m i c r o w a v e c o o k i n g time (Sahin et a l . , 2001). In c o m p a r i s o n to conventional heating m e t h o d s , Y a d a v et al., (2005) found that low-energy m i c r o w a v e irradiation e n h a n c e d results by up to 2.63 in a stable l i p a s e - c a t a l y z e d esterification  of adipic acid with various a l c o h o l s .  This  result  22  o c c u r r e d b e c a u s e of the greater recurrence of collision without any c h a n g e in activation e n e r g y of the two heating m o d e s ( Y a d a v et a l . , 2005). B o h r & B o h r (2000) s h o w e d that m i c r o w a v e irradiation c a n affect kinetics of the  folding  process  of  some  globular  proteins,  especially  beta-lactoglobulin  d e p e n d i n g on temperatures. A t a higher temperature the denaturation of the protein from its folded state is e n h a n c e d w h e r e a s at low temperatures the folding from the cold d e n a t u r e d p h a s e of the protein is e n h a n c e d . A t higher temperatures a negative temperature gradient is required for the denaturation p r o c e s s itself, w h i c h indicates that the effects of the m i c r o w a v e s are nonthermal.  T h i s c o n c l u s i o n supports the  idea that coherent topological excitations c a n exist in proteins (Bohr & Bohr, 2000). It w o u l d a p p e a r , therefore, that the application of m i c r o w a v e s is suitable for a wide range of biotechnological applications, for e x a m p l e , protein aggregation a n d protein s y n t h e s i s a n d s u b s e q u e n t l y would h a v e implications for biological s y s t e m s a s well.  2.3.7.3 Blanching and Microwave Blanching T h e browning of potatoes is d u e to e n z y m a t i c activity. prevented  by blanching which  But this c a n be  m a y a l s o involve adding antioxidants  such as  p o t a s s i u m bisulphite or a s c o r b i c acid (Severini et al., 2001). Dipping the potatoes in boiling water is the most c o m m o n method of blanching a n d is a l s o u s e d to d e c r e a s e the reducing s u g a r content at the surface of the product.  T h e r e are s e v e r a l  d i s a d v a n t a g e s w h e n using the boiling water blanching method s u c h a s s o l u b l e solid loss, a d e c r e a s e in firmness a n d high water c o n s u m p t i o n .  B l a n c h i n g of potatoes  h a s a l s o b e e n performed by s t e a m a n d m i c r o w a v e s (Severini et a l . , 2001).  23  2.4  T h e Potato  2.4.1 Measuring Potato P P O Activity P P O activity in potatoes continues to i n c r e a s e throughout tuber d e v e l o p m e n t but is highest on a fresh weight b a s i s in d e v e l o p i n g tubers ( T h y g e s e n et a l . , 1995). P P O is present a s a small multigene family in potato a n d e a c h g e n e h a s a specific temporal a n d spatial pattern of e x p r e s s i o n ( T h y g e s e n et al., 1995).  2.4.2 Potato Blanching It is c o n c e i v a b l e that low temperature blanching of w h o l e potatoes prior to minimal p r o c e s s i n g m a y possibly be a potential treatment to control e n z y m a t i c browning in sliced or pre-peeled potatoes ( Y e m e n i c i o g l u , 2002).  Yemenicioglu  (2002) found that there w a s no loss in f i r m n e s s a s well a s no browning o n the potato p e e l s , e y e s or e v e n infected a r e a s w h e n he b l a n c h e d R u s s e t potatoes for up to 60 min at 50° C . A s well, low temperature blanching for 4 5 min did not a p p e a r to c a u s e a significant reduction in crude polyphenol o x i d a s e activity.  But increasing the time  c a u s e d s o m e different results. T h e activity a n d specific activity of the e n z y m e w e r e r e d u c e d by 2 7 - 4 5 % and 2 2 - 4 3 % respectively w h e n the heating time w a s e x t e n d e d to 6 0 min ( Y e m e n i c i o g l u , 2002).  Slight browning on the p e e l s a n d e y e s of the  potatoes a s well a s r e d u c e d f i r m n e s s resulted w h e n heating time w a s e x t e n d e d to 7 5 min. Y e m e n i c i o g l u (2002), c o n c l u d e d that the browning w a s d u e to the sharp drop in the K m that c a u s e d the activation of the P P O . It is c l e a r that e n z y m a t i c  24  browning c a t a l y z e d by polyphenol o x i d a s e is a major p r o b l e m in the p r o c e s s i n g of pre-peeled or sliced potatoes.  minimal  In his study Y e m e n i c i o g l u (2002),  pointed out that sulphites h a v e s u c c e s s f u l l y b e e n u s e d to eliminate browning but b e c a u s e of the a d v e r s e health effects c a u s e d by sulphites, c h e m i c a l s s u c h a s a s c o r b i c acid, erythorbic  acid a n d citric acid have b e e n e m p l o y e d to  browning but are less effective.  control  B e s i d e s n e e d i n g less time m i c r o w a v e drying h a s  the potential for producing better quality dried potatoes (Bouraoui et a l . , 1994).  25  3  HYPOTHESIS AND O B J E C T I V E S  3.1  Hypothesis 1. V M blanching will i n c r e a s e the inactivation rate of P P O in the R u s s e t t potato p u r e e or supernatant relative to the s a m e temperature by c o n d u c t i v e heating. 2.  D - v a l u e s for the inactivation of P P O are different w h e n m i c r o w a v e heated than w h e n conduction h e a t e d .  3. D-value is replaced by Activation E n e r g i e s . 4.  M i c r o w a v e blanching is faster than s t e a m a n d hot water blanching inactivating in terms of P P O in w h o l e R u s s e t t potato fries (shorter time at s a m e temperature).  3.2  Objectives 1. T o m e a s u r e the e n z y m e activities of P P O of m i c r o w a v e b l a n c h e d R u s s e t t potato p u r e e s a n d c o m p a r e them with conventionally b l a n c h e d ( R u s s s e t t potato purees) at various temperatures a n d times. 2. T o m e a s u r e the D-values, Z - v a l u e s a n d activation e n e r g i e s of P P O of m i c r o w a v e a n d conduction heated (Russett potato trials) at v a r i o u s temperatures.  26  4. M A T E R I A L S AND M E T H O D S  4.1  Materials P o t a t o e s ( S o l a n u m t u b e r o s u m L. cv. R u s s e t t Burbank) w e r e p u r c h a s e d from  a local supermarket. All reagents w e r e of food grade. C a t e c h o l , a p o l y p h e n o l ( P P ) a n d a c o m m o n reactant for the P P O a s s a y a n d polyvinylpyrrolidone ( P V P P ) w e r e p u r c h a s e d from the S i g m a C h e m i c a l C o m p a n y (St. L o u i s , Mo.). P V P P w a s u s e d a s a n agent to r e m o v e extraneous P P s from the potato s a m p l e s that might otherwise interfere with the P P O a s s a y .  S o d i u m p h o s p h a t e ( m o n o b a s i c a n d dibasic) w a s  p u r c h a s e d from the F i s h e r Scientific C o m p a n y (Fair L a w n , N.J.).  4.2  Sample Preparation 3 0 0 g of potatoes (Russett) w e r e prepared for e a c h blanching p r o c e s s for  a p p a r a t u s 1 a n d 2. P o t a t o e s w e r e p e e l e d , sliced into s m a l l p i e c e s a n d then p l a c e d in 6 0 0 m L of 0.2 M S o d i u m P h o s p h a t e (Fisher Scientific C o m p a n y , N.J.) with a p H of 6.5 (4°C), b l e n d e d in a W a r i n g blender to a fairly uniform c o n s i s t e n c y a n d then further h o m o g e n i z e d using a n experimental h o m o g e n i z e r for 30 s e c o n d s at a s p e e d of six. T h e p H w a s m e a s u r e d using a F i s h e r A c c u m e t M o d e l 4 2 0 Digital (pH/ion) p H meter.  T h e h o m o g e n a t e w a s then immediately centrifuged (Sorvall R C 5 B 6 g ,  M a n d e l Scientific C o m p a n y Ltd., U S A ) at 12,000 x g at 4°C, filtered through B u c h n e r funnel a n d the filtrate w a s u s e d for the heat treatments.  a  F o r a p p a r a t u s 3,  27  w h o l e potatoes w e r e cut into fries (5mm wide, 5 m m thick a n d up to 10 c m in length), (200 g) a n d then b l a n c h e d either in hot water (steam kettle) or a m i c r o w a v e field either with v a c u u m or without v a c u u m (Figure 4.1).  4.3 Overview of Methods A p p a r a t u s 1: T h i s apparatus (Figure 4.1) c o n s i s t e d of a modified m i c r o w a v e o v e n (Figure 4.2) rated 700 W , 2 4 5 0 M H z , within which a n a u g m e n t e d g l a s s dessicator was placed. o n e litre.  T h e g l a s s v e s s e l w a s d e s i g n e d with a n internal v o l u m e of  It w a s c o n n e c t e d to a pump (Figure 4.3) allowing the circulation of the  solution to enter and exit in a c l o s e d loop (Figure 4.4). A t h e r m o c o u p l e w a s p l a c e d on the exterior part of the continuous flow loop to record the temperature on a d a t a logger.  T h e temperature of the particular experiment w a s d e t e r m i n e d to be the  a v e r a g e temperature  readings from the data logger.  Pressure changes were  a c h i e v e d by connecting the top of the g l a s s c h a m b e r to a n exterior v a c u u m p u m p . T h e previously mentioned circulating p u m p w a s adjusted to give a flow rate of 3ml_ per s e c o n d .  28  G  =1  Vacuum Pump  Computer Recording Temperature  (t)  Sample Port 3X  Circulating Pump Microwave Oven  1  Figure 4 . 1 : S c h e m a t i c d i a g r a m of a v a c u u m m i c r o w a v e a p p a r a t u s .  29  Figure 4 . 3 : T h e pump (Cole P a r m e r Instrument C o m p a n y , W A , U S A ; m o d e l N o . 26130).  A p p a r a t u s 2:  T h e a p p a r a t u s (Figure 4.5) c o n s i s t e d of a n E S , 2 4 5 0 M H z  m i c r o w a v e reactor (Figure 4.6 a n d 4.7) with a temperature/pressure control s y s t e m , w h i c h w a s operated by a Microsoft c o m p u t e r software  program.  The  quartz  m i c r o w a v e reaction v e s s e l w a s c o n n e c t e d to a peristaltic p u m p ( M a n d e l Scientific C o m p a n y , Ltd., B e l , F r a n c e ; M o d e l N o . M 3 1 2 ) , to allow for the filling a n d emptying of the g l a s s v e s s e l through Teflon tubing. A l s o , the pump w a s u s e d to obtain s a m p l e s during a n experiment. 300mL,  which  was  T h e v e s s e l w a s d e s i g n e d with a v o l u m e of approximately s e a l e d from  the  outside  environment  and  contained  a  temperature recorder, cooling finger a n d a safety valve. T h e E S (Figure 2.2.5) had a built-in m a g n e t i c stirring d e v i c e that w a s located underneath the bottom of the glass vessel.  B y placing a m a g n e t i c spin bar in the v e s s e l , efficient stirring w a s  g u a r a n t e e d a n d uniform heating of the s a m p l e solution w a s a c h i e v e d . A s c h e m a t i c representation of the apparatus is s h o w n below:  o  I '  I Computer  Microwave Reactor System  Figure 4 . 5 : S c h e m a t i c d i a g r a m of the E t h o s Synth m i c r o w a v e s y s t e m .  31  Figure 4.6: Front view of m i c r o w a v e reactor (Milestone M i c r o w a v e Laboratory Systems, Boston, U S A ) .  Figure 4.7: T o p view of m i c r o w a v e reactor (Milestone M i c r o w a v e Laboratory Systems, Boston, U S A ) .  A p p a r a t u s 3: P a r a m e t e r s s u c h a s temperature, time, P P O activities a n d moisture content w e r e m e a s u r e d for all of the blanching e x p e r i m e n t s using the industrial s i z e d V M D (Figure 4.8).  Figure 4.8: V a c u u m m i c r o w a v e drying ( V M D ) m a c h i n e ( E n w a v e , V a n c o u v e r , Canada)  4.3.1  Heat Treatments T h e r e w e r e three heat treatments, d e s c r i b e d s e p a r a t e l y below:  V a c u u m M i c r o w a v e : 6 0 0 ml_ aliquots of the o . 2 M p h o s p h a t e buffer (pH 6.5) w e r e circulated within a v a c u u m c h a m b e r a s previously d e s c r i b e d ( A p p a r a t u s 1) at 7 0 0 W of m i c r o w a v e power. T h e v a c u u m pump w a s adjusted to control the boiling point a n d equilibrium temperature of the circulating buffer.  U p o n a c h i e v e m e n t of  equilibrium, the circulating pump w a s set at a flow rate of 3ml_/s. W h e n the target  33  temperature, e.g., 55°C w a s r e a c h e d , a blank s a m p l e w a s t a k e n . T h e n the 50ml_, 4°C s a m p l e P P O filtrate w a s injected into the circulating buffer through the s a m p l i n g port.  S i n c e the rate of water evaporation through the v a c u u m p u m p in the v a c u u m  m i c r o w a v e w a s experimentally m e a s u r e d to be 11ml_/min, 11ml_ of distilled water w a s injected every minute that the experiment continued s o that the  reactant  concentration levels w e r e maintained. S a m p l e s for e n z y m e determination w e r e less that 1 m L e a c h a n d were taken at 1 minute  intervals for the duration  of the  experiment.  E t h o s S y n t h M i c r o w a v e R e a c t o r : A 2 0 0 m L aliquot of 0 . 2 M p h o s p h a t e buffer solution (pH 6.5) w a s p l a c e d in the g l a s s reactor v e s s e l of the E S m i c r o w a v e reactor w h i c h h a s a temperature/pressure control s y s t e m operated by a c o m p u t e r software program.  T e m p e r a t u r e w a s controlled by this program by adjusting the m i c r o w a v e  power output of the o v e n . A s with the V M , w h e n the target temperature (between 40-70°) w a s r e a c h e d , a 20ml_, blank s a m p l e w a s t a k e n , a 2 0 m L , 4°C filtrate aliquot of P P O filtrate w a s p u m p e d into the reaction v e s s e l through the valve at the top of the g l a s s v e s s e l . S i n c e there w a s not a n exterior v a c u u m p u m p a n d the s y s t e m w a s c l o s e d , the v o l u m e remained the s a m e throughout the experiment, e x c e p t for taking out the 1ml_ s a m p l e s u s e d for m e a s u r i n g P P O activity.  W a t e r Bath: circulated within  A 6 0 0 m L aliquot of the 0 . 2 M p h o s p h a t e buffer (pH 6.5) w a s  a v a c u u m c h a m b e r a s d e s c r i b e d in A p p a r a t u s 1.  The  only  difference b e t w e e n the V M treatment a n d the water bath treatment w a s that the  34  water bath treatment had the g l a s s reaction v e s s e l i m m e r s e d in a water bath, w h e r e a s , the V M treatment had the v e s s e l contained in the m i c r o w a v e cavity.  4.3.2  Enzymatic Activity Determination T h e P P O of the heat treatment s a m p l e s (1ml_) w a s d e t e r m i n e d by m e a s u r i n g  the rate of i n c r e a s e in a b s o r b a n c e over time at 4 2 0 n m using a U V - 1 6 0 visible recording spectrophotometer ( S h i m a d z u 24, T e k s c i e n c e H a d l e y , O a k v i l l e , O N . , C a n a d a ) at 25°C using a F i s h e r V e r s a - B a t h water bath for 50 s e c o n d s .  The assay  c o n s i s t e d of a 0 . 2 M p h o s p h a t e buffer, p H 6.5, 0 . 1 0 M substrate (catechol) a n d an e n z y m e s a m p l e taken from an experimental trial.  T h e a s s a y w a s p r e p a r e d by  mixing 1.25ml_ buffer, 200ul_ of substrate a n d then adding 50ul_ ( e n z y m e ) of the experimental s a m p l e .  4.3.3  Kinetic Studies T h e thermal inactivation of e n z y m e s , a s with other c h e m i c a l reactions, h a s  been  s u c c e s s f u l l y d e s c r i b e d with respect to temperature  d e p e n d e n c e by  the  A r r h e n i u s equation:  K = k b  k: b  b 0  e  E a / R T  (2)  rate constant at a temperature T  k o: rate constant at a reference temperature To b  35  E : activation energy (J/mol) a  R:  g a s constant (8.314 J / m o l * K )  T:  absolute temperature (K)  B y graphing In k v e r s u s 1/7, the s l o p e c a n be obtained a n d is e q u a l to - E / R . a  Therefore, the activation energy c a n be calculated for specific reaction. T h e D-value or d e c i m a l reduction time (i.e., the time required to c h a n g e the concentration of reactants or products by 90%) c a n be obtained from the s e m i logarithm curve a s the time taken to traverse o n e log c y c l e a n d is related by the equation below:  D = 2.303/k  (3)  Furthermore, it c a n be a s s u m e d that the D v a l u e follows a semi-logarithmic relationship with temperature a s illustrated below:  Log (D:/D ) = T - T i ) / z 2  2  w h e r e D<\ = d e c i m a l reduction time at T i D ;  2  (4)  = d e c i m a l reduction at T , a n d z = 10. 2  A l s o , z is called the z - v a l u e .  4.3.4. Statistical Analysis A two-tailed t-test w a s performed b e t w e e n all the heat treatment D-values a n d s l o p e s from the z - v a l u e c u r v e s a n d activation e n e r g i e s . T h e level of c o n f i d e n c e required for significance w a s s e l e c t e d to be p<0.05.  36  5.  R E S U L T S AND DISCUSSION  5.1  P P O Enzyme A s s a y and P P O Characteristics A g e n e r a l e n z y m e study w a s c o m p l e t e d to study more extensively the P P O  a s s a y a n d to a d d to the previous work d o n e on this e n z y m e . T h e r e d o e s not s e e m to be a set standard P P O a s s a y kit that c a n be found in the literature; therefore, it a p p e a r e d appropriate to study a P P O a s s a y procedure in depth. T h i s work includes s o m e structural a n d g e n e r a l characteristics of the e n z y m e , p r o p o s e d m e c h a n i s m of P P O , a l o n g with s o m e a s s a y t e c h n i q u e s a n d experimental a s s a y results that c a n be u s e d for the P P O a s s a y . A l s o , included here is a n a n a l y s i s of a journal article which e x a m i n e d m e t h o d s to estimate total P P O activity in potatoes ( H s u et a l . , 1988). T h e m e t h o d s s e l e c t e d from the P P O a s s a y a n a l y s i s in this chapter w e r e u s e d in the kinetic a n a l y s i s a n d pilot plant e x p e r i m e n t s d i s c u s s e d later in this thesis. A b s o r p t i o n spectrophotometry is u s e d extensively in the literature to obtain P P O activities of plant material w h i c h indicates the p r e s e n c e of e n z y m a t i c browning (Mui et a l . , 2002). determine  C o n t i n u o u s spectrophotometric a s s a y s are widely u s e d to  P P O activity a n d s o m e kinetic a s s a y s m e a s u r e the a p p e a r a n c e of  q u i n o n e s p r o d u c e d by P P O e n z y m a t i c activity ( E s p i n et al., 1996). S i n c e there are m a n y substrates that P P O could u s e to c o m p l e t e a browning reaction, the literature s h o w s m a n y different P P O a s s a y s .  T h e majority of m e t h o d s that h a v e b e e n u s e d  recently involve m e a s u r i n g spectrophotometrically, a product of a P P O c a t a l y z e d reaction.  37  T h e method c h o s e n here involves c a t e c h o l (a diphenol) w h i c h forms ob e n z o q u i n o n e in the p r e s e n c e of o x y g e n a n d P P O . T h e o - b e n z o q u i n o n e c o m p o u n d a b s o r b s best at 4 2 0 nm. T h e a s s a y t a k e s p l a c e at a temperature of 25°C a n d in a 0 . 2 M s o d i u m p h o s p h a t e buffer with a p H of 6.5. A v o l u m e of 2 5 microliters of the e n z y m e extract w a s u s e d . reasons.  T h e s e a s s a y c o m p o n e n t s w e r e c h o s e n for various  Firstly, the substrate, c a t e c h o l w a s u s e d for the P P O a s s a y b e c a u s e of  potato P P O ' s relatively high activity for c a t e c h o l a s a substrate (Whitaker, 1994). P r e v i o u s s t u d i e s w h i c h utilized potatoes to perform absorption spectrophotometry P P O a s s a y s are few in number; for instance, B a r u a h a n d S w a i n , 1 9 5 9 a n d V a m o s V i g y a z o et a l . , 1973 u s e d substrates other than c a t e c h o l in the  P P O assay.  H o w e v e r , c a t e c h o l w a s u s e d here s i n c e it w a s u s e d in m a n y p a p e r s s u c h a s M u i et al., 2 0 0 2 , E s p i n et al., 1996 and Whitaker, 1994. A l s o , P P O c a n c a t a l y z e reactions with m o n o p h e n o l s , but this is a s m a l l contribution in the potato P P O a s s a y w h e n using a diphenol s u c h a s c a t e c h o l (Whitaker, 1994). T h e s o d i u m p h o s p h a t e buffer is relatively standard a n d is u s e d in n u m e r o u s P P O a s s a y s in the literature. A p H of 6.5 w a s u s e d s i n c e it is within the range of optimum P P O activity a n d within the normal p H range inside the plant cell. P r o c e d u r e s previously d e s c r i b e d w e r e u s e d to d e v e l o p a P P O a s s a y method u s e d here fore potatoes (Hsu et al., 1984; M u i et al., 2002).  T h e a s s a y included  1 0 m M c a t e c h o l a s the substrate, 0.2 M s o d i u m p h o s p h a t e buffer (pH 6.5) at 25°C. T h e v o l u m e s u s e d in the first a s s a y c o n s i s t e d of 2 5 u L of the supernatant (enzyme), 200ul_ of c a t e c h o l a n d 0 . 7 7 5 m L of p h o s p h a t e buffer, which a d d e d to a total a s s a y v o l u m e of 1.0mL.  In following a s s a y s , appropriate a s s a y constituent v o l u m e s were  38  u s e d in a c c o r d a n c e of keeping a standard b a s e l i n e of potato P P O activity  from  s a m p l e variations from d a y to day. After e x a m i n i n g the literature on the five m e t h o d s d e s c r i b e d for estimation of the total activity homogenate:  of polyphenol  o x i d a s e ( P P O ) activity  from  the  crude  1) Polyvinylpyrrolidone ( P V P P ) absorption method 2)  potato  Ammonium  sulphate precipitation method 3) A c e t o n e precipitation method 4) D i a l y s i s method a n d 5) S e p h a d e x G - 2 5 chromatography, the S e p h a d e x G - 2 5 w a s s h o w n to be the best at estimating total P P O activity (Hsu et a l . , 1988).  T h e y c o n c l u d e d that this  method w a s best at removing the e n d o g e n o u s phenolic c o m p o u n d s in the crude h o m o g e n a t e , s i n c e oxidized p h e n o l s irreversibly  inhibit  P P O catalysis.  These  c o m p o u n d s s h o u l d be r e m o v e d b e c a u s e a n e x c e s s in p h e n o l s m a y c a u s e p r o b l e m s in the a s s a y s u c h a s i n c r e a s e d non-linearity.  In addition, it h a s b e e n s h o w n that  there is a rapid inactivation of P P O during the oxidation of c a t e c h o l , thus the initial reaction rate is usually linear for a very short time ( - 3 0 to 90s) (Whitaker, 1994). T h e g o a l of the present m i c r o w a v e e x p e r i m e n t s w a s to m e a s u r e the relative P P O activities between different blanching treatments a n d not to m e a s u r e the total a m o u n t s of P P O , reported earlier ( H s u et al., 1988).  T h e method c h o s e n w a s the I  P V P P absorption method (Hsu et al., 1988) a n d this method w a s preferable for various r e a s o n s .  A s u m m a r y of the P P O potato a s s a y s performed is included in  T a b l e s 5.1 a n d 5.2.  39  T a b l e 5 . 1 : P P O a s s a y s of the P V P P treated c r u d e h o m o g e n a t e . A s s h o w n b e l o w , the P V P P treated crude h o m o g e n a t e w a s diluted a n d reaction rate w a s m e a s u r e d by c h a n g e of a b s o r b a n c e over the c h a n g e in time. E a c h e n z y m e dilution a s s a y w a s d o n e in triplicate.  F o r a s a m p l e g r a p h i c a l r e p r e s e n t a t i o n , p l e a s e s e e figure 5.2. Reaction rate (change of ABS over time [s])  Enzyme Dilution Undiluted 2x 4x 5x 10x 20x  1  2  3.465E-03 1.849E-03 9.643E-04 7.429E-04 3.314E-04 1.800E-04  Volume of undiluted Enzyme Double x2 Quadruple x4 Octuple x8  6.074E-03 9.662E-03 1.104E-02  3.667E-03 1.750E-03 8.633E-04 6.194E-04 2.604E-04 8.039E-05  3 3.550E-03 1.787E-03 9.190E-04 6.857E-04 3.000E-04 8.359E-05  AVRG 3.561 E-03 1.795E-03 9.155E-04 6.827E-04 2.973E-04 1.147E-04  St. Dev. 1.014E-04 5.002E-05 5.059E-05 6.181E-05 3.558E-05 5.661 E-05  4.505E-03 7.433E-03 1.061E-02  4.667E-03 7.160E-03 1.060E-02  5.082E-03 8.085E-03 1.075E-02  8.629E-04 1.373E-03 2.512E-04  Multiplied by DF 3.561 E-03 3.591 E-03 3.662E-03 3.413E-03 2.973E-03 2.293E-03  40  T a b l e 5.2: T a b l e s u m m a r i z i n g reaction rate w h e n the substrate concentration is varied. Substrate (catechol) Concentration Volume [M] [ML]  a  c  b  AVRG  St. DEV.  1.00  500 uL  2.813E-03  2.487E-03  2.540E-03  2.613E-03  1.749E-04  0.80  400 uL  4.049E-03  3.641 E-03  3.947E-03  3.879E-03  2.123E-04  0.60  300ML  5.843E-03  5.577E-03  4.240E-03  5.220E-03  8.591E-04  0.40  200 uL  5.043E-03  5.905E-03  6.964E-03  5.971 E-03  9.622E-04  0.30  150 uL  7.879E-03  7.793E-03  6.414E-03  7.362E-03  8.221 E-04  0.20  100 uL  8.540E-03  7.793E-03  6.473E-03  7.602E-03  1.047E-03  0.10  50 uL  4.886E-03  6.463E-03  4.821 E-03  5.390E-03  9.298E-04  0.05  25 uL  4.140E-03  4.088E-03  3.605E-03  3.944E-03  2.950E-04  0.01  5MI  2.253E-03  2.189E-03  2.058E-03  2.167E-03  9.940E-05  A s indicated in T a b l e 5.1, the e n z y m e concentration w a s varied while the other a s s a y conditions r e m a i n e d the s a m e (volumes r e m a i n e d u n c h a n g e d ) a s the standard a s s a y a b o v e . T h e a v e r a g e reaction rate of the P P O w a s b a s e d on three s e p a r a t e s a m p l e m e a s u r e m e n t s (triplicate).  Illustrated in T a b l e 5.1, the 2x, 4 x a n d  5x dilutions s h o w e d proportional reaction rates.  F o r e x a m p l e , w h e n the e n z y m e  concentration d o u b l e s , there is a l s o a doubling of reaction rate w h i c h s h o w e d consistent a s s a y s .  I would p r o p o s e that any of t h e s e dilutions could be u s e d , but  looking c l o s e l y at all t h e s e g r a p h s , I would c h o o s e the undiluted e n z y m e or the 2x dilution of the e n z y m e for future e x p e r i m e n t s b e c a u s e t h e s e g r a p h s indicated more of a linear e n z y m e reaction rate curve that the other dilutions.  41  A l s o , at the bottom of T a b l e 5.1, the v o l u m e of the undiluted e n z y m e w a s altered in t h e s e a s s a y s .  F o r instance, the d o u b l e (2x) had a v o l u m e of 5 0 u L of the  e n z y m e extract a n d therefore, 2 5 u L less buffer. A s s e e n from the a v e r a g e reaction rate, adding double the v o l u m e of e n z y m e extract, the reaction w a s not d o u b l e d . C o n s e q u e n t l y , w e d o not s e e a c o r r e s p o n d i n g doubling of reaction rate with a doubling of e n z y m e v o l u m e .  T h e inactivation of the P P O here is not d u e to a n  instability of the e n z y m e b e c a u s e of substrate, p H , or temperature conditions; the inactivation, most likely, is c a u s e d by product formation of o - b e n z o q u i n o n e a n d its combination with P P O to form a covalent linkage in or p e r h a p s n e a r the active site (Whitaker, 1994). T h e P V P P absorption method improves the linearity of the P P O a s s a y (see Figure 5.2) in contrast to the crude h o m o g e n a t e (see Figure 5.1), w h i c h w a s not treated by a n y of the five m e t h o d s c h o s e n by H s u et a l . , 1988.  42  -ABS  •Linear (ABS)  Figure 5.1: S a m p l e graphical representation of the crude potato h o m o g e n a t e P P O a s s a y . T h i s w a s a n undiluted e n z y m e s a m p l e . T h i s a s s a y included 50 4L of the e n z y m e extract (supernatant), 0.20 ml_ of 0 . 2 M c a t e c h o l , a n d 1.250 ml_ of the 0 . 2 M s o d i u m p h o s p h a t e buffer. T h e reaction took p l a c e at 25°C a s the substrate a n d the buffer w e r e kept on a water bath. T h e a b s o r b a n c e w a s taken at 4 2 0 nm every 5 s e c o n d s for 120 s e c o n d s . 3.370E-03X + 1.668E-02  120  —•— ABS  Linear (ABS)  Figure 5.2: S a m p l e graphical representation of the P V P P treated c r u d e potato homogenate P P O assay. T h i s w a s a n undiluted e n z y m e s a m p l e a n d a s s a y conditions w e r e the s a m e a s Figure 2 . 3 . 1 . T h e linearity improved w h e n c o m p a r e d to the untreated crude h o m o g e n a t e in Figure 5.1.  43  A s s e e n in Figure 5.2, this a s s a y p r o d u c e s a nearly linear a s s a y .  T h e r is 2  f a v o u r a b l e b e c a u s e of its proximity to 1.0. In other w o r d s , the r indicates that more 2  than 9 9 % of the variability in A o w a s a c c o u n t e d for by the e n z y m e activity. 4 2  In addition, the time required to p r o c e s s o n e crude h o m o g e n a t e s a m p l e w a s lower for P V P P , c o m p a r e d to the other m e t h o d s (Hsu et a l . , 1988).  In addition,  w h e n taking into account the v o l u m e s in my experiment of the c r u d e h o m o g e n a t e (200ml_ or more), for instance, the S e p h a d e x G - 2 5 c h r o m a t o g r a p h y requires 4 0 minutes to p r o c e s s 2ml_ of the crude h o m o g e n a t e ; multiply this by 100 a n d it t a k e s approximately 66 hours to p r o c e s s the crude h o m o g e n a t e for just o n e experiment. Alternatively, the P V P P method requires approximately 8 hours for the s a m e s a m p l e number.  Furthermore, the A c e t o n e a n d Dialysis m e t h o d s w e r e e v e n more time  c o n s u m i n g than the S e p h a d e x G - 2 5 ( H s u et al., 1988). T h e only m e t h o d other than the S e p h a d e x G - 2 5 chromatography that w a s c o m p a r a b l e to the P V P P method in terms of time w a s the a m m o n i u m sulphate precipitation m e t h o d , but this method routinely b r o w n e d during the isolation of P P O ( H s u et al., 1988).  Next, the specific  activity of the P V P P treated crude h o m o g e n a t e did s h o w better or similar results w h e n c o m p a r e d to the other m e t h o d s u s e d to p r o c e s s the crude h o m o g e n a t e (Hsu et al., 1988).  Lastly, the P V P P absorption method did reduce the p h e n o l s by 2 6 %  (Hsu et al., 1988), which s e e m e d to contribute in the i n c r e a s e of linearity in the P P O a s s a y method. A s just previously m e n t i o n e d , all of the m e t h o d s improved the a s s a y results a s c o m p a r e d to not treating the crude potato extract by removing the e n d o g e n o u s  44  phenols.  H o w e v e r , filtering the  crude extract  through  PVPP  appropriate for the v o l u m e s u s e d in the experiment (~200mL).  s e e m e d to Using  be  PVPP  provided a more convenient a n d time s a v i n g method that a l s o did not alter the c o m p o s i t i o n of the potato extract c o m p o u n d a s c o m p a r e d to the other m e t h o d s of excluding the e n d o g e n o u s p h e n o l s . Finally, it w a s determined by statistical a n a l y s i s using t-tests, that 50 s e c o n d s w a s sufficient to characterize the P P O a s s a y (Figure 5.3).  0  10  20  30  40  50  time [s] •  ABS  Linear (ABS)  Figure 5.3: S a m p l e graphical representation of the P V P P treated c r u d e potato h o m o g e n a t e P P O a s s a y . T h i s w a s a n undiluted e n z y m e s a m p l e . T h e first 50 s e c o n d s of e a c h s a m p l e a s s a y w a s statistically c h o s e n to determine the s l o p e .  A s s e e n in T a b l e 5.2, the substrate concentration w a s varied with a fixed concentration of the e n z y m e (25ul_)> T h i s data w a s u s e d to estimate K m a n d V m a x by non-linear r e g r e s s i o n below in Figure 5.4 w h i c h plots the velocity of the P P O  45  c a t a l y z e d reaction v e r s u s varying substrate (catechol) concentrations.  Vmax was  calculated to be 7.8 E-03 (A of A / s ) by finding the m a x i m u m of the c u r v e a n d then from this result, K m w a s estimated to be 0.05 M .  0.009 -i  1  S u b s t r a t e [M]  Figure 5.4: S h o w n here is the velocity of the P P O c a t a l y z e d reaction v e r s u s varying substrate (catechol) concentrations. T h e curve obtained w a s u s e d to calculate V m a x and K m .  5.2  Heat Treatments with Experimental Microwave Apparatus Heat treatment duration w a s b a s e d on preliminary studies.  d e c r e a s e d quickly with time w h e n temperatures e x c e e d e d 60°C.  P P O activity Therefore, the  time w a s kept to 20 minutes for a total of 20 s a m p l e s for e x p e r i m e n t s a b o v e 60°C. Furthermore, not all s a m p l e s w e r e a n a l y z e d , if there w a s a total loss of P P O activity before 20 minutes.  F o r instance, if the 7  t h  and 8  t h  s a m p l e s s h o w e d no P P O  activities, then the rest of s a m p l e s 9-20 w e r e not e x a m i n e d for P P O activities.  For  heat treatments below 60°C, the time of the experiment w a s 4 0 minutes, while  46  s a m p l e s w e r e taken every 2 minutes for a total of 20 s a m p l e s . All s a m p l e s from the heat treatments w e r e a s s a y e d in triplicate for the P P O activity. A first order reaction w a s a s s u m e d s i n c e the P P O a s s a y reaction rate w a s proportional to the reactant (catechol). T h i s w a s supported by the d a t a g e n e r a t e d in this study. T h e extensive literature dealing with polyphenol o x i d a s e (1.10.3.1 o-diphenol: oxygen  oxidoreductase,  hereafter  referred  to  as  ODO=o-diphenol  oxidase)  d e s c r i b e s a relatively s m a l l n u m b e r of activity a s s a y methods. T h e great majority of t h e s e m e t h o d s are b a s e d upon the following principles: a) M a n o m e t r i c m e a s u r e m e n t of o x y g e n uptake d u e to e n z y m e action, polarographic or potentiometric a s s a y of the s a m e ; b) M e a s u r e m e n t of the colour intensity of the c o m p o u n d s p r o d u c e d from the substrate by e n z y m e action;  c) Indirect m e a s u r e m e n t of e n z y m e action by a s s e s s i n g the d e c r e a s e of the amount of reducing s u b s t a n c e a d d e d to the e n z y m e - substrate system.  T h e c h a n g e in a b s o r b a n c e of the a s s a y mixture at 4 2 0 n m w a s u s e d to m e a s u r e e n z y m a t i c browning reaction rate. T h e r e w e r e 3 different heat treatments s i n c e this study wanted to s h o w first, the c o m p a r i s o n between a conventional heat treatment  s u c h a s hot water a n d a m i c r o w a v e heat treatment  (VM and E S ) .  S e c o n d l y , w e w a n t e d to s h o w the c o m p a r i s o n of a constant m i c r o w a v e field heat treatment (VM) a n d a variable m i c r o w a v e field heat treatment ( E S ) . T h e browning  47  rate at e a c h e x p e r i m e n t a l t e m p e r a t u r e followed first-order reaction kinetic, i.e., the log of a b s o r b a n c e i n c r e a s e d apparently linearly with time. F o r a s u m m a r y of the D - v a l u e s , z - v a l u e s a n d E , a n d a s s o c i a t e d p r e c u r s o r s a  for the three heating m e t h o d s at the s a m e temperature, s e e ( T a b l e 2.3.2.1).  T a b l e 5.3. T e m p e r a t u r e d e p e n d e n c e of the rate c o n s t a n t a n d activation e n e r g y for water  bath, v a c u u m microwave and  Ethos Synth microwave  reactor at  various  temperatures. °C  1/°K  40.2 42.8 51.1 55.91 59.8 60.99 64.3 40.99 49.51 56.65 59.93 67.2 40 50 55 60 65  3.167*10" 3.193*1fT* 3.086*10" 3.040*10" 3.005*10" 2.994*10" 2.959*10" 3.185*10" 3.101*10" 3.034*10" 3.004*10" 2.939*10" 3.195*10" 3.096*10" 3.049*10" 3.003*10" 2.959*10"  Slope of log A B S vs. time [min ] 1.27*10"* 2.25*10" 1.16*10"" 3.03*10" 3.53*10"" 4.16*10"" 6.84*10"" 1.02*10"" 1.47*10" 2.02*10" 2.84*10" 6.46*10" 2.50*10"" 5.18*10" 6.80*10" 8.67*10" 2.35*10"  k [min ]  log k  0.0029 0.0051 0.026 0.069 0.081 0.095 0.157 0.023 0.033 0.046 0.065 0.148 0.033 0.119 0.156 0.199 0.541  -2.5376 -2.2924 -1.5850 -1.1611 -1.0915 -1.0222 -0.8041 -1.6382 -1.4814 -1.3372 -1.1870 -0.8297 -1.4814 -0.9244 -0.8068 -0.7011 -0.2668  1  Slope of log k vs. 1/°K  D value [min]  Z valu e[°F]  E [kJ/m ol]  -5.88*10  J  18.58  489  -3.12*10  3  33.89  259  -4.65*10  3  788.9 445.3 86.1 33.0 28.3 24.0 14.6 98.2 68.2 49.5 35.2 15.5 40.0 19.3 14.7 11.5 4.26  22.67  387  a  1  Water Bath  VM  ES Microwav e Reactor  J  J  a  3  3  3  3  3  3  3  3  3  3  3  3  3  3  2  2  2  2  2  2  2  1  S p e c i f i c D - v a l u e s of t h e s e three heat treatments w e r e o b t a i n e d from the log c h a n g e of a b s o r b a n c e o v e r time.  T h e negative reciprocal s l o p e s of the r e g r e s s e d  straight line of log D - v a l u e s at different t e m p e r a t u r e s g a v e the c o r r e s p o n d i n g z v a l u e s (Figure 5.5) for the three different heat treatments.  48  time [min] - * - log slope  — Linear (log slope)  Figure 5.5: E t h o s Synth R e a c t o r at 60°C  time [min] - • - l o g slope — L i n e a r (log slope)  Figure 5.6: M i c r o w a v e at 4 0 ° C  49  time [min] - * log slope  — Linear (log slope)  Figure 5.7: Hot water bath at 60°C  T h e s e D-value c u r v e s c a n be u s e d to obtain z - v a l u e s w h i c h are s h o w n in Figure 5.8. temperature.  It w a s evident that P P O destruction rates i n c r e a s e d with increasing Furthermore, both the V M a n d the E S reactor D-value c u r v e s w e r e  significantly different from the hot water bath D-value c u r v e s (p<0.05).  50  3.5 3  4 2.5  *  0.5  A  0  -I  ,  ,  35  40  45  ,  ,  ,  ,  1  50  55  60  65  70  Temperature •  EOS  B  microwave  A  hotwaterbath  EOS —— "microwave  -  -  :  hotwaterbai  Figure 5.8: T h e D-value curve of the E S reactor, the V M a n d the hot water bath. T h e z v a l u e is e q u a l to the negative reciprocal s l o p e . T h e e q u a t i o n s for e a c h line are: E S synth reactor: y=-0.0354x+3.0628; R = 0 . 9 2 4 9 ; z=28.5 °C, V M : y=-0.0295x+3.2678; R = 0 . 9 3 0 4 ; z=33.89 °C; Hot water bath: y=-0.0538x=4.6339; R = 0 . 9 5 1 4 ; z=18.58 °C. 2  2  2  T h e temperature sensitivity of c h e m i c a l reactions is d e p e n d e n t upon the activation e n e r g y ( E ) . A s a g e n e r a l rule, c h e m i c a l reactions tend to g o faster at a  higher temperatures.  Increasing the temperature  i n c r e a s e s the fraction of the  m o l e c u l e s sharing the E , thus the k d e c r e a s e s a n d reaction rate i n c r e a s e s . a  In this  experiment, the A r r h e n i u s equation w a s u s e d to explain the relationship b e t w e e n reaction rate a n d temperature.  S i n c e a D-value is e q u a l to 2.303//c, k v a l u e s could  be obtained a n d the A r r h e n i u s relationship c a n n o w b e e x p l o r e d a n d is s h o w n graphically in Figure 5.9.  51  O.OE+00 2.90  E-03  -5.0E-01 -  -1.0E+00  ro -1.5E+00 -|  o  -2.0E+00 A  -2.5E+00  -3.0E+00 Temperature [1/K] •  Ethos  hotwaterbath  VMD  Ethos  • hotwaterbath  VMD  Figure 5.9: A r r h e n i u s relationship for the E S reactor, the V M a n d the hot water bath. T h e s l o p e is u s e d to calculate the activation e n e r g i e s for e a c h treatment. T h e e q u a t i o n s for e a c h line are: V M : y=3.123E03x+8.224; R = 0 . 9 1 8 0 ; E = 2 5 9 K J / m o l , E S reactor: y = - 4 . 6 5 8 E 0 3 x + 1 . 3 4 2 E 0 1 ; R = 0 . 9 5 6 ; E = 3 8 7 K J / m o l , Hot water bath: y=-5.88E03x+1.66201; R = 0 . 9 5 3 2 ; E = 4 8 9 K J / m o l . 2  a  2  a  2  a  T h e greater reaction rate a c h i e v e d by the v a c u u m m i c r o w a v e s y s t e m could p e r h a p s be explained by the superheating effect. temperature  of  a  solution  in a  microwave  S u p e r h e a t i n g o c c u r s a s the  rises rapidly,  teaching  temperatures for a very short time interval ( - 1 / 1 0 of a s e c o n d ) . reaction rate might i n c r e a s e quickly during t h o s e fluctuations.  very  high  Therefore, the  It w a s a l s o noticed  that at lower temperatures, the D-values for the V M a n d E S reactor w e r e greatly r e d u c e d a s c o m p a r e d to the hot water bath; h e n c e , s u p e r h e a t i n g might h a v e b e e n occurring at t h e s e temperatures.  T h e superheating at t h e s e lower temperatures  52  might  be  caused  temperatures,  by the  which  difficulty  led to  of  controlling  high temperature  the  vacuum  fluctuations.  pump  at  Unfortunately,  lower the  s u p e r h e a t i n g p h e n o m e n o n could not be exactly recorded in this e x p e r i m e n t a s s u p e r h e a t i n g w a s very fast a n d difficult to m e a s u r e with the e x p e r i m e n t a l a p p a r a t u s u s e d in the heat treatments.  5.3  Industrial Sized V a c u u m Microwave S h o w n below in T a b l e 5.4 is a s u m m a r y of the blanching e x p e r i m e n t s  c o n d u c t e d in the Pilot Plant at T h e University of British C o l u m b i a F o o d S c i e n c e Department.  Included below in T a b l e 5.4 are four figures; o n e being a s a m p l e  picture from o n e of the blanching e x p e r i m e n t s a n d the other three being graphical representations of the parameters from the blanching e x p e r i m e n t s . P o w e r w a s kept at 7 1 O W for the microwave s i n c e , in preliminary studies, this d e g r e e of power w a s sufficient to blanch the fires. A s indicated in T a b l e 5.4, the m i c r o w a v e (w/o v a c u u m ) fries blanch had activities that w e r e relatively high a n d did not d e c r e a s e throughout the p r o c e s s . Moisture content a n d activity w e r e both an a v e r a g e of two replications.  53  T a b l e 5.4: T h e b l a n c h i n g of potato fries using v a r i o u s treatments. T h e temperature, moisture content (%wb), time a n d P P O activity are s h o w n for e a c h treatment.  Treatment (blanch)  wave vacui  E  2 ~ o 5 s |  » > S 2 o  ?  Hot wat Blanchii  CP  E 1 to >  MC [%wb]  Temp. 1 [°C]  Temp. 2 [°C]  76.3 81.5 83.2 85.0 88.8 81.6 64.9 85.1 80.1 82.1 81.6 79.0 84.5 81.3 79.4 80.6 78.5 79.2 80.0 81.4 84.1 81.5 84.3 87.8 80.4 84.7 83.7 84.4 85.8 80.4 85.9 85.1  45.7 52.9 58.2 63.9 68.4 73.1 73.1 83.2 76 83.1 85.1 87.5 38.8 37.6 41.3 39.7 42.8 41.6 43.5 45.9 63.2 74.6 83.9 64.8 76 85.8 63.7 74.2 86.1 65.4 75.9 83.8  46.3 53.1 57.8 64.1 69.6 74.9 73.9 82.8 76 82.9 86.9 86.5 39.7 40.2 40.7 41.3 42.2 42.8 44.5 45.2 66.8 75.4 86.1 65.2 74 84.2 66.3 75.8 83.9 64.6 74.1 86.2  Average Temperature [°C] 46 53 58 64 69 74 73.5 83 76 83 86 87 39.5 38.9 41.0 40.5 42.5 42.2 44.0 45.5 65 75 85 65 75 85 65 75 85 65 75 85  Time [min] 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.5 2.5  5.0  7.5  10.0  Activity [Aabs/min @ 420nm] 0.2095 0.1686 0.0753 0.0852 0.0268 0.0198 0.0077 0.0049 0.0036 0.0000 0.0004 0.0000 0.3426 0.3612 0.3350 0.3395 0.3238 0.3284 0.2649 0.2661 0.0709 0.0459 0.0010 0.0690 0.0297 0.0000 0.0618 0.0021 0.0000 0.0560 0.0019 0.0000  P l e a s e s e e F i g u r e 5.10 for a flowchart of t h e s e e x p e r i m e n t s s h o w n in T a b l e 5.4.  54  Potatoes  Peel  Slice  Weigh 200g for treatments  Hot water blanch  Microwave (71 OW)  no vacuum  vacuum  Measure P P O activity and moisture  Figure 5.10: M i c r o w a v e potato fries.  M o r e data w a s collected for the m i c r o w a v e (w/o the v a c u u m ) , s i n c e the temperatures for the m i c r o w a v e (w the v a c u u m ) w e r e too low for the a m o u n t of time the fries w e r e b l a n c h e d . M o r e o v e r , a n experiment  with the  m i c r o w a v e that ran for 2.5  minutes  included five data points; e a c h of t h e s e points represented a s e p a r a t e 2 0 0 g batch.  55  T h i s w a s the c a s e s i n c e the temperature could not be m e a s u r e d internally by the m i c r o w a v e a n d thus, the m i c r o w a v e  had to be s t o p p e d a n d the  temperature  recording w a s taken with an infrared thermometer. F o r a graphical representation, p l e a s e s e e F i g u r e s 5.11 a n d 5.12.  a  5  time [min] —A  Temperature 1  x~  Temperature 2  MC 1 —o— MC 2  •  Activity 1 -a  Activity 2  Figure 5.11: G r a p h i c a l representation of the m i c r o w a v e (w/o v a c u u m ) blanch data from T a b l e 4 . 1 .  56  0.40  90  1  1.5  t i m e [min] Temperature 1  •Hx-Temperature 2  X  MC 1  Activity 1  Activity 2  Figure 5.12: G r a p h i c a l representation of the m i c r o w a v e (w/ v a c u u m ) b l a n c h data from T a b l e 4 . 1 . T h e s a m p l e s moisture content d e c r e a s e s slightly, w h i c h c a n b e attributed to the increasing temperature. T h e e n z y m e activity d e c r e a s e s over time. Blanching temperature and activity versus time  o £  50 0.04 =  time [min] •Temp rep 1 • B -Temp rep 2  Temp re 3 — A c t rep 1  • Act rep 2  Act rep 3  Figure 5.13: G r a p h i c a l representation of the Hot water blanch data from T a b l e 4 . 1 . T h e moisture content c h a n g e varies slightly b e t w e e n runs but d o e s not s h o w a n y consistent trend. T h e temperature d o e s not c h a n g e over time in all three runs. T h e e n z y m e activity d e c r e a s e s at different d e g r e e s for e a c h temperature level, but it d e c r e a s e s distinctly.  57  6.  CONCLUSIONS  It s e e m s presently fitting to u s e the electromagnetic s p e c t r u m ( E M ) to a c h i e v e o n e of the main g o a l s in this thesis - the food p r o c e s s i n g of potatoes, in this c a s e , using m i c r o w a v e s in the p r o c e s s of blanching to stop food deterioration. M i c r o w a v e s are a natural p h e n o m e n o n a s w i t n e s s e d by the blanketing of m i c r o w a v e radiation of earth from all directions in s p a c e .  W e are, in fact, dealing  with the natural p h e n o m e n o n that the E M s p e c t r u m is with w a v e l e n g t h s that are significantly larger than that of visible light. W e are utilizing a different portion of the E M s p e c t r u m than what our a n c e s t o r s u s e d to p r o c e s s food.  M o s t importantly, w e  c a n utilize the benefits that m i c r o w a v e s h a v e to offer within the food a r e n a . T o provide a c l o s e r observation of m i c r o w a v e b l a n c h i n g , with a n d without vacuum,  it w a s n e c e s s a r y to c o n s i d e r the wealth of information  in e x i s t e n c e  regarding the examination of polyphenol o x i d a s e ( P P O ) , w h i c h is c o n s i d e r e d o n e of the major factors of e n z y m a t i c browning. First of all, the experiments performed in the Pilot Plant in the Department of F o o d S c i e n c e , w e r e practical in terms of c o n s u m e r w a n t s a n d n e e d s . T h e R u s s e t t Backer's  potato, formed  in the  s h a p e of fries, a s e x p e c t e d to  be e a t e n  by  c o n s u m e r s , g i v e s immediate a n d viable applicability. H e r e , w e h a v e the opportunity of e x a m i n i n g the potato more in its native form, not c h e m i c a l l y disturbed or reformed into a n e w potato product. fries, a blanching method  Furthermore, by using the m i c r o w a v e to p r o c e s s  or application c a n be found  and  utilized  in  future  installations.  58  After observing the m a c r o s c o p i c effects of m i c r o w a v e blanching in the Pilot Plant, a c l o s e r look/step w a s undertaken to study the m i c r o w a v e effect, particularly in a v a c u u m , environment using a unique continuous flow method for the v a c u u m m i c r o w a v e (VM) a n d hot water bath.  A l s o , a third a p p a r a t u s ( M i c r o w a v e Reactor)  no v a c u u m - w a s u s e d . M o r e specifically, the c h e m i c a l kinetics of P P O w a s studied in this novel m i c r o w a v e setting to initially investigate thermal a n d potentially n o n thermal effects that m i c r o w a v e s could p o s s e s s against a biological c o m p o n e n t s u c h as an enzyme.  T h e potato w a s o n c e again u s e d in this m i c r o w a v e field, but the  potato w a s relatively purified from its new form into a very liquid form, a puree.  The  puree form of the potato w a s s e l e c t e d for s e v e r a l r e a s o n s : firstly, the puree, w h i c h w a s previously blended a n d h o m o g e n i z e d with a p h o s p h a t e buffer,  was  then  centrifuged a n d subsequently the supernatant w a s filtered with ( P V P P ) , being fairly mobile.  In other words, the viscosity of this liquid w a s low a n d c o u l d e a s i l y m o v e  throughout the V M apparatus.  S e c o n d l y , the literature attests to the difficulty  of  purifying P P O , let alone obtaining e n o u g h of a relatively purified s a m p l e to u s e in the three experimental d e v i c e s . In addition, p u r c h a s i n g the pure form of P P O w a s too e x p e n s i v e to provide all the data n e c e s s a r y to obtain kinetic c u r v e s from the three apparatus.  The  P P O kinetics,  destruction  values,  (D-values),  Z-values  and  activation e n e r g i e s ( E ) w e r e c o m p a r e d using m i c r o w a v e s in a v a c u u m , hot water in A  a v a c u u m , a n d variable m i c r o w a v e s without a v a c u u m .  Moreover, since P P O  destruction d o e s not usually o c c u r at temperatures below 50°C, it w a s c o n s i d e r e d that the v a c u u m microwave could be u s e d below t h e s e t e m p e r a t u r e s , d o w n to approximately  40°C  thereby,  hopefully  illustrating  a  non-thermal  effect.  59  C o n s e q u e n t l y , it w a s profoundly difficult to m e a s u r e a n isolated athermal m i c r o w a v e effect on the e n z y m e P P O . At the s a m e time, it w a s d e c i d e d that a m o r e in-depth view w a s required regarding P P O , w h i c h would include a c l o s e r e x a m i n a t i o n at the P P O a s s a y , w h i c h is s u b s e q u e n t l y intended to m e a s u r e the a m o u n t of P P O in a particular s a m p l e .  A l s o , P P O had to be purified to a greater e x t e n d .  PVPP  (mentioned a b o v e ) w a s c h o s e n for u s e s i n c e it s e e m e d the most viable a n d logical treatment.  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