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

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KINETICS 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) during V A C U U M M I C R O W A V E B L A N C H I N G by Zoran Miladinovic B . S c . ( H u m a n Nutr i t ion), T h e Un ive rs i t y of Br i t ish 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 Un ive rs i t y of Br i t ish 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 R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( F o o d S c i e n c e ) 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 F e b r u a r y 2 0 0 6 © Z o r a n M i l a d i n o v i c , 2 0 0 6 A B S T R A C T V a c u u m microwave (VM) blanching dec reases enzymat ic food browning at low temperatures (between ~40-55°C) when compared to convent ional process ing methods. The impact and reaction of microwave blanching on polyphenol ox idase ( P P O ) of the Russe t potato was investigated and compared with the effects of convent ional convect ive heating. In order to c losely examine P P O kinetics in a microwave field and to establ ish a P P O assay for use in further investigations, potato puree plus polyvinylpolypyrrol idone ( P V P P ) was centrifuged and filtered to produce a treatment ready suspens ion . Who le potatoes were cut into French fry strips and b lanched using a pi lot-sized V M dehydrator. Th is w a s compared to a s team kettle blanch to determine the blanch effects on physical form. Finally, two different microwave treatments were utilized along with a heated water bath in order to compare the microwave heated potato suspens ion samp les with convent ional heating. Microwave treatments used a 700 W , 2450 M H z vacuum microwave oven and an Ethos Synth ( E S - M R ) microwave reactor set at 2450 M H z , which dropped the microwave power level from 700 W to below 100 W after reaching the desired temperature. Exper iments in all three treatments were conducted under controlled temperatures between 40°C and 70°C. The dec imal reduction va lues (D-value) of P P O were reduced by heating in vacuum microwave treatments as compared to heating in a water bath at temperatures between 40°C and 57°C. At these temperatures, the D-values, z-va lues and activation energies (E a ) for the P P O model reaction were significantly lower (p < 0.05) for the V M and the E S than the heated water bath. The results suggest the ex is tence of an alternative inactivation mechan ism for P P O when heated in a microwave field compared to a water bath at low process ing temperatures. T A B L E OF CONTENTS A B S T R A C T II T A B L E OF CONTENTS IV LIST OF T A B L E S VI LIST OF FIGURES VII LIST OF S Y M B O L S AND ABBREVIATIONS VIII A C K N O W L E D G E M E N T S X 1. INTRODUCTION 1 2. L ITERATURE REVIEW 3 2.1 General Introduction / Central Concept of Thesis 3 2.2 Food Spoilage / Deterioration and Browning 6 2.2.1 Introduction 6 2.2.2 Enzymatic Browning 6 2.2.2.1 Introduction 6 2.2.2.2 P P O as Indicator of Enzymatic Browning 8 2.2.2.3 PPO Characteristics 9 2.3 Physical Processing / Microwaves 13 2.3.1 Introduction 13 2.3.2 Enzymes and Radiation / Heat 13 2.3.3 Enzymes in Solution 15 2.3.4 Novel Microwave Interactions 15 2.3.5 EM Radiation and the EM Spectrum 17 2.3.6 Microwave Heating 18 2.3.6.1 Introduction 18 2.3.6.2 Factors Affecting Microwave Heating 19 2.3.7 Microwave Applications 20 2.3.7.1 Introduction 20 2.3.7.2 Effects of Microwaves on Food and Enzymes 21 2.3.7.3 Blanching and Microwave Blanching 23 iv 2.4 The Potato 24 2.4.1 Measuring Potato PPO Activity 24 2.4.2 Potato Blanching 24 3 HYPOTHESIS AND OBJECTIVES 26 3.1 Hypothesis 26 3.2 Objectives 26 4. MATERIALS AND METHODS 27 4.1 Materials 27 4.2 Sample Preparation 27 4.3 Overview of Methods 28 4.3.1 Heat Treatments 33 4.3.2 Enzymatic Activity Determination 35 4.3.3 Kinetic Studies 35 4.3.4. Statistical Analysis 36 5. R E S U L T S AND DISCUSSION 37 5.1 PPO Enzyme Assay and PPO Characteristics 37 5.2 Heat Treatments with Experimental Microwave Apparatus 46 5.3 Industrial Sized Vacuum Microwave 53 6. CONCLUSIONS 58 7. R E F E R E N C E S 61 LIST O F T A B L E S Tab le 5.1 P P O assays of the P V P P treated crude homogenate 40 Tab le 5.2 Tab le summariz ing reaction rate when the substrate concentrat ion is varied 41 Tab le 5.3 Temperature dependence of the rate constant and activation energy for water bath, vacuum microwave and Ethos Synth microwave reactor at various temperatures 48 Tab le 5.4 The blanching of fries using var ious treatments 54 vi LIST O F F I G U R E S Figure 2.1: Flowchart to show the logical progression of this thesis 5 Figure 2.2: P P O catalyzed reaction that a lso includes copper as a co-factor 7 Figure 4 .1 : Schemat ic diagram of a vacuum microwave apparatus 29 Figure 4 .2 : Mic rowave oven 29 Figure 4 .3 : The pump 30 Figure 4.4: Internal Mechan ism of the Microwave apparatus for vacuum microwave process ing of liquids 30 Figure 4 .5 : Schemat ic diagram of the Ethos Synth microwave sys tem 31 Figure 4.6: Front view of microwave reactor 32 Figure 4.7: Top view of microwave reactor 32 Figure 4.8: V a c u u m microwave drying (VMD) machine 33 Tab le 5.1: P P O a s s a y s of the P V P P treated crude homogenate 40 Tab le 5.2: Tab le summariz ing reaction rate when the substrate concentrat ion is varied 41 Figure 5.1: Samp le graphical representation of the crude potato homogenate P P O a s s a y 43 Figure 5.2: Samp le graphical representation of the P V P P treated crude potato homogenate P P O assay 43 Figure 5.3: Samp le graphical representation of the P V P P treated crude potato homogenate P P O assay 45 Figure 5.4: S h o w n here is the velocity of the P P O cata lyzed reaction ve rsus varying substrate (catechol) concentrat ions 46 Tab le 5.3. Temperature dependence of the rate constant and activation energy for water bath, vacuum microwave and Ethos Synth microwave reactor at var ious temperatures 48 Figure 5.5: Ethos Synth Reactor at 60°C 49 Figure 5.6: Microwave at 40°C 49 Figure 5.7: Hot water bath at 60°C 50 Figure 5.8: The D-value curve of the E S reactor, the V M and the hot water bath. The z value is equal to the negative reciprocal s lope 51 Figure 5.9: Arrhenius relationship for the E S reactor, the V M and the hot water bath. The s lope is used to calculate the activation energ ies for each treatment 52 Tab le 5.4: The blanching of potato fries using var ious treatments 54 Figure 5.10: Microwave potato fries 55 Figure 5.11: Graphica l representation of the microwave (w/o vacuum) blanch data from Table 4.1 56 Figure 5.12: Graph ica l representation of the microwave (w/ vacuum) b lanch data from Tab le 4.1 57 Figure 5.13: Graph ica l representation of the Hot water blanch data from Tab le 4 .1 . The moisture content change varies slightly between runs but does not show any consistent trend 57 VII LIST OF SYMBOLS AND ABBREVIATIONS Symbols °C Degrees Ce ls ius °F Degrees Fahrenheit D-value T ime required to change the concentrat ion of reactants or products by 9 0 % E a Activation energy K Kelvin Log Logarithm M Mole/l i ter m M Millimole/liter fjL Microliter cm Cent imeter nm Nanometer M H z Megaher tz s seconds h Hour W Watts z Temperature change required to change the dec imal reduction time by a factor of 10 Abbreviations A N O V A Ana lys is of Var iance E M Electro-magnet ic E S - M R Ethos Synth Microwave Reactor P E R Protein Eff iciency Ratio P P O Polyphenol ox idase P V P P Polyvinylpolypyrrol idone V M V a c u u m microwave V M B V a c u u m microwave blanching V M D V a c u u m microwave drying ACKNOWLEDGEMENTS I would like to take this opportunity to thank the many people who ass is ted and supported me during this long term endeavour. First of al l , I want to express my deepest gratitude and thanks to Dr. Tim Durance, my supervisor, for his encouragement , advice and continued support. I would a lso like to thank the other members of the supervisory committee; Dr. David Kitts and Dr. Bruce Todd for their constructive recommendat ions and ass is tance. In addit ion, I would like to extend my s incere appreciat ion to Mr. She rman Y e e and M s Valer ie Skura for their technical ass is tance and invaluable adv ice during the course of this thesis. A s wel l , a huge thank you to Paras too Y a g h m a e e and others in the lab for answer ing quest ions when I didn't know what one plus one w a s anymore and to d i scuss topics related to this thesis. A spec ia l thank you to my family as well as to Katrin and S lobodan for their help with the computer. 1. INTRODUCTION Convent ional methods of drying foods such as air drying have been used for hundreds of years. However, certain preservation methods produce some undesirable characterist ics, one being enzymat ic browning. B lanching is usual ly an effective method used to prevent enzymat ic browning. Rad ian energy (microwaves) combined with a vacuum environment forms the basis of V a c u u m Microwave Blanching (VMB) which will be d iscussed with respect to polyphenol ox idase ( P P O ) and blanching. The objectives of the proposed research include: determine if a non-thermal denaturing effect on polyphenol ox idase may occur in the V M B process and to provide a blanching application (VMB) . T h e s e objectives, hopefully, will be ach ieved with the experimental apparatus that our laboratory has created that al lows the testing of the effects of V a c u u m Microwave Drying (VMD) on the enzyme P P O . Accord ing to some studies, there may be a reduction in both time and temperature for the inactivation of P P O using a microwave field treatment as compared to a water bath treatment. In a normal microwave field, the temperature r ises to a point where P P O begins to denature; however, in a vacuum microwave f ield, the temperature might not reach the denaturing point found in convent ional heating due to the reduction in the boiling point of water at reduced pressure. Non-thermal effects, therefore, may become dominant during V M D P P O inactivation. S o m e ev idence for the non-thermal effects of microwaves on enzymes w a s reported for the enzyme pectin methyl es terase in orange juice (Tajchakavit et a l . , 1995). The 1 polyphenol ox idase assay results for samp les b lanched in different ways will provide a quantitative analys is of how microwave energy in a vacuum environment might interact with polyphenol ox idase. The signi f icance of this research lies in the fact that V M B provides a relatively new method of process ing foods by blanching them. A s relatively few studies have examined microwave blanching, this study may enrich future research. Un less eaten fresh, food preservat ion is vital to maintain food integrity and quality. O n e method of food preservation is blanching, which is a p rocess used to destroy undesirable enzymat ic activity in vegetables and fruits prior to further process ing and conventionally includes a hot water or s team medium. A major purpose in blanching fresh foods is to prevent enzymat ic browning during storage which mainly occurs when the enzyme P P O is exposed to 0 2 in the p resence of polyphenols (PP) , which are abundant in plant materials. M ic rowaves can a lso be used to blanch food materials and destroy P P O . Specif ical ly, the method of heating (microwave vs. hot water) and its effects on P P O were investigated in this study. Absorpt ion spectrometry has been successfu l ly used in severa l studies to determine the rate of enzymat ic browning by measur ing the P P O activity of the macerated fruits at an absorbance of 420 nm (P izzocaro et a l . , 1993; A lme ida and Noguei ra , 1995). Furthermore, the heat resistance of enzymes can be character ized by D-values and z-va lues of different p rocesses which then can be compared to see the effect iveness (decrease in enzymat ic browning) of different heating methods. The absorbance at 420 nm of heat treated samp les in this study was used to measure enzymat ic browning reaction rate; extent of formation of the 2 coloured products, quinones, and the measurement of colour changes due to the p resence of soluble pre-melanoidins. Accord ing to Mayer and Harel (1979), colour formation is likely due to both the formation of low molecular weight compounds , i.e. quinones, and to the presence of melanoids with high molecular weights. The measured change in concentration was consequent ly used to obtain parameters such as D-values, z -va lues and activation energies 2. LITERATURE REVIEW 2.1 General Introduction / Central Concept of Thesis Food processing is a critical field in Food Sc ience , being essent ia l for practical production and distribution of many foods. Techno logy plays an important role in the food system of today s ince the world's population is increasing at a rapid rate and it is therefore, impossible to feed everybody with non-processed foods. At the s a m e time, however, people do not want foods to be altered excess ive ly . For example , when chemica ls are added to preserve food or more importantly, when the taste is changed with a physical process, foods may be less acceptab le to consumers . In this thesis, therefore, a physical p rocess , blanching, which conventional ly occurs in a s team or hot water medium, w a s investigated. A blanching medium used in this study involved a vacuum microwave environment which represents a new technology in food processing that will hopefully be cons idered in the future study of Food Sc ience . 3 Blanching is an establ ished process , widely used in the food industry, for instance, to prevent enzymat ic browning. The major enzyme assoc ia ted with enzymat ic browning is polyphenol ox idase ( P P O ) . The amount of this enzyme in a food can be measured by means of an assay . The potato (white baker 's potato) was chosen from several candidate vegetables as the food that would be used to measure changes in P P O due to blanch treatments. Finally, convent ional p rocesses were compared to this new technological process, V a c u u m Microwave Blanching (VMB) . The flowchart shows a graphical representation of the conceptua l idea behind this thesis. (Figure 2.1). 4 Browning has a negative influence on vegetable quality Prevent enzymat ic browning by process ing S o m e processes not as effective as others Therefore, choose a process to maximize food's quality by measur ing a variable, in this case , the amount of enzyme destroyed Microwave and vacuum P r o c e s s Benefic ial s ince microwave heating (radiant) is faster than convent ional heating (conduction). A l so , lower processing temperatures may be obtained in vacuum microwave heating Figure 2 .1 : Flowchart to show the logical progression of this thesis. 2.2 Food Spoilage / Deterioration and Browning 2.2.1 Introduction Food in today's consumer consc ious society is constantly under scrutiny regarding its nutritional value or lack of it. Daily we hear of reports condemning certain foods as being unhealthy or even inedible. Today 's consumer cons iders fresh food to be preferable for var ious perceived reasons, such as it being healthier or having a high nutritional value. However, it s e e m s increasingly difficult within our temporary North Amer ican culture to eat fresh food consistently and on a regular bas is . Therefore, the need ar ises to prevent, or delay food spoi lage and deterioration, which can be caused by var ious factors, such as oxidat ion, micro-organism growth, non-enzymat ic and enzymat ic browning. The focus of this thesis will be enzymat ic browning and its prevention, in particular, using the potato as the model food. 2.2.2 Enzymatic Browning 2.2.2.1 Introduction General ly , the browning of foods is undesirable and reduces not only the appearance but the nutritional value of that particular food. In some c a s e s , such as coffee and tea, the brown appearance is desi red and a required asset . However, in 6 vegetables and fruits, browning and/or brown spots reduce consumer appea l , thereby decreas ing the monetary value of the food. Speci f ical ly, browning of foods, cata lyzed by enzymes , is referred to as enzymat ic browning. For a significant portion of the thesis, the d iscuss ion will include enzymat ic browning involving the enzyme, polyphenol ox idase ( P P O ) . For the schemat ic enzyme reaction d iagram, p lease s e e Figure 2.2. 0 \ J + 2 H 2 0 Figure 2.2: P P O catalyzed reaction that also includes copper (cuprous) as a co -factor (Whitaker, 1994) Polyphenol ox idase ( P P O ) has a d ichotomous role in the plant and animal k ingdoms. P P O is present in all plants, with particularly higher concentrat ions, for example , in potato tubers, mushrooms, bananas and apples. P P O plays a critical role in human functions, for example, in eye, hair and skin pigmentat ion and is specif ical ly critical in the formation of the exoske le tons of insects. Furthermore, P P O is instrumental in the curing of tea and coffee. T o b a c c o is simi lar to tea and coffee in this respect. The colour of prunes and raisins is attributed to P P O also. The main d isadvantage of P P O is due to its detrimental browning effects on bruised and broken plant t issues. 7 2.2.2.2 PPO as Indicator of Enzymatic Browning Enzymat ic browning caused by P P O has been studied extensively in fruits and vegetables and is responsib le for the enzymat ic browning of these fresh horticultural products, following bruising, cutting or other d a m a g e to the cell (Mart inez & Whitaker, 1995). In general , browning results from both enzymat ic and non-enzymat ic oxidation of phenol ic compounds . Browning usual ly impairs the sensory propert ies of products because of the assoc ia ted changes in colour, f lavour and texture softening (Martinez & Whitaker, 1995). Unfavourable enzymat ic browning occurs in many plants and vegetables and is of a great concern to Food Technologis ts and processors . The discolourat ion of browning, however, is not a chemica l quality defect (Jeon et al . , 1996), but is less appeal ing to consumers and therefore, reduces the market value of the fruits and vegetables. P P O is the major cause of enzymat ic browning in higher plants (Thygesen et al . , 1995). P P O catalyzes the convers ion of monophenols to o-diphenols and o-dihydroxyphenols to o-quinones (Thygesen et al . , 1995). Black or brown pigment deposi ts result when the quinine products polymerize and react with amino acid groups of cel lular proteins. A s a result, P P O activity c a u s e s cons iderab le economic and nutritional loss in the commerc ia l production of fruits and vegetables. Browning can be prevented by var ious methods: exc lus ion of molecular oxygen, methylation of the phenols with o-methylase, addition of reducing agents 8 such as ascorbate, bisulfite, thiols, which prevent the accumulat ion and polymerizat ion of o-benzoquinone, utilizing metal complexing agents such as sodium fluoride and az ide which inactivate the enzyme by reacting with the essent ia l copper, heat treatment which thermally destructs the enzyme, lowering the pH to below pH 4.5 and using competit ive inhibitors such as sodium benzoate. The reducing agent L-ascorb ic acid is an example of a subs tance which prevents browning by reducing the o-benzoquinone back to o-diphenol as rapidly as it is formed. Therefore, no browning occurs as long as ascorb ic acid is present. A s well , ascorb ic acid has a direct effect on polyphenol ox idase, especia l ly in the presence of micromolar copper ions. 2.2.2.3 PPO Characteristics Polyphenol ox idase ( P P O ) ( E C 1.14.18.1) is ubiquitous in the plant k ingdom (Mart inez and Whitaker, 1995). A wide range of molecular weight P P O s exist in different plant t issues and even in the s a m e plant, which can have multiple forms of this enzyme (Richardson and Hyslop, 1994). Vegetab les , such as potatoes are suscept ib le to discoloration during handling and processing and undergo enzymat ic browning as a result of cellular disruption and an e x c e s s of oxygen in the presence of P P O (Mui et al . , 2002). P P O ' s that exist in the animal k ingdom are the tyros inases. In fact, the unit definition for P P O in enzyme manuals usual ly states L-tyrosine as a substrate for the standard P P O assay . P P O s have both a monopheno lase and a d iphenolase (i.e., catechol) activity, of which, the latter will be 9 the focus in this study. A l so to clarify, P P O has been cal led pheno lase, catechol ox idase, ca techo lase, c reso lase and tyrosinase (Mui et a l . , 2002). A spec ia l quality of P P O is that it can catalyze two separate and varied types of reactions, both involving phenol ic compounds . The first reaction involves the hydroxylation of monophenols by P P O to give o-diphenols. This reaction is a lso often referred to as c reso lase activity, s ince p-cresol is often used as the substrate (Whitaker, 1994). The second reaction (Figure 1.2) will be the one util ized in this thesis which involves the process of oxidation. Both procedures can be used to measure P P O activity. A by-product of both these procedures is the formation of melanin because of the O2 uptake of oxidation of the p-diphenol and of subsequent nonenzyme-cata lyzed reactions. It is important to note that all polyphenol ox idases have activity on o-diphenols. It has been reported, for example , that polyphenol ox idases from banana and tea leaves have activity on o-diphenols exclusively but not activity on hydroxylate monophenols (Whitaker, 1994). Polyphenol ox idases from potato and apple, however, reflect both types of activity. Catecho l is the substrate most often used in the a s s a y of P P O activity on o-diphenols, therefore, referred to somet imes as ca techo lase activity. Even though ca techo lase activity of P P O can be measured independently of c reso lase activity by the use of o-diphenols as substrates, compl icat ions can ar ise from the rapid inactivation of the enzyme during reaction and inhibition of activity at high substrate concentrat ions. Th is inactivation of the enzyme, however, is not due to the instability of the enzyme to the pH, or the temperature used, or the inactivation caused by catechol . 10 Instead, this inactivation is caused by the reaction of an intermediate product, o-semibenzoqu inone free radical. B e c a u s e of this free-radical oxidat ion, copper is thereby re leased and the active site subsequent ly destroyed (Whitaker, 1994). This p rocess is cal led reaction inactivation. Methods using manometr ic, polarographic, chronometr ic and spectrophotometr ic analys is can be used to follow the activity of polyphenol ox idase on o-diphenols such as catechol . It can be expected that different results are derived depending upon the method used. For example , the manometr ic method results in the lowest P P O activity because of the difficulty of obtaining true initial rates. A s well , the nonlinear response and flattening of the curve of enzymat ic activity are due to limitations placed on the rate of incorporation of O2 into the solution by shaking. Us ing chronometr ic and spectrophotometr ic methods resulted in the s a m e outcome a s low enzyme concentrat ions (Whitaker, 1994). The polarographic method, which measures 0 2 uptake, g ives a l inear result with enzyme concentrat ion. A surprising result, however, was that the rate of 0 2 uptake was larger than that found by the manometr ic method. It would appear that these two a s s a y s are the preferred methods because of the similar initial rates obtained by both the polarographic and spectrophotometr ic methods. Po lyphenol ox idases that originate from different sources a lso differ substantial ly in speci f ic substrate requirements, particularly for potato and peach . In addit ion, the activities on monophenols are substantial ly lower than on o-diphenols which is not unusual for polyphenol ox idases . It has been establ ished that a number of polyphenol ox idases catalyze both o-hydroxylation of monopheno ls and dehydrogenat ion of o-diphenols (Whitaker, 1994). The ratio of these two activities, 11 however, differs depending on the method of preparation. Th is presented a concern with mushroom polyphenol ox idase until it was clearly establ ished that this enzyme exists in severa l multiple molecular forms which do not affect the s a m e activities on p-cresol and catechol (Whitaker, 1994). B a s e d on the enzyme isolated from var ious sources , it has been shown that P P O has a pH optimum that is between 5.0 - 7.0, with a sharp dec rease in activity below 4.5 (Mayer and Harel , 1979). P P O is located in the chloroplast thylakoid membranes of plant t issues (Mart inez and Whitaker, 1995). The enzyme, which cata lyzes the reaction in which phenols (i.e. catechol) are oxidized to qu inones, requires two copper metal ions (cuprous state) for activity as well as molecular oxygen. The two main P P O inhibitors are benzo ic ac id, which competes with the phenol ic substrate, diethyldithiocarbamate which interacts specif ical ly with the copper cofactor in the enzyme. The enzyme itself is an oxygenase ; crude extracts of mushroom P P O show multiplicity (different forms of the enzyme) and the M W of the predominant form has been often reported to be between 116,000 and 128,000 Daltons. There are four tetramers, each approximately 28 kDa to 32 k D a . There has been little s u c c e s s in purifying and character iz ing the entire structure and amino acid sequence of many P P O s . However, the amino acid s e q u e n c e s of P P O from mushroom, Neurospora crassa and Streptomyces glaucesens, were found to have little dif ference in coding sequence , demonstrat ing the c lose evolutionary relationship between the enzymes from different sources (Mayer and Hare l , 1979). The extinction coefficient of P P O is 24.9 at an absorbance of 280 nm. Bes ides using oxygen to catalyze the dehydrogenat ion of catechols to qu inones, the 12 quinones rapidly polymerize to form insoluble brown compounds known as melanins (Mart inez and Whitaker, 1995). 2.3 Physical Processing / Microwaves 2.3.1 Introduction W h e n d iscuss ing physical p rocess ing, it is important to include chemical ly p rocessed foods. It appears that the public wants less material added to their food; ideally, the fewer ingredients the better and also, ingredients that are known and famil iar to the consumer are preferred. Therefore, a method such as V a c u u m Microwave Blanching (VMB) , which util izes a vacuum environment along with microwaves, can be seen as a possib le alternative for the addit ion of chemica l P P O inhibitors. The vacuum provides a lower p rocess temperature environment than convent ional blanching and the microwaves provide the energy to support heating. 2.3.2 Enzymes and Radiation / Heat Browning reactions in fruits and vegetables are a ser ious problem for the food industry (e.g. mushroom processing). The principal enzyme responsib le for the browning reaction is polyphenol ox idase ( P P O ) . A microwave appl icator was des igned and used to study mushroom P P O inactivation (Rodr iguez-Lopez et al . , 1999). The effects of microwaves and convent ional heating on the kinetics of the monopheno lase and d iphenolase activities of P P O were studied. Convent iona l and microwave treatments produced different enzyme intermediates with different 13 stability and kinetic properties. Cons iderab le time was saved with microwave inactivation of the enzyme compared with the time needed when convent ional hot-water treatment was used, possibly resulting in enhanced quality and greater profitability. A s well , the short exposure time required for samp les irradiated with microwaves is very important for maintaining the quality of mushrooms (Rodr iguez-Lopez et al. ,1999). The fast microwave treatment typically results in an increase in antioxidant content and a considerable dec rease in browning. Microwave energy may be a viable alternative to hot water blanching for the avo idance of browning. The most restrictive factor for the appl icat ion of microwave heating techniques, however, is the temperature gradients generated within some samp les during microwave heating (Rodr iguez-Lopez et a l . , 1999). Rodr iguez-Lopez et a l . , (1999) elucidated in their study the heating inactivation kinetics of mushroom P P O using 2450 M H z microwave radiation. Microwave energy irradiation of P P O at 2450 M H z was found to cause a significant loss in the d ipheno lase activity of mushroom P P O (Sanchez -Hernandez et a l . , 1999). Thermal inactivation was irreversible in all cases , which appears to suggest that this loss of activity was due to a change in the overall conformation of the enzyme. W h e n s low blanching methods were used , the enzyme remained active for a longer time in the p resence of its phenol substrates. The fast microwave treatment, however, resulted in an increase in antioxidant content and a considerable dec rease in browning. Th is technique also permitted a rapid temperature increase compared to more convent ional blanching 14 techniques, which has demonstrated a large gradient of permanent enzyme inactivation. 2.3.3 Enzymes in Solution Irreversible inactivation resulted when the effect of microwave (f=10.4 G H z ) irradiation on a thermostable enzyme w a s tested. The activity of enzymat ic solut ions w a s compared to that of a sample heated in a water bath at the s a m e temperature. E n z y m e concentrat ion, microwave power level, and exposure time were factors that affected the residual activity of the exposed samp les . W h e n concentrat ions were above 50 ug/ml. microwave effects d isappeared (La C a r a et al . , 1999). T h e s e results were not consistent following water bath heating using identical temperatures and duration t imes. The results from microwave heating raw milk from cows and goats in a cont inuous flow unit up to temperatures ranging from 73.1 to 96.7 degrees C , indicate that this cont inuous microwave process may be an efficient method for the pasteurizat ion of milk (Vil lamiel et a l . , 1996). The outcome of the heat treatments was calculated by measur ing protein denaturat ion, lactose isomerizat ion and the total bacterial count. 2.3.4 Novel Microwave Interactions Hernandez-Infante et al. , (1998) found that microwaving destroyed trypsin inhibitors just as was observed in beans cooked using the convent ional method. But Protein Eff iciency Ratio ( P E R ) for raw s e e d s with low content of anti-nutrients (faba 15 beans, peas , ch ickpeas and lentils), were not affected. In compar ison to microwave-heated dry soybeans , microwave-heated soaked soybeans had a higher amount of destroyed trypsin inhibitors as well as a higher P E R . B e c a u s e microwave heating of the common beans failed to demol ish hemagglut inins and trypsin inhibitors, their digestibility and P E R values were poor (Hernandez-Infante et al . , 1998). Microwave heating, therefore, presents an adequate method for destroying hemagglut in ins and trypsin inhibitors without compromis ing protein quality of most legume seeds . The only except ion to this would be common beans that preserved anti-nutritional subs tances . V u k o v a et al . , (2004) reported that microwave exposure inf luences enzyme complexes . Their studies investigating microwave fields produced s o m e stabil izing and prolonged effects on the acety lchol inesterase ( A C h E ) activity in skeletal musc le fractions from frog skeletal musc les . The effects of cont inuous microwaves (2.45 G H z ) of different field intensity on acety lchol inesterase activity and protein conformation were examined. Dif ferences were found between samp les irradiated with a low-intensity microwave field and samp les exposed to high-intensity microwave (Vukova et al . , 2004). A n augmentat ion of random coi ls, amorphous structures and (3-sheets were revealed in the samp les irradiated with a low-intensity microwave field, whereas the changes were less noticeable in the samp les exposed to high-intensity microwaves. Expos ing frog skeletal musc le fraction to microwaves (2.45 G H z ) results in intensity-dependent, non-thermal and prolonged modification of acety lchol inesterase activity. 16 Beck et al . , (2002) used the microwave oven to thaw fresh f rozen p lasma used for transfusions. Th is proved to be a more superior, gentler and quicker method compared to using the water bath and water bag sys tem. A n understandable concern was whether microwaves might have a negative impact on the virus-inactivated p lasma by enhanc ing the activation procedure or impairing the proteins which had been initiated by the inactivation process, therefore reducing hemostat ic activity. There were, however, no significant di f ferences found in the hemostat ic parameters which could be attributed to the thawing method (Beck et a l . , 2002). In addit ion, the microwave oven is suited for thawing of not only fresh frozen p lasma but virus-inactivated p lasma as well which because of the shorter thawing t ime, is an added bonus particularly in a mass ive transfusion situation. 2.3.5 EM Radiation and the EM Spectrum Microwave energy is one form of electromagnet ic energy which can be v iewed as transmitted as waves , and can penetrate food and is eventual ly converted to heat. Microwaves, like all e lectromagnet ic radiation, have an electr ic component as well as a magnet ic component . The microwave wave is character ized by wavelengths between 1mm to 1m (Fel lows, 1987). Mic rowaves are produced at speci f ied f requency bands, for instance, 2450 M H z in household microwaves and somet imes 896 M H z in Europe and 915 M H z in the U S A (Fel lows, 1987). The depth of penetration into a food is inversely related to f requency. In general , lower-frequency microwaves, therefore, penetrate more deeply than higher f requency microwaves. Other factors, such as dielectr ics a lso 17 inf luence microwave penetration. The depth of penetration of microwaves is determined by the loss factor of the food and the wavelength of f requency of the microwaves as indicated in the equation below: x [m]: depth of penetration A [m]: wavelength in space e : dielectric constant £ : loss factor x = (1) 2ne* Greater penetration and more uniform heating is, therefore, obtained using longer wavelengths (896 M H z and 915 MHz) , with foods that have lower loss factors, or with smal ler p ieces of food. However, deep penetration into food is not necessar i ly the main requirement and the wavelength of microwaves is chosen to suit the required application (Fel lows, 1987). 2.3.6 Microwave Heating 2.3.6.1 Introduction In convent ional or surface heating, the process time is limited by the rate of heat f low to the body of the material from the surface as determined by its specif ic heat, thermal conductivity, density and/or viscosity. Sur face heating is not only slow, but a lso non-uniform with the sur faces, edges and corners being much hotter than 18 the inside of the material. Consequent ly , the quality of convent ional ly heated materials is variable and frequently inferior to the desired result. Imperfect heating causes product rejections, wasted energy and extended process t imes that require large production areas devoted to ovens. Large p ieces of equipment are s low to respond to needed temperature changes , take a long time to warm up and have high heat capaci t ies and radiant losses. Their s luggish performance makes them slow to respond to change in production requirements making their control difficult, subjective and expens ive. Converse ly , with microwaves, heating the vo lume of a material at substantial ly the s a m e rate is possib le. Th is is known as volumetric heating. Energy is transferred through the material electro-magnetical ly, not as a thermal heat flux. Therefore, the rate of heating is not limiting and the uniformity of heat distribution is greatly improved. Heating t imes can be reduced to less than one percent of that required, using conventional techniques. S ince the beginning of the use of microwaves in chemistry, athermal effects have been suggested (Stuerga et al . , 1996). Many publications in chemistry claim speci f ic or athermal effects of microwave heating to explain results obtained (Stuerga e t a l . , 1996). 2.3.6.2 Factors Affecting Microwave Heating The molecular structure of water consis ts of an oxygen atom with a partial negative charge separated from hydrogen atoms with partial posit ive charges. This 19 forms an electric dipole. W h e n a rapidly oscil lating electric field is appl ied to a food, d ipoles in the water are reoriented with each change in the field direction. The number of dipoles and the changes induced by the electric field determine the dielectric constant £ of a food. Th is is the ratio of the capac i tance of the food to the capac i tance of air (or in s o m e c a s e s a vacuum). The var ious distortions and deformations to the molecular structure, caused by re-al ignment of the dipoles, d issipate the appl ied energy as heat. There is a delay of a fraction of a microsecond before the dipoles respond to changes in the electric field, which is termed the relaxation time. This is inf luenced by the viscosity of the food and is, therefore, dependent on temperature. For example , when water changes its state to ice, the dielectric constant falls and cont inues to dec rease as the ice is further coo led. Consequent ly , ice is more transparent to microwaves than water and f rozen foods that have a significant amount of moisture absorb energy more intensely as they thaw (Fel lows, 1987). 2.3.7 Microwave Applications 2.3.7.1 Introduction Variabil i ty in colour and lack of colour stability are major problems exper ienced with processed fruit products (de A n c o s et al . , 1999). Undesi rab le sensory and biochemical changes during handl ing, process ing and storage of fruit products result from enzymat ic browning and from non-enzymat ic react ions (Maillard mechan isms) . Development of browning or discolorat ion, off-flavours and nutritional 20 damage were attributed to the action of enzymes polyphenol ox idase and perox idase. The use of microwave energy to inactivate enzymes prior to process ing fruits and vegetables is not a common practice. The potential advantages of the use of microwave energy when compared with convent ional heat-blanching are: (1) volumetric heating resulting in a reduced temperature gradient; (2) inactivation of enzyme complexes and (3) avo idance of the leaching of vi tamins, f lavours, pigments, carbohydrates and other water-soluble components . A s wel l , extensive studies have shown equal or better retention of some vitamins ( B ^ B 2 , B 6 , C , and folic acid) after microwave heating compared with convent ional heating (Watanabe et a l . , 1998). Humans have used the p rocess of drying foods for ages . Today, however, we have the potential of drying foods more efficiently, safely and quickly. O n e dehydrat ion technology used today combines microwave heat transfer with vacuum drying (Durance, 2000). This dehydrat ion method uses mechan ica l and electromagnet ic dev ices permitting the drying of foods with less heat and oxygen damage than by air-drying (Durance, 2000). 2.3.7.2 Effects of Microwaves on Food and Enzymes After x-raying the structural and dynamical effects of microwave fields on tetragonal single crystals of hen egg-white lysozyme, We issenborn et a l . , (2005) found distinct results between using high microwave power levels and lower microwave levels. W h e r e a s high microwave power levels led to increased, but recoverable lattice defects because of the evaporat ion of crystal water, local ized 21 reproducible changes in the mean-square d isp lacements occurred at lower microwave power levels. In some c a s e s the B factors even dec reased when microwave power was increased. In this c a s e there was no ev idence of large microwave-dr iven d isp lacements of structural subunits in the protein which might be expected if microwaves were to be absorbed by protein vibrations. The effects of microwaves on protein dynamics and structure, therefore, are meagre when microwaves link non-thermally to globular proteins at functioning hydration levels (Weissenborn et al . , 2005). The relationship between protein denaturation and textural changes were investigated by Sah in et al . , (2001), by cooking an uncooked trout (Onchorhyncus mykiss) in a microwave oven using 20, 40 and 6 0 % power levels for 10, 20, 30, 40 s. A s microwave power increased, texture degradat ion was reduced because of the proteolytic enzymes . W h e n time and/or microwave power increased, proteolytic activity dec reased , indicating an increase in enzyme inactivation. Four conclus ive results were observed: 1) 6 0 % power and 20 s were the opt imum cooking condit ions, 2) proteolytic enzymes were effective especia l ly on myos in , 3) a correlation of (r-0.973) was establ ished between the variation of texture and proteolytic activity, and 4) most of the fatty ac ids remained in tact during the microwave cooking time (Sahin et a l . , 2001). In compar ison to convent ional heating methods, Y a d a v et al . , (2005) found that low-energy microwave irradiation enhanced results by up to 2.63 in a stable l ipase-cata lyzed esterification of adipic acid with var ious a lcohols. Th is result 22 occurred because of the greater recurrence of coll ision without any change in activation energy of the two heating modes (Yadav et a l . , 2005). Bohr & Bohr (2000) showed that microwave irradiation can affect kinetics of the folding process of some globular proteins, especia l ly beta-lactoglobulin depending on temperatures. At a higher temperature the denaturation of the protein from its folded state is enhanced whereas at low temperatures the folding from the cold denatured phase of the protein is enhanced . At higher temperatures a negative temperature gradient is required for the denaturation process itself, which indicates that the effects of the microwaves are nonthermal. This conclus ion supports the idea that coherent topological excitat ions can exist in proteins (Bohr & Bohr, 2000). It would appear, therefore, that the appl icat ion of microwaves is suitable for a wide range of biotechnological appl icat ions, for example, protein aggregat ion and protein synthesis and subsequent ly would have implications for biological sys tems as well . 2.3.7.3 Blanching and Microwave Blanching The browning of potatoes is due to enzymat ic activity. But this can be prevented by blanching which may a lso involve adding antioxidants such as potass ium bisulphite or ascorb ic acid (Severini et al . , 2001). Dipping the potatoes in boiling water is the most common method of blanching and is a lso used to dec rease the reducing sugar content at the surface of the product. There are severa l d isadvantages when using the boiling water blanching method such as soluble solid loss, a dec rease in f i rmness and high water consumpt ion. B lanching of potatoes has a lso been performed by s team and microwaves (Severini et a l . , 2001). 23 2.4 The Potato 2.4.1 Measuring Potato PPO Activity P P O activity in potatoes cont inues to increase throughout tuber development but is highest on a fresh weight bas is in developing tubers (Thygesen et a l . , 1995). P P O is present as a smal l mult igene family in potato and each gene has a speci f ic temporal and spatial pattern of express ion (Thygesen et al . , 1995). 2.4.2 Potato Blanching It is conceivab le that low temperature blanching of whole potatoes prior to minimal process ing may possibly be a potential treatment to control enzymat ic browning in s l iced or pre-peeled potatoes (Yemenic ioglu, 2002). Yemenic iog lu (2002) found that there was no loss in f i rmness as well as no browning on the potato peels, eyes or even infected areas when he b lanched Russe t potatoes for up to 60 min at 50° C . A s well, low temperature blanching for 45 min did not appear to cause a significant reduction in crude polyphenol ox idase activity. But increasing the time caused s o m e different results. The activity and specif ic activity of the enzyme were reduced by 27 -45% and 22 -43% respectively when the heating time was extended to 60 min (Yemenic ioglu, 2002). Slight browning on the peels and eyes of the potatoes as well as reduced f i rmness resulted when heating t ime w a s extended to 75 min. Yemenic iog lu (2002), conc luded that the browning was due to the sharp drop in the K m that caused the activation of the P P O . It is c lear that enzymat ic 24 browning cata lyzed by polyphenol ox idase is a major problem in the minimal process ing of pre-peeled or sl iced potatoes. In his study Yemen ic iog lu (2002), pointed out that sulphites have successfu l ly been used to el iminate browning but because of the adverse health effects caused by sulphites, chemica ls such as ascorb ic ac id, erythorbic acid and citric acid have been employed to control browning but are less effective. Bes ides needing less time microwave drying has the potential for producing better quality dried potatoes (Bouraoui et a l . , 1994). 25 3 HYPOTHESIS AND OBJECTIVES 3.1 Hypothesis 1. V M blanching will increase the inactivation rate of P P O in the Russet t potato puree or supernatant relative to the same temperature by conduct ive heating. 2. D -va lues for the inactivation of P P O are different when microwave heated than when conduct ion heated. 3. D-value is replaced by Activat ion Energ ies . 4. Microwave blanching is faster than s team and hot water blanching inactivating in terms of P P O in whole Russet t potato fries (shorter t ime at s a m e temperature). 3.2 Objectives 1. To measure the enzyme activities of P P O of microwave b lanched Russet t potato purees and compare them with conventional ly b lanched (Russset t potato purees) at various temperatures and t imes. 2. To measure the D-values, Z-va lues and activation energ ies of P P O of microwave and conduction heated (Russett potato trials) at var ious temperatures. 26 4. MATERIALS AND METHODS 4.1 Materials Potatoes (Solanum tuberosum L. cv. Russet t Burbank) were purchased from a local supermarket. Al l reagents were of food grade. Catecho l , a polyphenol (PP) and a common reactant for the P P O a s s a y and polyvinylpyrrol idone ( P V P P ) were purchased from the S igma Chemica l C o m p a n y (St. Louis, Mo.). P V P P was used as an agent to remove extraneous P P s from the potato samp les that might otherwise interfere with the P P O assay . Sod ium phosphate (monobasic and dibasic) was purchased from the Fisher Scientif ic C o m p a n y (Fair Lawn, N.J.). 4.2 Sample Preparation 300g of potatoes (Russett) were prepared for each blanching p rocess for apparatus 1 and 2. Potatoes were peeled, sl iced into smal l p ieces and then placed in 600mL of 0.2 M Sod ium Phosphate (Fisher Scientif ic Company , N.J.) with a pH of 6.5 (4°C), b lended in a War ing blender to a fairly uniform cons is tency and then further homogen ized using an exper imental homogenizer for 30 seconds at a speed of six. The pH was measured using a F isher Accume t Mode l 420 Digital (pH/ion) pH meter. The homogenate was then immediately centrifuged (Sorval l R C 5B 6g, Mande l Scienti f ic Company Ltd., U S A ) at 12,000 x g at 4°C, filtered through a Buchner funnel and the filtrate was used for the heat treatments. For apparatus 3, 27 whole potatoes were cut into fries (5mm wide, 5mm thick and up to 10 cm in length), (200 g) and then blanched either in hot water (steam kettle) or a microwave field either with vacuum or without vacuum (Figure 4.1). 4.3 Overview of Methods Appara tus 1: Th is apparatus (Figure 4.1) consis ted of a modif ied microwave oven (Figure 4.2) rated 700 W , 2450 M H z , within which an augmented g lass dess icator was p laced. The g lass vesse l was des igned with an internal vo lume of one litre. It was connected to a pump (Figure 4.3) allowing the circulation of the solution to enter and exit in a c losed loop (Figure 4.4). A thermocouple w a s p laced on the exterior part of the cont inuous flow loop to record the temperature on a data-logger. The temperature of the particular experiment was determined to be the average temperature readings from the data logger. P ressure changes were ach ieved by connect ing the top of the g lass chamber to an exterior vacuum pump. The previously mentioned circulating pump was adjusted to give a f low rate of 3ml_ per second . 28 G Vacuum Pump =1 ( t ) Computer Recording Temperature Sample Port 3 X Microwave Oven 1 Circulating Pump Figure 4 .1 : Schemat ic d iagram of a vacuum microwave apparatus. 29 Figure 4 .3 : The pump (Cole Parmer Instrument Company , W A , U S A ; model No. 26130). Appara tus 2: The apparatus (Figure 4.5) consisted of an E S , 2450 M H z microwave reactor (Figure 4.6 and 4.7) with a temperature/pressure control sys tem, which was operated by a Microsoft computer software program. The quartz microwave reaction vesse l was connected to a peristaltic pump (Mandel Scientif ic Company , Ltd., Be l , France; Mode l No. M312) , to allow for the filling and emptying of the g lass vesse l through Teflon tubing. A l so , the pump was used to obtain samp les during an experiment. The vesse l was des igned with a vo lume of approximately 300mL, which was sea led from the outside environment and contained a temperature recorder, cool ing finger and a safety valve. The E S (Figure 2.2.5) had a built-in magnet ic stirring dev ice that was located underneath the bottom of the g lass vesse l . By placing a magnet ic spin bar in the vesse l , efficient stirring was guaranteed and uniform heating of the sample solution was ach ieved. A schemat ic representat ion of the apparatus is shown below: o I I Microwave ' Reactor System Computer Figure 4.5: Schemat ic d iagram of the Ethos Synth microwave sys tem. 31 Figure 4.6: Front view of microwave reactor (Mi lestone Mic rowave Laboratory Sys tems, Boston, U S A ) . Figure 4.7: Top view of microwave reactor (Mi lestone Mic rowave Laboratory Sys tems, Boston, U S A ) . Appara tus 3: Parameters such as temperature, time, P P O activities and moisture content were measured for all of the blanching exper iments using the industrial s i zed V M D (Figure 4.8). Figure 4.8: V a c u u m microwave drying (VMD) machine (Enwave, Vancouve r , Canada) 4.3.1 Heat Treatments There were three heat treatments, descr ibed separately below: V a c u u m Microwave: 600 ml_ aliquots of the o .2Mphosphate buffer (pH 6.5) were circulated within a vacuum chamber as previously descr ibed (Apparatus 1) at 700 W of microwave power. The vacuum pump was adjusted to control the boiling point and equil ibrium temperature of the circulating buffer. Upon ach ievement of equil ibrium, the circulating pump was set at a flow rate of 3ml_/s. W h e n the target 33 temperature, e.g., 55°C was reached, a blank sample was taken. Then the 50ml_, 4°C sample P P O filtrate was injected into the circulating buffer through the sampl ing port. S ince the rate of water evaporat ion through the vacuum pump in the vacuum microwave w a s experimental ly measured to be 11ml_/min, 11ml_ of distil led water was injected every minute that the experiment continued so that the reactant concentrat ion levels were maintained. S a m p l e s for enzyme determination were less that 1mL each and were taken at 1 minute intervals for the duration of the experiment. Ethos Synth Microwave Reactor : A 200mL aliquot of 0 .2M phosphate buffer solution (pH 6.5) was placed in the g lass reactor vesse l of the E S microwave reactor which has a temperature/pressure control sys tem operated by a computer software program. Temperature was controlled by this program by adjusting the microwave power output of the oven. A s with the V M , when the target temperature (between 40-70°) was reached, a 20ml_, blank sample was taken, a 20mL, 4°C filtrate aliquot of P P O filtrate was pumped into the reaction vesse l through the valve at the top of the g lass vesse l . S ince there was not an exterior vacuum pump and the sys tem was c losed , the vo lume remained the s a m e throughout the experiment, except for taking out the 1ml_ samp les used for measur ing P P O activity. Water Bath: A 600mL aliquot of the 0.2M phosphate buffer (pH 6.5) was circulated within a vacuum chamber as descr ibed in Appara tus 1. The only dif ference between the V M treatment and the water bath treatment w a s that the 34 water bath treatment had the g lass reaction vesse l immersed in a water bath, whereas , the V M treatment had the vesse l contained in the microwave cavity. 4.3.2 Enzymatic Activity Determination The P P O of the heat treatment samp les (1ml_) was determined by measur ing the rate of increase in absorbance over time at 420nm using a U V - 1 6 0 visible recording spectrophotometer (Sh imadzu 24, Teksc ience Hadley, Oakvi l le , O N . , Canada ) at 25°C using a Fisher Ve rsa -Ba th water bath for 50 seconds . The assay consis ted of a 0.2M phosphate buffer, pH 6.5, 0 .10M substrate (catechol) and an enzyme samp le taken from an experimental trial. The a s s a y w a s prepared by mixing 1.25ml_ buffer, 200ul_ of substrate and then adding 50ul_ (enzyme) of the exper imental sample . 4.3.3 Kinetic Studies The thermal inactivation of enzymes , as with other chemica l react ions, has been successfu l ly descr ibed with respect to temperature dependence by the Arrhenius equat ion: K b = k b 0 e E a / R T (2) k b: rate constant at a temperature T k bo: rate constant at a reference temperature To 35 E a : activation energy (J/mol) R: gas constant (8.314 J/mol*K) T: absolute temperature (K) By graphing In k versus 1/7, the s lope can be obtained and is equal to - E a / R . Therefore, the activation energy can be calculated for speci f ic reaction. The D-value or decimal reduction time (i.e., the time required to change the concentrat ion of reactants or products by 90%) can be obtained from the semi -logarithm curve as the time taken to traverse one log cycle and is related by the equat ion below: D = 2.303/k (3) Furthermore, it can be assumed that the D value fol lows a semi- logari thmic relationship with temperature as illustrated below: Log (D:/D2) = T 2 -T i ) / z (4) where D<\ = dec imal reduction time at T i ; D 2 = dec imal reduction at T 2 , and z = 10. A l so , z is cal led the z-value. 4.3.4. Statistical Analysis A two-tailed t-test was performed between all the heat treatment D-values and s lopes from the z-value curves and activation energies. The level of conf idence required for s igni f icance was selected to be p<0.05. 36 5. RESULTS AND DISCUSSION 5.1 PPO Enzyme Assay and PPO Characteristics A general enzyme study was completed to study more extensively the P P O assay and to add to the previous work done on this enzyme. There does not s e e m to be a set standard P P O assay kit that can be found in the literature; therefore, it appeared appropriate to study a P P O a s s a y procedure in depth. Th is work includes s o m e structural and general characterist ics of the enzyme, proposed mechan ism of P P O , a long with some assay techniques and experimental assay results that can be used for the P P O assay . A l so , included here is an analys is of a journal article which examined methods to est imate total P P O activity in potatoes (Hsu et a l . , 1988). The methods selected from the P P O a s s a y analys is in this chapter were used in the kinetic analys is and pilot plant exper iments d i scussed later in this thesis. Absorpt ion spectrophotometry is used extensively in the literature to obtain P P O activities of plant material which indicates the presence of enzymat ic browning (Mui et a l . , 2002). Cont inuous spectrophotometr ic a s s a y s are widely used to determine P P O activity and some kinetic a s s a y s measure the appearance of qu inones produced by P P O enzymat ic activity (Espin et al . , 1996). S i nce there are many substrates that P P O could use to complete a browning react ion, the literature shows many different P P O assays . The majority of methods that have been used recently involve measur ing spectrophotometrical ly, a product of a P P O cata lyzed reaction. 37 The method chosen here involves catechol (a diphenol) which forms o-benzoquinone in the presence of oxygen and P P O . The o-benzoquinone compound absorbs best at 420 nm. The assay takes p lace at a temperature of 25°C and in a 0 .2M sodium phosphate buffer with a pH of 6.5. A vo lume of 25 microliters of the enzyme extract was used. T h e s e assay components were chosen for var ious reasons. Firstly, the substrate, catechol was used for the P P O a s s a y because of potato P P O ' s relatively high activity for catechol as a substrate (Whitaker, 1994). Prev ious studies which utilized potatoes to perform absorpt ion spectrophotometry P P O a s s a y s are few in number; for instance, Baruah and Swa in , 1959 and V a m o s -V igyazo et a l . , 1973 used substrates other than catechol in the P P O assay . However, catechol was used here s ince it was used in many papers such as Mui et al . , 2002, Esp in et al . , 1996 and Whitaker, 1994. A l so , P P O can cata lyze reactions with monopheno ls , but this is a smal l contribution in the potato P P O a s s a y when using a diphenol such as catechol (Whitaker, 1994). The sodium phosphate buffer is relatively standard and is used in numerous P P O a s s a y s in the literature. A pH of 6.5 w a s used s ince it is within the range of opt imum P P O activity and within the normal pH range inside the plant cel l . P rocedures previously descr ibed were used to develop a P P O a s s a y method used here fore potatoes (Hsu et al . , 1984; Mui et al . , 2002). The a s s a y included 10mM catechol as the substrate, 0.2 M sodium phosphate buffer (pH 6.5) at 25°C. The vo lumes used in the first assay consis ted of 25uL of the supernatant (enzyme), 200ul_ of catechol and 0.775mL of phosphate buffer, which added to a total assay vo lume of 1.0mL. In following assays , appropriate assay constituent vo lumes were 38 used in accordance of keeping a standard basel ine of potato P P O activity from sample variat ions from day to day. After examining the literature on the five methods descr ibed for est imation of the total activity of polyphenol ox idase ( P P O ) activity from the crude potato homogenate: 1) Polyvinylpyrrol idone ( P V P P ) absorpt ion method 2) Ammon ium sulphate precipitation method 3) Ace tone precipitation method 4) Dia lys is method and 5) S e p h a d e x G-25 chromatography, the Sephadex G-25 was shown to be the best at estimating total P P O activity (Hsu et a l . , 1988). They conc luded that this method was best at removing the endogenous phenol ic compounds in the crude homogenate, s ince oxidized phenols irreversibly inhibit P P O catalysis. T h e s e compounds should be removed because an e x c e s s in phenols may c a u s e problems in the a s s a y such as increased non-linearity. In addit ion, it has been shown that there is a rapid inactivation of P P O during the oxidation of catechol , thus the initial reaction rate is usually l inear for a very short t ime ( -30 to 90s) (Whitaker, 1994). The goal of the present microwave exper iments was to measure the relative P P O activities between different blanching treatments and not to measure the total amounts of P P O , reported earlier (Hsu et al . , 1988). The method chosen was the I P V P P absorpt ion method (Hsu et al . , 1988) and this method w a s preferable for var ious reasons. A summary of the P P O potato a s s a y s performed is included in Tab les 5.1 and 5.2. 39 Table 5.1: P P O assays of the P V P P treated crude homogenate. A s shown below, the P V P P treated crude homogenate was diluted and reaction rate was measured by change of absorbance over the change in time. E a c h enzyme dilution a s s a y was done in triplicate. For a sample graphical representat ion, p lease see figure 5.2. Reaction rate (change of ABS over time [s]) Enzyme Dilution 1 2 3 AVRG St. Dev. Multiplied by DF Undiluted 3.465E-03 3.667E-03 3.550E-03 3.561 E-03 1.014E-04 3.561 E-03 2x 1.849E-03 1.750E-03 1.787E-03 1.795E-03 5.002E-05 3.591 E-03 4x 9.643E-04 8.633E-04 9.190E-04 9.155E-04 5.059E-05 3.662E-03 5x 7.429E-04 6.194E-04 6.857E-04 6.827E-04 6.181E-05 3.413E-03 10x 3.314E-04 2.604E-04 3.000E-04 2.973E-04 3.558E-05 2.973E-03 20x 1.800E-04 8.039E-05 8.359E-05 1.147E-04 5.661 E-05 2.293E-03 Volume of undiluted Enzyme Double x2 6.074E-03 4.505E-03 4.667E-03 5.082E-03 8.629E-04 Quadruple x4 9.662E-03 7.433E-03 7.160E-03 8.085E-03 1.373E-03 Octuple x8 1.104E-02 1.061E-02 1.060E-02 1.075E-02 2.512E-04 40 Table 5.2: Tab le summariz ing reaction rate when the substrate concentrat ion is var ied. Substrate (catechol) a b c AVRG St. DEV. Concentration [M] Volume [ML] 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 Tab le 5.1, the enzyme concentrat ion was varied while the other a s s a y condit ions remained the s a m e (volumes remained unchanged) as the standard a s s a y above. The average reaction rate of the P P O was based on three separate sample measurements (triplicate). Illustrated in Tab le 5.1, the 2x, 4x and 5x dilutions showed proportional reaction rates. For example , when the enzyme concentrat ion doubles, there is a lso a doubl ing of reaction rate which showed consistent assays . I would propose that any of these dilutions could be used, but looking c losely at all these graphs, I would choose the undiluted enzyme or the 2x dilution of the enzyme for future exper iments because these graphs indicated more of a l inear enzyme reaction rate curve that the other dilutions. 41 A lso , at the bottom of Tab le 5.1, the volume of the undiluted enzyme was altered in these assays . For instance, the double (2x) had a vo lume of 50uL of the enzyme extract and therefore, 25uL less buffer. A s seen from the average reaction rate, adding double the vo lume of enzyme extract, the reaction w a s not doubled. Consequent ly , we do not see a corresponding doubl ing of reaction rate with a doubl ing of enzyme volume. The inactivation of the P P O here is not due to an instability of the enzyme because of substrate, p H , or temperature condit ions; the inactivation, most likely, is caused by product formation of o-benzoquinone and its combinat ion with P P O to form a covalent l inkage in or perhaps near the active site (Whitaker, 1994). The P V P P absorption method improves the linearity of the P P O assay (see Figure 5.2) in contrast to the crude homogenate (see Figure 5.1), which w a s not treated by any of the five methods chosen by Hsu et a l . , 1988. 42 -ABS •Linear (ABS) Figure 5.1: Samp le graphical representation of the crude potato homogenate P P O assay . This was an undiluted enzyme sample . Th is assay included 50 4L of the enzyme extract (supernatant), 0.20 ml_ of 0 .2M catechol , and 1.250 ml_ of the 0 .2M sodium phosphate buffer. The reaction took p lace at 25°C as the substrate and the buffer were kept on a water bath. The absorbance was taken at 420 nm every 5 seconds for 120 seconds . 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 crude potato homogenate P P O assay . Th is was an undiluted enzyme samp le and assay condit ions were the same as Figure 2.3.1. The linearity improved when compared to the untreated crude homogenate in Figure 5.1. 43 A s seen in Figure 5.2, this assay produces a nearly l inear assay . The r2 is favourable because of its proximity to 1.0. In other words, the r2 indicates that more than 9 9 % of the variability in A 4 2 o was accounted for by the enzyme activity. In addit ion, the time required to p rocess one crude homogenate samp le was lower for P V P P , compared to the other methods (Hsu et a l . , 1988). In addit ion, when taking into account the vo lumes in my experiment of the crude homogenate (200ml_ or more), for instance, the S e p h a d e x G-25 chromatography requires 40 minutes to process 2ml_ of the crude homogenate; multiply this by 100 and it takes approximately 66 hours to process the crude homogenate for just one experiment. Alternatively, the P V P P method requires approximately 8 hours for the s a m e sample number. Furthermore, the Ace tone and Dialysis methods were even more time consuming than the Sephadex G-25 (Hsu et al . , 1988). The only method other than the S e p h a d e x G-25 chromatography that was comparab le to the P V P P method in terms of t ime was the ammonium sulphate precipitation method, but this method routinely browned during the isolation of P P O (Hsu et al . , 1988). Next, the speci f ic activity of the P V P P treated crude homogenate did show better or similar results when compared to the other methods used to process the crude homogenate (Hsu et al . , 1988). Lastly, the P V P P absorpt ion method did reduce the phenols by 2 6 % (Hsu et al . , 1988), which seemed to contribute in the increase of linearity in the P P O a s s a y method. A s just previously ment ioned, all of the methods improved the a s s a y results as compared to not treating the crude potato extract by removing the endogenous 44 phenols. However, filtering the crude extract through P V P P s e e m e d to be appropriate for the vo lumes used in the experiment (~200mL). Us ing P V P P provided a more convenient and time saving method that a lso did not alter the composi t ion of the potato extract compound as compared to the other methods of excluding the endogenous phenols. Finally, it was determined by statistical analys is using t-tests, that 50 seconds was sufficient to character ize the P P O a s s a y (Figure 5.3). 0 10 20 30 40 50 time [s] • A B S Linear (ABS) Figure 5.3: Samp le graphical representation of the P V P P treated crude potato homogenate P P O assay . This was an undiluted enzyme sample . The first 50 seconds of each sample assay was statistically chosen to determine the s lope. A s seen in Tab le 5.2, the substrate concentrat ion was varied with a fixed concentrat ion of the enzyme (25ul_)> This data was used to est imate K m and V m a x by non-l inear regression below in Figure 5.4 which plots the velocity of the P P O 45 cata lyzed reaction versus varying substrate (catechol) concentrat ions. V m a x was calculated to be 7.8 E-03 (A of A /s ) by finding the max imum of the curve and then from this result, K m was est imated to be 0.05 M. 0.009 -i 1 Substrate [M] Figure 5.4: Shown here is the velocity of the P P O catalyzed reaction versus varying substrate (catechol) concentrat ions. The curve obtained was used to calculate V m a x and K m . 5.2 Heat Treatments with Experimental Microwave Apparatus Heat treatment duration was based on preliminary studies. P P O activity dec reased quickly with time when temperatures exceeded 60°C. Therefore, the time was kept to 20 minutes for a total of 20 samp les for exper iments above 60°C. Furthermore, not all samples were ana lyzed, if there was a total loss of P P O activity before 20 minutes. For instance, if the 7 t h and 8 t h samp les showed no P P O activities, then the rest of samp les 9-20 were not examined for P P O activities. For heat treatments below 60°C, the time of the experiment was 40 minutes, while 46 samp les were taken every 2 minutes for a total of 20 samples . Al l samp les from the heat treatments were assayed in triplicate for the P P O activity. A first order reaction was a s s u m e d s ince the P P O a s s a y reaction rate was proportional to the reactant (catechol). This was supported by the data generated in this study. The extensive literature deal ing with polyphenol ox idase (1.10.3.1 o-diphenol: oxygen oxidoreductase, hereafter referred to as ODO=o-d ipheno l oxidase) descr ibes a relatively smal l number of activity a s s a y methods. The great majority of these methods are based upon the following principles: a) Manometr ic measurement of oxygen uptake due to enzyme act ion, polarographic or potentiometric assay of the same; b) Measurement of the colour intensity of the compounds produced from the substrate by enzyme act ion; c) Indirect measurement of enzyme action by assess ing the dec rease of the amount of reducing subs tance added to the enzyme - substrate sys tem. The change in absorbance of the a s s a y mixture at 420nm was used to measure enzymat ic browning reaction rate. There were 3 different heat treatments s ince this study wanted to show first, the compar ison between a convent ional heat treatment such as hot water and a microwave heat treatment (VM and E S ) . Second ly , we wanted to show the compar ison of a constant microwave field heat treatment (VM) and a variable microwave field heat treatment (ES) . T h e browning 47 rate at each experimental temperature fol lowed first-order reaction kinetic, i.e., the log of absorbance increased apparently linearly with time. For a summary of the D-values, z-va lues and E a , and assoc ia ted precursors for the three heating methods at the s a m e temperature, see (Table 2.3.2.1). Tab le 5.3. Temperature dependence of the rate constant and activation energy for water bath, vacuum microwave and Ethos Synth microwave reactor at var ious temperatures. °C 1/°K Slope of log ABS vs. time [min1] k [min1] log k Slope of log k vs. 1/°K D value [min] Z valu e[°F] E a [kJ/m ol] Water Bath 40.2 3.167*10" J 1.27*10"* 0.0029 -2.5376 -5.88*10 J 788.9 18.58 489 42.8 3.193*1fT* 2.25*10" 3 0.0051 -2.2924 445.3 51.1 3.086*10" J 1.16*10"" 0.026 -1.5850 86.1 55.91 3.040*10"a 3.03*10" 0.069 -1.1611 33.0 59.8 3.005*10"3 3.53*10"" 0.081 -1.0915 28.3 60.99 2.994*10" 3 4.16*10"" 0.095 -1.0222 24.0 64.3 2.959*10" 3 6.84*10"" 0.157 -0.8041 14.6 VM 40.99 3.185*10"3 1.02*10"" 0.023 -1.6382 -3.12*10 3 98.2 33.89 259 49.51 3.101*10"3 1.47*10"2 0.033 -1.4814 68.2 56.65 3.034*10"3 2.02*10" 2 0.046 -1.3372 49.5 59.93 3.004*10"3 2.84*10" 2 0.065 -1.1870 35.2 67.2 2.939*10" 3 6.46*10" 2 0.148 -0.8297 15.5 ES Microwav e Reactor 40 3.195*10"3 2.50*10"" 0.033 -1.4814 -4.65*10 3 40.0 22.67 387 50 3.096*10"3 5.18*10"2 0.119 -0.9244 19.3 55 3.049*10"3 6.80*10" 2 0.156 -0.8068 14.7 60 3.003*10"3 8.67*10"2 0.199 -0.7011 11.5 65 2.959*10" 3 2.35*10"1 0.541 -0.2668 4.26 Spec i f ic D-values of these three heat treatments were obtained from the log change of absorbance over time. The negative reciprocal s lopes of the regressed straight line of log D-values at different temperatures gave the corresponding z va lues (Figure 5.5) for the three different heat treatments. 48 time [min] - * - log slope — Linear (log slope) Figure 5.5: Ethos Synth Reactor at 60°C time [min] - • - l og slope — L i n e a r (log slope) Figure 5.6: Microwave at 40°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 curves can be used to obtain z -va lues which are shown in Figure 5.8. It was evident that P P O destruction rates increased with increasing temperature. Furthermore, both the V M and the E S reactor D-value curves were significantly different from the hot water bath D-value curves (p<0.05). 50 3.5 3 4 2.5 * 0.5 A 0 -I , , , , , , 1 35 40 45 50 55 60 65 70 Temperature • EOS B microwave A hotwaterbath EOS —— "microwave - - : hotwaterbai Figure 5.8: The D-value curve of the E S reactor, the V M and the hot water bath. The z value is equal to the negative reciprocal s lope. The equat ions for each line are: E S synth reactor: y=-0.0354x+3.0628; R 2 =0.9249; z=28.5 °C, V M : y=-0.0295x+3.2678; R 2 =0.9304; z=33.89 °C; Hot water bath: y=-0.0538x=4.6339; R 2 =0.9514; z=18.58 °C. The temperature sensitivity of chemica l reactions is dependent upon the activation energy (E a ) . A s a general rule, chemica l react ions tend to go faster at higher temperatures. Increasing the temperature increases the fraction of the molecu les shar ing the E a , thus the k dec reases and reaction rate increases. In this experiment, the Arrhenius equation was used to explain the relationship between reaction rate and temperature. S ince a D-value is equal to 2.303//c, k va lues could be obtained and the Arrhenius relationship can now be explored and is shown graphical ly in Figure 5.9. 51 O.OE+00 2.90 -5.0E-01 --1.0E+00 ro -1.5E+00 -| o -2.0E+00 A -2.5E+00 -3.0E+00 E-03 • Ethos VMD Temperature [1/K] hotwaterbath Ethos VMD • hotwaterbath Figure 5.9: Arrhenius relationship for the E S reactor, the V M and the hot water bath. The s lope is used to calculate the activation energies for each treatment. The equat ions for each line are: V M : y=3.123E03x+8.224; R 2 =0.9180; E a =259 KJ /mo l , E S reactor: y=-4.658E03x+1.342E01; R 2 =0.956; E a =387 KJ /mo l , Hot water bath: y=-5.88E03x+1.66201; R 2 =0.9532; E a =489 KJ /mo l . The greater reaction rate ach ieved by the vacuum microwave sys tem could perhaps be explained by the superheat ing effect. Superheat ing occurs as the temperature of a solution in a microwave rises rapidly, teaching very high temperatures for a very short t ime interval ( -1/10 of a second) . Therefore, the reaction rate might increase quickly during those fluctuations. It w a s a lso noticed that at lower temperatures, the D-values for the V M and E S reactor were greatly reduced as compared to the hot water bath; hence, superheat ing might have been occurr ing at these temperatures. The superheat ing at these lower temperatures 52 might be caused by the difficulty of controlling the vacuum pump at lower temperatures, which led to high temperature fluctuations. Unfortunately, the superheat ing phenomenon could not be exactly recorded in this exper iment as superheat ing was very fast and difficult to measure with the exper imental apparatus used in the heat treatments. 5.3 Industrial Sized Vacuum Microwave Shown below in Table 5.4 is a summary of the blanching exper iments conducted in the Pilot Plant at The University of British Co lumb ia Food Sc ience Department. Included below in Tab le 5.4 are four f igures; one being a sample picture from one of the blanching exper iments and the other three being graphical representat ions of the parameters from the blanching exper iments. Power was kept at 71OW for the microwave s ince, in preliminary studies, this degree of power was sufficient to blanch the fires. A s indicated in Tab le 5.4, the microwave (w/o vacuum) fries blanch had activities that were relatively high and did not dec rease throughout the process . Moisture content and activity were both an average of two replications. 53 Table 5.4: The blanching of potato fries using various treatments. The temperature, moisture content (%wb), time and P P O activity are shown for each treatment. Treatment (blanch) MC [%wb] Temp. 1 [°C] Temp. 2 [°C] Average Temperature [°C] Time [min] Activity [Aabs/min @ 420nm] 76.3 45.7 46.3 46 0.5 0.2095 81.5 52.9 53.1 53 0.1686 83.2 58.2 57.8 58 1.0 0.0753 E 85.0 63.9 64.1 64 0.0852 wave vacui 88.8 68.4 69.6 69 1.5 0.0268 wave vacui 81.6 73.1 74.9 74 0.0198 2 ~ o 5 64.9 73.1 73.9 73.5 2.0 0.0077 s | 85.1 83.2 82.8 83 0.0049 80.1 76 76 76 2.5 0.0036 82.1 83.1 82.9 83 0.0000 81.6 85.1 86.9 86 3.0 0.0004 79.0 87.5 86.5 87 0.0000 84.5 38.8 39.7 39.5 0.5 0.3426 81.3 37.6 40.2 38.9 0.3612 » E 79.4 41.3 40.7 41.0 1.0 0.3350 > 1 S to 80.6 39.7 41.3 40.5 0.3395 78.5 42.8 42.2 42.5 1.5 0.3238 2 > o 79.2 41.6 42.8 42.2 0.3284 80.0 43.5 44.5 44.0 2.5 0.2649 81.4 45.9 45.2 45.5 0.2661 84.1 63.2 66.8 65 2.5 0.0709 81.5 74.6 75.4 75 0.0459 84.3 83.9 86.1 85 0.0010 87.8 64.8 65.2 65 5.0 0.0690 CP ? 80.4 76 74 75 0.0297 Hot wat Blanchii 84.7 85.8 84.2 85 0.0000 Hot wat Blanchii 83.7 63.7 66.3 65 7.5 0.0618 Hot wat Blanchii 84.4 74.2 75.8 75 0.0021 85.8 86.1 83.9 85 0.0000 80.4 65.4 64.6 65 10.0 0.0560 85.9 75.9 74.1 75 0.0019 85.1 83.8 86.2 85 0.0000 P l e a s e see Figure 5.10 for a flowchart of these exper iments shown in Tab le 5.4. 54 Hot water blanch Potatoes Peel Sl i ce Weigh 200g for treatments Microwave (71 OW) no vacuum vacuum Measure P P O activity and moisture Figure 5.10: Microwave potato fries. More data was col lected for the microwave (w/o the vacuum), s ince the temperatures for the microwave (w the vacuum) were too low for the amount of time the fries were b lanched. Moreover , an experiment with the microwave that ran for 2.5 minutes included five data points; each of these points represented a separate 200g batch. 55 This was the c a s e s ince the temperature could not be measured internally by the microwave and thus, the microwave had to be stopped and the temperature recording was taken with an infrared thermometer. For a graphical representation, p lease see Figures 5.11 and 5.12. a 5 time [min] —A Temperature 1 x~ Temperature 2 M C 1 — o — M C 2 • Activity 1 -a Activity 2 Figure 5.11: Graphica l representation of the microwave (w/o vacuum) blanch data from Tab le 4 .1 . 56 90 0.40 T e m p e r a t u r e 1 1 1.5 t ime [min] • H x - T e m p e r a t u r e 2 X M C 1 Activity 1 Activity 2 Figure 5.12: Graph ica l representation of the microwave (w/ vacuum) b lanch data from Tab le 4 .1 . The samples moisture content dec reases slightly, which can be attributed to the increasing temperature. The enzyme 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: Graphica l representation of the Hot water blanch data from Tab le 4 .1 . The moisture content change var ies slightly between runs but does not show any consistent trend. The temperature does not change over time in all three runs. The enzyme activity dec reases at different degrees for each temperature level, but it dec reases distinctly. 57 6. CONCLUSIONS It s e e m s presently fitting to use the electromagnet ic spectrum (EM) to ach ieve one of the main goals in this thesis - the food process ing of potatoes, in this case , using microwaves in the process of blanching to stop food deterioration. M ic rowaves are a natural phenomenon as wi tnessed by the blanketing of microwave radiation of earth from all directions in space . W e are, in fact, deal ing with the natural phenomenon that the E M spectrum is with wavelengths that are significantly larger than that of visible light. W e are utilizing a different portion of the E M spectrum than what our ancestors used to process food. Most importantly, we can utilize the benefits that microwaves have to offer within the food arena. To provide a c loser observat ion of microwave blanching, with and without vacuum, it w a s necessary to cons ider the wealth of information in ex is tence regarding the examinat ion of polyphenol ox idase ( P P O ) , which is cons idered one of the major factors of enzymat ic browning. First of al l , the exper iments performed in the Pilot Plant in the Department of Food Sc ience , were practical in terms of consumer wants and needs. The Russet t Backer 's potato, formed in the shape of fries, as expected to be eaten by consumers , g ives immediate and viable applicability. Here, we have the opportunity of examining the potato more in its native form, not chemical ly disturbed or re-formed into a new potato product. Furthermore, by using the microwave to process fries, a blanching method or application can be found and utilized in future installations. 58 After observing the macroscop ic effects of microwave blanching in the Pilot Plant, a c loser look/step was undertaken to study the microwave effect, particularly in a vacuum, environment using a unique cont inuous flow method for the vacuum microwave (VM) and hot water bath. A l so , a third apparatus (Microwave Reactor) no vacuum - was used. More specif ical ly, the chemica l kinetics of P P O was studied in this novel microwave setting to initially investigate thermal and potentially non-thermal effects that microwaves could p o s s e s s against a biological component such as an enzyme. The potato was once again used in this microwave 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 was selected for several reasons: firstly, the puree, which was previously blended and homogenized with a phosphate buffer, w a s then centrifuged and subsequent ly the supernatant was filtered with ( P V P P ) , being fairly mobi le. In other words, the viscosity of this liquid was low and could easi ly move throughout the V M apparatus. Second ly , the literature attests to the difficulty of purifying P P O , let alone obtaining enough of a relatively purified sample to use in the three exper imental dev ices. In addit ion, purchasing the pure form of P P O was too expens ive to provide all the data necessary to obtain kinetic curves from the three apparatus. The P P O kinetics, destruction va lues, (D-values), Z -va lues and activation energies (E A ) were compared using microwaves in a vacuum, hot water in a vacuum, and variable microwaves without a vacuum. Moreover , s ince P P O destruction does not usually occur at temperatures below 50°C, it was cons idered that the vacuum microwave could be used below these temperatures, down to approximately 40°C thereby, hopefully illustrating a non-thermal effect. 59 Consequent ly , it was profoundly difficult to measure an isolated athermal microwave effect on the enzyme P P O . At the s a m e time, it was dec ided that a more in-depth v iew w a s required regarding P P O , which would include a c loser examinat ion at the P P O assay , which is subsequent ly intended to measure the amount of P P O in a particular sample . A lso , P P O had to be purified to a greater extend. P V P P (mentioned above) was chosen for use s ince it seemed the most viable and logical treatment. In addition, Kmax and V m a x were experimental ly obtained to increase the amount of kinetics data on P P O . 60 7. R E F E R E N C E S Alme ida , M . E . M . and Nogueira, J . N . 1995. 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