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Kinetics of Dissolved Oxygen Consumption and Deoxygenation of Pineapple Juice and Model Solutions Using a Thin Film Enzy...

Permanent Link: http://ufdc.ufl.edu/UFE0041798/00001

Material Information

Title: Kinetics of Dissolved Oxygen Consumption and Deoxygenation of Pineapple Juice and Model Solutions Using a Thin Film Enzyme Reactor
Physical Description: 1 online resource (68 p.)
Language: english
Creator: Ponagandla, Narsi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ascorbicacid, catalase, dissolvedoxygen, enzymereactor, glucoseoxidase, immobilization
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Dissolved oxygen (DO) affects the quality of fruit juices by reacting with ascorbic acid and flavor components. In this study experiments were run at 5.0, 13.0, 21.5, 30.5 or 40.0 degreeC for both DO measurement and Ascorbic acid (AA) measurement. First order, second order and 0.5 order kinetic mechanisms were considered. First order model fit better initial consumption of DO in AA solutions and pineapple juices. First order reaction rate constants ranged between 1.03 x 10-5 to 2.97 x 10-4 s-1 for 5masculine ordinalC and 40 masculine ordinalC, respectively for 28 mM AA solutions and 9.58 x 10-6 to 1.08 x 10-4 s-1 for 13masculine ordinalC and 40 masculine ordinalC, respectively for 2.8 mM AA solutions and between 8.89 x 10-6 to 4.75 x 10-5 s-1 for 13masculine ordinalC and 40 masculine ordinalC, respectively for pineapple juices. In both AA solutions and pineapple juices the reaction rate of DO consumption followed Arrhenius behavior. The activation energy (Ea) for DO consumption was calculated as 67.7 plus or minus 4.2, 67.12 plus or minus 6.72 and 48.62 plus or minus 2.6 kJ?mol-1?K-1 for 28 mM, 2.8 mM AA solutions and pineapple juices respectively. Temperature did not show a significant difference in decrease in the rate of AA degradation between 13masculine ordinalC and 40masculine ordinalC. An enzyme reactor consisting of Glucose oxidase - Catalase complex immobilized in electrochemically generated poly-o-phenylenediamine thin films deposited on the interior wall of a platinized platinum tube was proposed for deoxygenation of fruit juices. Reactors with GOx concentrations of 10 and 25 g L-1 showed better operational stability compared to 50 g L-1 where film detachment was observed. Increasing the retention time from 127.4 s to 382.4 s increased the % of DO removal from 63.6% to 91.6% in a 12-cm reactor. The effect of pH on reactor performance is negligible. We did not observe any effect of catalase (equivalent to two times GOx activity) on the performance of reactor. However, when increasing the catalase concentration from two times to five times the activity of GOx we did not observe any decrease in oxygen concentration. In both model solutions and pineapple juices approximately 90% of the DO was removed using a 12-cm enzyme reactor at a flow rate of 0.025 mL min-1 with a retention time of 6.4 min.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Narsi Ponagandla.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Reyes De Corcuera, Jose Ignacio.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041798:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041798/00001

Material Information

Title: Kinetics of Dissolved Oxygen Consumption and Deoxygenation of Pineapple Juice and Model Solutions Using a Thin Film Enzyme Reactor
Physical Description: 1 online resource (68 p.)
Language: english
Creator: Ponagandla, Narsi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ascorbicacid, catalase, dissolvedoxygen, enzymereactor, glucoseoxidase, immobilization
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Dissolved oxygen (DO) affects the quality of fruit juices by reacting with ascorbic acid and flavor components. In this study experiments were run at 5.0, 13.0, 21.5, 30.5 or 40.0 degreeC for both DO measurement and Ascorbic acid (AA) measurement. First order, second order and 0.5 order kinetic mechanisms were considered. First order model fit better initial consumption of DO in AA solutions and pineapple juices. First order reaction rate constants ranged between 1.03 x 10-5 to 2.97 x 10-4 s-1 for 5masculine ordinalC and 40 masculine ordinalC, respectively for 28 mM AA solutions and 9.58 x 10-6 to 1.08 x 10-4 s-1 for 13masculine ordinalC and 40 masculine ordinalC, respectively for 2.8 mM AA solutions and between 8.89 x 10-6 to 4.75 x 10-5 s-1 for 13masculine ordinalC and 40 masculine ordinalC, respectively for pineapple juices. In both AA solutions and pineapple juices the reaction rate of DO consumption followed Arrhenius behavior. The activation energy (Ea) for DO consumption was calculated as 67.7 plus or minus 4.2, 67.12 plus or minus 6.72 and 48.62 plus or minus 2.6 kJ?mol-1?K-1 for 28 mM, 2.8 mM AA solutions and pineapple juices respectively. Temperature did not show a significant difference in decrease in the rate of AA degradation between 13masculine ordinalC and 40masculine ordinalC. An enzyme reactor consisting of Glucose oxidase - Catalase complex immobilized in electrochemically generated poly-o-phenylenediamine thin films deposited on the interior wall of a platinized platinum tube was proposed for deoxygenation of fruit juices. Reactors with GOx concentrations of 10 and 25 g L-1 showed better operational stability compared to 50 g L-1 where film detachment was observed. Increasing the retention time from 127.4 s to 382.4 s increased the % of DO removal from 63.6% to 91.6% in a 12-cm reactor. The effect of pH on reactor performance is negligible. We did not observe any effect of catalase (equivalent to two times GOx activity) on the performance of reactor. However, when increasing the catalase concentration from two times to five times the activity of GOx we did not observe any decrease in oxygen concentration. In both model solutions and pineapple juices approximately 90% of the DO was removed using a 12-cm enzyme reactor at a flow rate of 0.025 mL min-1 with a retention time of 6.4 min.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Narsi Ponagandla.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Reyes De Corcuera, Jose Ignacio.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041798:00001


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1 KINETICS OF DISSOLVED OXYGEN CONSUMPTION AND DEOXYGENATION OF PINEAPPLE JUICE AND MODEL SOLUTIONS USING A THIN FILM ENZYME REACTOR By NARSI REDDY PONAGANDLA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FL ORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 N arsi Reddy P onagandla

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3 ACKNOWLEDGMENTS I would like to thank few people who were instrumental in completing this thesis success fully. Firstly, I would like to thank Dr. Jose Reyes for giving me the opportunity to work in his lab and for his valuable suggestions, encouragement and supervision. I would like thank Shelley Jones for helping me in reactor fabrication, my lab group Rosa lia Garcia, Michael Eisenmenger and Juan M anu e l for their suggestions I also would like to thank my committee members for their valuable suggestions Finally, I thank my mother and sisters for their relentless support

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Solubility of Oxygen ................................ ................................ ................................ 12 Effect of Dissolved Oxygen on Fruit Juices ................................ ............................. 12 Methods of Deaeration ................................ ................................ ............................ 14 Vacuum Deaeration ................................ ................................ .......................... 15 Gas Sparging ................................ ................................ ................................ ... 15 Membrane Deaerators ................................ ................................ ..................... 15 Enzymatic Deaeration ................................ ................................ ...................... 16 Immobilization of Enzymes ................................ ................................ ..................... 18 Limitations of Immobilization ................................ ................................ ............ 19 Applications of Immobilized Enzymes ................................ .............................. 19 Methods of Immobilization ................................ ................................ ...................... 20 Adsorption ................................ ................................ ................................ ........ 20 Covalent Bonding ................................ ................................ ............................. 20 Gel Entrapment ................................ ................................ ................................ 21 Polymer Entrapment ................................ ................................ ......................... 21 Encapsulation ................................ ................................ ................................ ... 22 Immobil ized Enzyme Reactors ................................ ................................ ............... 22 Packed Bed Reactor (PBR) ................................ ................................ .............. 22 Fluidized Bed Reactor (FBR) ................................ ................................ ............ 23 Measurement of Dissolved Oxygen ................................ ................................ ........ 25 Introduction ................................ ................................ ................................ ....... 25 Fiber Optic Oxygen Sensors ................................ ................................ ............ 26 Calibration ................................ ................................ ................................ ........ 27 Current Research ................................ ................................ ............................. 28 Advantages ................................ ................................ ................................ ...... 31 Limitations ................................ ................................ ................................ ........ 31 Gap in Knowledge ................................ ................................ ................................ .. 32 Objectives ................................ ................................ ................................ ............... 33 2 KINETICS OF DISSOLVED OXYGEN CONSUMPTION IN ASCORBIC ACID SOLUTIONS AND PINEAPPLE JUICES ................................ ................................ 34 Introduction ................................ ................................ ................................ ............. 34 Materials an d Methods ................................ ................................ ............................ 35

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5 DO Measurement ................................ ................................ ............................. 36 AA Measurement ................................ ................................ .............................. 36 Results and Dis cussion ................................ ................................ ........................... 37 Kinetics of DO Consumption in 28 mM, 2.8 mM AA Solutions and Pineapple Juices ................................ ................................ ................................ ............ 37 Measurement of Ascorbic Acid in 0.05 % AA Solutions and Pineapple Juices .. 44 Conclusions ................................ ................................ ................................ ............ 46 3 THIN FILM ENZYME REACTOR FOR DEOXYGENATION OF MODEL SOLUTIONS AND PINEAP PLE JUICE ................................ ................................ .. 48 Introduction ................................ ................................ ................................ ............. 48 Materials and Methods ................................ ................................ ............................ 49 Reactor Cl eaning ................................ ................................ .............................. 50 Reactor Platinization ................................ ................................ ........................ 50 Reactor Polymerization ................................ ................................ .................... 50 Mod el Orange Juice Solution ................................ ................................ ........... 51 Pineapple Juice Preparation ................................ ................................ ............. 51 Testing the Reactor ................................ ................................ .......................... 52 Measurement of Dissolved Oxygen ................................ ................................ .. 52 Results and Discussion ................................ ................................ ........................... 53 Effect of Enzyme Concentration ................................ ................................ ....... 53 Effect of Glucose Concentration ................................ ................................ ....... 55 Effect of Retention Time ................................ ................................ ................... 56 Reactor l ength ................................ ................................ ............................ 56 Flow r ate ................................ ................................ ................................ .... 57 Effect of pH ................................ ................................ ................................ ....... 58 Testing with Model Solutions and Fruit Juices ................................ .................. 59 Conclusions ................................ ................................ ................................ ............ 60 Over all Conclusions ................................ ................................ ............................... 61 Future Wo rk ................................ ................................ ................................ ............ 61 LIST OF REFERENCES ................................ ................................ ............................... 63 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 68

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6 LIST OF TABLES Table page 1 1 Immobilized enzyme reactors. ................................ ................................ ............ 24 1 2 Luminescence properties of oxygen sensitive dyes immobilized in sol gel matrixes. ................................ ................................ ................................ ............. 31 1 3 Comparison of different methods used to measure DO ................................ ...... 32 2 1 First order rate constants for dissolved oxygen (DO) consumption in 28 mM an d 2.8 mM ascorbic acid solutions ................................ ................................ .... 40 2 2 First order rate constants for dissolved oxygen (DO) consumption in pineapple juices. ................................ ................................ ................................ 43 3 1 Performance of 3 cm reactors at selected GOx concentrations in model orange juice solution determined as [DO] at the exit of the reactor .................... 54 3 2 Effect of flow rate on the performance of 2 5 g L 1 GOx reactor in 166 mM glucose solutions. ................................ ................................ ............................... 58 3 3 Effect of pH on the performance of 3 cm (GOx Cat) reactor in model orange juice solutions at a flow rate of 0.025 mL min 1 ................................ .................. 59 3 4 Performance of 12 cm (25 g L 1 GOx 2x Cat) reactor with model orange juice solution, glucose solution and pineapple juice ................................ .................... 60

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7 LIST OF FIGURES Figure page 1 1 Schematic diagram of an optical oxygen sensor ................................ ................ 26 1 2 Chemical structure of PtTFPP ................................ ................................ ............ 30 1 3 Chemical structure of PtOEP ................................ ................................ .............. 3 0 2 1 Dissolved oxygen measurement setup. ................................ .............................. 36 2 2 First order kinetic s of 2.8 mM AA solutions at 21.5 C. ................................ ........ 37 2 3 Second order kinetics of 2.8 mM AA solutions at 21.5 C ................................ ... 38 2 4 Half order kinetics of 2.8 mM AA solutions at 21.5 C ................................ ......... 38 2 5 Effect of temperature on first order rate constants of dissolved oxygen consumption in 28 mM ascorbic acid solutions. ................................ .................. 41 2 6 Arrhenius plot for rate constant of dissolved oxygen consumption in 28 mM ascorbic acid solutions ................................ ................................ ....................... 41 2 7 Effect of temperature on first order rate constants o f dissolved oxygen consumption in 2.8 mM ascorbic acid solutions. ................................ ................. 42 2 8 Arrhenius plot for rate constant of dissolved oxygen consumption in 2.8 mM ascorbic acid solutions ................................ ................................ ....................... 42 2 9 Effect of temperature on first order rate constants of dissolved oxygen consumption in pineapple juices. ................................ ................................ ........ 43 2 10 Arrhenius plot for rate cons tant of dissolved oxygen consumption in pineapple juices ................................ ................................ ................................ .. 44 2 11 Concentration of AA in 0.05% AA solutions at four different temperatures. ........ 45 2 12 Concentration of AA in pineapple juices at four different temperatures .............. 46 3 1 Enzyme immobilization setup. ................................ ................................ ............ 51 3 2 Setup for testing the reactor ................................ ................................ ............... 52 3 3 Effect of GOx concentration and glucose concentration on the amperometric response of 3 cm GOx reactors at a flow rate of 0.5 mL min 1 .......................... 55 3 4 Effect of glucose concentration on the amperometric response of a 3 cm, 25 g L 1 GOx reactor at a flow rate of 0.1 mL min 1 ................................ ............... 56

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8 3 5 Effect of reactor length on the performance of (25 g L 1 GOx 2X cat) reactors in model orange juice solution at a flow rate of 0.025 mL min 1 .......................... 57

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9 LIST OF ABBREVIATIONS DO Dissolved oxygen GOx Glucose oxidase GOx Cat Gl ucose oxidase C atalase L AA L Ascorbic acid PPD poly o phenylenediamine PBD Packed bed reactor

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Sci ence KINETICS OF DISSOLVED O XYGEN AND DEOXYGENATION OF PINEAPPLE JUICE AND MODEL SOLUTIONS USING A THIN FILM ENZYME REACTOR By N arsi Reddy P onagandla May 20 10 Chair: Jos I. Reyes De Corcuera Major: F ood S cience and Human Nutrition Dissolved oxygen (D O) affects the quality of fruit juices by reacting with ascorbic acid and flavor components. In this study e xperiments were run at 5 .0, 13.0, 21.5, 30.5 or 40.0 C for both DO measurement and Ascorbic acid (AA) measurement First order, second order and 0. 5 order kinetic me chanisms were considered. First order model fit better initial consumption of DO in AA solutions and pineapple juices. First order reaction rate constants ranged between 1.03 x 10 5 to 2.97 x 10 4 s 1 for 5 C and 40 C, respectively for 2 8 mM AA solutions and 9.58 x 10 6 to 1.08 x 10 4 s 1 for 13 C and 40 C, respectively for 2.8 mM AA solutions and between 8.89 x 10 6 to 4.75 x 10 5 s 1 for 13 C and 40 C, respectively for pineapple juices. In both AA solutions and pineapple juices the re action rate of DO consumption followed Arrhenius behavior. The activation energy ( E a ) for DO consumption w as calculated as 67.7 4.2 67. 12 6.72 and 48.62 2.6 1 1 for 28 mM, 2.8 mM AA solutions a nd pineapple juices respectively. Temperature did not show a significant difference in decrease in the rate of AA degradation between 13 C and 40 C. An enzyme reactor consist ing of Glucose oxidase Cat alase co mplex immobilized in electrochemically generated poly o phenylenediamine thin films deposited on the

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11 interior wall of a platinized platinum tube was proposed for deoxygenation of fruit juices Reactors with GOx concentrations of 10 and 25 g L 1 showed bet ter operational stability compared to 50 g L 1 where film detachment was observed. Increasing t he retention time from 127.4 s to 382.4 s increased the % of DO removal from 63.6 % to 91.6% in a 12 cm reactor. The effect of pH on reactor performance is neglig ible. W e did not observe any effect of catalase ( equivalent to two times GOx activity ) on the performance of reactor. However, when increasing the catalase concentration from two times to five times the activity of GOx we did not observe any decrease in o xygen concentration. In both model solutions and pineapple juice s approximately 90% of the DO was removed using a 12 cm enzyme reactor at a flow rate of 0.025 mL min 1 with a retention time of 6.4 min

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12 CHAPTER 1 INTRODUCTION Dissolv ed oxygen in the fruit juices causes number of problems. Dispersed oxygen is eas ier to remove than dissolved oxygen The most stable and abundant form of oxyg en is the molecular or diatomic triplet oxygen. Solubility of O xygen The solubility of oxygen in a solution is mainly dependent on three factors ( t emperature, p ressure and solutes concentration). The solubility decreases with increasing temperature, decreasing partia l pressure and with increase in solute concentration. The solubility of a gas dissolved in a liquid is described it states that the concentration of a gas dissolved in a liquid is proportional to the partial pressure of the gas in the vapor phase : (1 1) w here X A P A and H(T) are mole fraction of gas A in the liquid phase, partial (Gill and Menneer 1997) Effect of Dissolved O xygen on Fruit J uices Dissolved oxygen plays a vital ro le in the deterioration reactions of citrus products such as degradation of ascorbic acid and thus causes the non enzymatic browning of juices during the storage which results in the formation of off flavors (Rassis and Saguy 1995) Degr adation of ascorbic acid is one of the major causes of color and quality changes during storage Falade and others (2004) reported 16.25 % and 16.67 % ascorbic ac id loss in sweetened Julie and O gbomoso mango juices at 25 C after 12

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13 weeks. The loss of vitamin C in sweetened mango juices was attributed to both aerobic and an aerobic reactions. Rate of a scorbic acid degradation during storage increased with increasing temperature, presence of copper, iron and alkali, increased light exposure (Mack and others 1976; Singh and others 1976) and differences in pH. Difference in pH effect the number of ascorbic acid oxidation steps (1 or 2 steps). A s ynergistic relationship resulting in increased ascorbic acid degradation was reported between light and temperature. However, some contradicting data has been reported about the rate of ascorbic acid degradation in the literature Singh and others (1976) reported ascorbic acid degradation in liquid food as second order reaction. Mack and others (1976) reported oxygen uptake as a first order rate during the first 24 h in the same solution. Various studies on model solutions and citrus juices reported ascorb ic acid degradation as first order, zero order and second order kinetics; these contradictions in the results are due to the variations in the concentrations of DO. Garcia Torres and others (2009) reviewed t he interaction of dissolved oxygen consumpti on with various food components and reported that direct aerobic oxidation of L AA indirectly affects color and aroma profile. L ascorbic acid (L AA) degradation occurs by two consecutive or parallel pathways a erobic and anaerobic (Kennedy and others 1992; Johnson and others 1995; Manso and others 2001b; Baiano and others 2004) In the aerobic pathway, ascorbic acid in aqueous solution is easily oxidizes t o mono dehydroascorbate (MDHA) also called ascorbate free radical. MDHA can be reduce d back to L AA or two MDHA molecules can produce L AA and DHA. Dehydroascorbic acid (DHA) is unstable and

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14 undergoes irreversible hydrolytic ring cleavage to produce 2, 3 d iketogulonic acid in aqueous solution (E quation 1 2 and 1 3 ). (1 2 ) ( 1 3 ) Under anaerobic conditions, L A scorbic acid is degraded to furfural and rate is slower than aerobic degradation (Baiano and others 2004) Methods of Deaeration To prevent the deleterious effects due to DO fruit juices are often deaerated prior to pasteurization. Deoxygenation is accomplished by vacuum flash deaeration, gas sparging, oxygen scavengers, membrane deaerators and adding antioxidants using enzymes. Vacuum deaeration is the most commonly used method in the citrus industry. However, in this method essential oil and flavor compounds ar e also removed along with oxygen ( Braddock 1999) Gas sparging consists of displacing the DO with another gas such as nitrogen or carbon dioxide. Early studies on methods of deaeration indicate that in orange juice 99% of DO was removed using nit rogen sparging compared to 77% DO removed b y vacuum deaeration (Joslyn 1961) but significant amount of volatiles are removed in this process too Jordan and others (2003a) reported that the greatest losses in concentration of volatiles occurred during industrial deaeration. Membrane deaerators are most commonly used for water and waste water treatment and some other food products, but their application to fruit juices is limited (Cole and Genetell.Ej 1970) which may be due to the interference of pulp with the membrane. Garcia Torres and others (200 9) reviewed different deaeration methods and reported that vacuum H 2 0

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15 deaeration and gas sparging suitable to remove oxygen in fruit juices but they also removed important volatile aroma compounds. Vacuum D eaeration Vacuum deaeration is based on the reduction of pressure of the gas above the juice. Vacuum flash deaeration is the most commonly used method in the citrus industry. In this process, the juice is preheated to 50 60C and sprayed inside a vacuum vessel, where the juice will flash (boil). Oxygen and v olatile compounds are separated from the juice. The pressure in the vacuum chamber and the inlet temperature are adjusted so that the inlet temperature is 2 5C above the boiling p oint of the orange juice at that pressure (Ringblom 2004) Gas S parging Gas sparging or bubbling consists of displacing the dissolved oxygen with ano ther gas such as nitrogen, helium or carbon dioxide. The partial pressure of oxygen in the vapor phase is reduced by displacing the oxygen with another gas such as N 2 or CO 2 The gas can be bubbled into the liquid, meet in countercurrent with the liquid, o r the liquid can be sprayed into a vessel filled with gas. The height of the chamber, size of bubbles of gas and flow rates of gas and liquid determine the rate and extent of oxygen removal. The disadvantage of this deaeration process is that like vacuum dea e ration, it also remove s flavor volatile compo unds (Jordan and others 2003b) For this reason new techniques such as membrane and enzymatic deaerat ion are being developed Membrane D eaerato rs Membrane deaerators are widely used to remove oxygen from water, beer, wine and other particle free product Membrane deaer a tors consist of several hollow membrane fibers knitted into a fabric and wrapped around a cente r tube. L iquid flows

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16 into the cent er tube and is forced to pass radially through the membrane, vac uum or a swept gas are applied in order to remove the oxygen from the liquid. Cole and Genetell.Ej (1970) repor ted that 96% of DO was removed from water using hollow fiber membranes. Jiahui and others (2008) developed a hollow fiber m embrane system that removes DO in boiler feed water. The membrane was made using hydrophobic polypropylene The outer diameter of fiber was 300 m and the thickness was 100 m. The efficiency of deoxygenation decreased after a lo ng period of time T his was attributed to the membrane fouling which occurred due to organic matter and aluminum silicate in the feed water. There is a large body of research available on membrane deaerators and the ir application to water and pieces of equ ipment are commercially available. However, the use of these deaerators in the fruit juices was not successful This may be due to the interference of pulp with the membrane. Enzymatic D eaeration Glucose oxidase ( GO x EC: 1.13.4), which was discovered in 1928 by M uller in Aspergillus niger and P en i cillium glaucum D glucose to D glucono 1,5 lactone and hydrogen peroxide in aerobic conditions as shown in E quation 1 4 Catalase (Cat EC: 1.11.1.6 ) decomposes hydrogen peroxide t o water and oxygen as shown in E quation 1 5. 2C 6 H 12 O 6 + 2O 2 + 2H 2 O 2C 6 H 12 O 7 + 2H 2 O 2 ( 1 4 ) ( 1 5 ) Enzymatic deaeration of juices using GOx Cat in solu tion has been reported by (Sagi and Mannheim 1990; Parpinello and others 2002) F or each mole of oxidized glucose the enzym atic method removes half a mole of oxygen (Sagi and Mannheim glucose oxidase Catalase

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17 1990) The advantage of this system is that glucose is present in large excess with respect to DO in juices so there is no need to add any substrate. Some other commercial applications using GOx Cat i n food products include d esugaring of eggs, p rodu ction of reduced alcohol wine and p revention of browning in apple and pear purees. GOx Cat has been used to remove glucose from egg white during the commercial preparation of egg white powder in order to pr event non enzymatic browning during processing and storage (Sankaran and others 1989) GOx 92.3 g t 1 at 25 30 C, pH 5.5 7 was able to remove 95% of glucose from egg white and effectively suppressed the M aillard reac tion during processing and storage. They also found that the desugarized egg white maintained its initial functional characteristics with better smell and mobility than untreated egg white. Pickering and others (1998 ) observed low pH of grape juice was a limiting factor in the production of reduced alcohol wine using GOx Cat. Optimizing the process resulted in 87% conversion of glucose to D gluconic acid and was achieved by raising the pH of juice by adding calcium c arbonate. Parpinello and others (2002) studied use of GOx Cat sys tem to prevent non enzymatic browning in apple and pear purees and found that GOx was able to remove 99% oxygen in apple and pear purees which helped in preventing oxidation and browning. They also observed that the ascorbic acid prevented browning to a l arger extent than any other chemical. Ascorbic acid enhanced the activity of GOx Cat system in preventing browning in apple juice, suggesting a synergistic effect. Grape juice treated with GOx Cat and subjected HHP at 600 M P a improved the sensory propertie s and also helped in c olor stabilization of juice (Castellari

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18 and others 2000) Ough (1975) reported that GOX C at effectively removed DO from white table and rose wines. Hydrogen peroxide was reduced by sulfur dioxide rather than catalase Immobilization of E nzymes Advantages of Immobilized E nzymes Enzymes are catal ysts that increase the rate of reaction without being unchanged. They do this by lowering the activation energy of reactions. Although the cost of major processing enzymes has decreased considerably, enzyme costs are still important in the food and pharmac eutical industries E nzyme s can be chemically or physically immobilized to an insoluble support so that it can be physically reclaimed from the reaction medium thus decreasing enzyme costs Like all proteins enzymes are affected by temperature, pH and inhi bitors. Immobilization often mitigates such effects increasing the reusability of enzymes and further decreasing operation costs The first type immobilization technique based on adsorption was developed by Nelson and Griffin in 1916 After that several d ifferent immobilization techniques were developed. Up to now more than 5 000 publications and patents are available on different enzyme immobilization techniques. Different supports and techniques can be used for immobilization of enzymes One of the imp ortant property is t he support material should have high affinity for proteins. Availability of reactive functional groups for direct reaction with proteins or for chemical modifications and non toxicity (Agullo and others 2003) The selection of suitable enzymatic preparation depends on enzyme properties and the purpose of its application. galactosidases are generally used for the hydrolysis of lactose in milk and sweet whey w galactosidases are used for acidic whey

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19 hydrolysis because fungal enzymes are more t hermostable than yeast enzymes The supports and techniques are chosen in such way that maximum enzyme activity, stab ilit y and durability are achieved Lim itations of I mmobilization Immobilization of enzyme has several advantages. However, some limitations are also associated with immobilization. After immobilization e nzymes are restricted in movement so d ecrease of enzy me activity has been reported compared free enzymes However, the extent of decrease depends mainly on the immobilization method and the source of enzyme. One of the easiest ways of immobilizing enzymes is through adsorption however, the main limitation is l eakage or desorption of the enzyme f rom the matrix Applications of Immobilized E nzymes There are numerous applications available using the immobilization of enzymes. Immobilized enzymes are widely used in the different fields. On e of the most important application s of immobilized enzymes is in the p roduction of high fructose corn syrup. Production of cheese using immobilized enzymes has been considered as promising step for the rationale use of rennin Grosova and others (2008) reported application of i mmo gala ctosidase in the hydr olysis of milk and whey lactose. Baticz and Tomoskozi (2002) developed an immobilized cholesterol oxidase reactor for d etermination of tot al c holesterol content in foods Parpinello and others (2002) Pickering and others (1998) reported different app lications immobilized glucose oxidase catalase system for d esugaring of eggs to prevent non enzymatic browning, p ro duction of reduced alcohol wine and p revention of browning in apple and pear purees. Immobilized enzyme biosensors are tested for d etermina tion of Ascorbic acid in human

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20 serum using ascorbic oxidase immobilized in poly o phenylenediamine film. There are some applications using immobilized enzymes in active packaging. Kothapalli and others (2007) studied GOx in low density poly ethylene using UV polymerization M ethods of I mmobilization Several methods of enzyme immobiliz ation have been developed aiming at prevent ing loss of enzyme activity and maximizing activity A good understanding of the active site of the enzyme is often critical in the selection of the met hod of immobilization Several immobilization techniques are avail able based on the application. These include a dsorption covalent bonding by tethering or cross linking e ncapsulation, and entrapment in gels or polymer s Adsorption Adsorption is the simplest and oldest way of immobilizing the enzyme. The e nzyme is mixed with the support material under appropriate conditions and is adsorbed onto the support material usually through hydrogen bonding hydrophobic interactions; Several support materials are available for immobilization like cellulose, collagen, calcium carbonate etc. However this type of immobilization is dependent on experimental conditions like pH, temperature, ionic strength. Therefore, changes in these conditions can result in enzyme desorp tion of the support material. For example, i f ion ion inter action is predominant with very little hydrogen bonding ionic strength changes then a small shift in pH could result in the desorption of the enzyme. Covalent B onding This is one of the most intensely studied immobilization technique. Here formation of co valent bond between enzyme and the support matrix takes place. Typically enzyme s covalent ly bind to a re active group on the support material (typically amino,

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21 carboxy or hydroxy derivatized surfaces) through the reactive groups on side chains of its amino acids or with terminal amino and carboxyl groups of the polypeptide chains. Also, i nter molecular cross linking of enzymes by bi functional or multi functional reagents have been widely used The most common reagent used for cross linking is gluteraldehyd e C holesterol oxidase was immobilized on amino modified Kieselgel 100 polymer beads by cross linking with gluter aldehyde for the determination of total cholesterol in food by flow injection analysis. Gel E ntrapment Entrapment of enzymes into the semi per meable gel or enclosing the enzyme semi permeable polymer membrane. Polyacrylamide is one of the most commonly used matrix for entrapment of enzymes. A number of procedures for the entrapment of enzymes in polyacrylamide gels have been published Polymer E ntrapment Conventional procedures for immobilizing enzymes like cross linking, covalent bonding and entrapment in gels or membranes suffer from a poor reproducibility and a poor spatially controlled deposition. The immobilization of enzymes in electroch emical polymers is gaining importance. Unlike, conventional immobilization methods, the electrochemical formation of polymer layers of controlled thickness gives a reproducible and non manual procedure for sensor fabrication. The electrochemical method inv olves the entrapment of biomolecules in organic layers during their electro deposition on an electrode surface. Most of the polymers used in electrochemical generation are conducting polymers such as polyacetylene, polythiophene, polyaniline, polyindole an d polypyrrole. In comparison to the physical entrapment of enzymes within polymer films such as polypyrrole, polythiophene, polyacetylene or polyaniline, this approach

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22 preserves a better access of substrate to the immobilized molecules and fa cilita t es macr omolecular interactions. Encapsulation Microencapsulation focuses upon maintaining the solution environment around the enzyme rather than physical or chemical forces necessary for immobilization. Formation of polymeric membrane around an enzyme solution t o make a microsphere requires a great deal of technology. The diameter of the microspheres can range from several microns to several thousand microns and the membrane thickness can go from hundreds of Angstroms to several microns. Immobilized Enzyme R eact ors The objective of an enzymatic reactor is to allow enzyme and substrate to come into contact for a sufficient period of time for reaction to t ake place; after that enzyme can be recovered at the end of the reaction and further the processed product is n ot contaminated with enzyme (Roig and others 1987) Immobilization gives more surface area available for reaction per unit volume thus improving the rate of liquid to solid mass transport of the substrate. The selection of proper reactor system depen ds on the type of immobilization and the type of process (Roy and Gupta 2003) Some enzyme reactors developed for food applications are discussed below Packed Bed R eactor (PBR) In a packed bed reactor the immobilized particles are held in a column and substrate is pumped through in plug flow mode The flow rate profile across a transverse cross section of the column is perfectly flat. Systems like this are called plug flow reactors. The flow of substrate c an be either from top or bottom and the column can be either vertical or horizontal. The main disadvantage of packed bed reactor is that

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23 temperature or pH is not controlled properly especially in reactors with diameter s greater than 15 cm Zhou and Chen (2001) and others immobilize galactosidase on graphite surfaces by gluteraldehyde. They used this PBR for continuous hydrolysis of lactose in skim milk solution. At flow rate of 7 mL min 1 90% conversion of lactose was achieved within a residence time of 15 minutes. Ovsejevi and o thers (1998) studied the galactosidase on to activated agarose gel by thiosulfinate. The mini reactor was used for the lactose hydrolysis. Whey permeate at a flow rate of 7 mL h 1 was fed continuously and a 90% conversion was observed. Illanes and others (1999) designed an immobilized enzyme reactor using immobilized lactase and glucose isomerase in sequential packed bed reactor for continuous production of fructose syrup from whey permeates. Baticz and Tomoskozi ( 2002) d eveloped an immobilized enzyme reactor to determine the total cholesterol content in foods using cholesterol oxidase. The Enzyme was immobilized on Amino modified Kieselgel 100 polymer beads by cross linking with gluteraldehyde. They reported that the immo bilized reactor was stable for two months. The diameter, length of the reactor and the flow rate were optimized to get the best possible results. Fluidized Bed R eactor (FBR) In a fluidized bed reactor the immobilized enzyme particles are fluidized. The par ticles become suspended in the substrate stream. Immobilized enzyme particles density should be sufficiently high to prevent them from being blown out of the reactor. Otherwise larger diameter particles have to be used (Roy and others 2000) This rea ctor is quite efficient at mixing catalyst with the substrate Coughlin and others (1978) galactosidase was adsorbed on porous alumina and cross linked with gluteraldehyde.

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24 Roy and Gupta (2 003) designed a FBR by immobilizing the enzyme on epichlorohydrin activated cellulose beads. The reactor with recirculation of substrate was used for hydrolysis of lactose in milk whey and whole milk. Ninety four percent of the conversion of the lactose i n milk whey was observed. However, the conversion in whole milk did not cross 60% this was attributed to the fat molecules that impairs the performance of the fluidized bed. The main disadvantage of this type of rectors is that they are difficult to scale up so their use is restricted to small scale but for high cost products (Roy and others 2000) Table 1 1 Immobilized enzyme reactors. Type Application Advantages Limitations References Packed bed reactor To determine the total cholesterol in foods Lactose hydrolysis in milk. Continuous production of fructose syrup from whey permeate. The immobilized enzyme reactor values are in comparable with GC values. These reactors are being used in the industry. Temperature or pH is not controlled properly especially in reactors of >15cm diameter. (Zhou and Chen 2001) (Baticz and Tomoskozi 2002) (Ovsejevi and others 1998) (Baticz and Tomoskozi, 2002) (Illanes and others 1999) Fluidized bed reactor Hydrolysis of lactose in milk whey Ninety four percent conversion of lactose was observed in milk whey. Density of immobilized enzyme particles should be high or diameter should be large. Difficult to scale up. Hydrolysis of la ctose did not cross 60% in whole milk. (Roy and others 2000) (Coughlin and others 1978) (Roy and Gupta 2003)

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25 M easurement of Dissolved Oxygen Introduction The US geological survey (USGS) standard field methods to measure dissolved oxygen are amperometric, luminescence, w inkler titration, an d colorimetric methods Several limitations were associated with some of these methods. Industry standard technique for measurement of DO is conventional Clark type electrode (amperometric method). Limitations associated with electrochemical probe include drifts in calibration due to oxygen consumption by the electrode, need for turbulent flow to prevent the formation of concentration gradients and electrical interference. The most recently developed methods are solid sate luminescence methods based on fluorescence peak quenching or multi frequency phase shift. The most common flu o rophores are ruthenium or platinum complexes (Lee and Okura 1997) because they are excited and emit at wavelengths in the visible light range an d they have high sensitivity to oxygen and large stokes shifts. Ruthenium complex based sensors are the most commonly used and are used for aqueous liquids and vapors. There are some commercially available ruthenium based sensors that can be used for the d etermination of DO in organic liquids and vapors. Another way of measuring oxygen using luminescent based sensor system is using phase modulation techniques. The phase shift between the exciting and the emitted light source is correlated to DO concentrati on ( Barnes and others 1990; Jiang and others 2002) Phase measurements are more stable and essentially independent on the luminescence dye concentration and other factors whi ch affect the intensity signal (Ogurtsov and P apkovsky 1998)

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26 Fiber Optic O xygen S ensors The principle of operation of this type of sensors is the fluorescence quenching of a flu o rophore When a flu o rophore in its excited state collides with oxygen molecules then the energy is transfer r ed to the oxy gen molecule in a non radiative transfe r, decreasing f luorescence intensity. The higher the number of collisions the greater is the fluorescence quenching. The flu o rophore is usually a ruthenium or platinum complex. Ruthenium complex based sensors are the most commonly used and are used for aqueous liquids and vapors. Optic sensors having Platinum complex as a flu o rophore is used for the measurement of low levels of DO. These sensors have high sensitivity compared to other optical sensors. There are some c ommercially available ruthenium based sensors that can be used for the determination of DO in organic liquids and vapors. Figure 1 1. Schematic d iag ram of an optical oxygen sensor Exciting radiation Fluorescent radiation Fluorophore Optic fiber Probe Filters LED / laser CCD array Len s

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27 Working The pulsed blue LED (laser emission diode) sends light at 475 nm through one optical fiber. The light excites flu o rophore which is immobilized at the tip of the probe. The excited Ru complex fluor esces and emits energy at 600 nm ; The Pt complex energy is emitted at 650 nm. The emitted light is collected throu gh a second optical fiber to the spectrometer. An A nalog to digital converter converts this analog data to digital data and is typically collected and displayed by a computer program. Calibration Calibration of optical sensors is based on the following Ste rn Volmer equation (1 6) Where I o and I are the luminescence intensities in the absence and presence of oxygen respectively, K S is the Stern Volmer constant, [ O 2 ] is the oxygen concentration, k is the diffusion dependent bimolecular quenching constant and o is the excited state lifetime of the fluorophore in absence of O 2 The plot of Io/I vs. [ O 2 ] is a straight line with slope equal to K s and an intercept of 1 (McDonagh and others 1998) However sometimes, Stern Volmer plots are non linear due to microscopic heterogeneity in the structure of the sample matrix. This heterogeneity is explained by the presence of sites with different quenching constants, called, quencher easy and quencher difficult accessible sites (Shin and others 2002) When Stern Volmer plot is not linear, a second order polynomial calibration is recommended (1 7 )

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28 Where A and B are temperature dependent coefficients. B is analogous to the K s constant in Stern Volmer equation. When the probe does not have an overcoat it fluoresces in all directions. In the presence of a reflec ting surface or backscattering medium the intensity of the emitted light is greater than in transparent, non reflective media So when calibrating a probe (not having overcoat) standards should be in the same state of matter as sample and also must have sa me refractive index as sample. So one cannot calibrate in gas and correlate it to liquids. Current Research Optical oxygen sensors consist of an oxygen sensitive dye entrapped in a matrix with a high permeability to oxygen. The flu o rophore is deposited usi ng sol gel immobilization technique. The use of sol gel immobilization has several attractive features these include ambient processing conditions, chemical inertness and stability of the sol gel, high optical transparency, ease of use and the ability to t ailor the structure of the matrix through contr ol of the processing parameters (Campbell and Uttamchandani 2004) Various precursor compounds are available fo r preparation of sol gel structures. The most common ones are tetraethoxysilane (TEOS) and tetramethoxysilane (TMOS). However ORMOSILs (organically modified silicates) are a class of precursors that have received increasing attention over the years. These compounds are of the form RSi(OR)n, where R is an alkyl group covalently attached to the silicon and is not present in the non modified precursors. Alkyl groups in ORMOSILs have poor affinity towards water. This hydrophobicity has been the reason for incre asing response of ORMOSILs than for non mediated precursors such as TEOS since oxygen permeation is generally greater in hydrophobic media. The ORMOSILs also stabilize very quickly which makes them to use in harsh environments soon after preparation.

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29 Stabi lity and response time of the sensor depends on both the quenching rate and matrix characteristics. For example density, viscosity and h ydrophobicity. Many Luminescent dyes have been tested as oxygen sensing probes. However Ruthenium and platinum complexe s (Lee and Okura 1997) are the most commonly u sed because of v isible light absorption h igh sensitivity to oxygen and l shifts In an attempt to increase the sensitivity, photostability and response time of the sensors several methods have been tried over the years and are broadly classifi ed into three categories. 1. Modification of the sensor system: Klimant and wolfbeis ( 1995 ) proposed optical isolation of the sensing layer to minimize photo bleaching of the dye. Black silicone, black t eflon and titania were used as isolation layers which m ade the sensor more photo stable than those with only a sensing layer. 2. Modification of the existing oxygen sensitive dyes: Earlier in optical oxygen sensors the matrix was doped with ruthenium complexes since this particular dye has a long excited lifetime (5.3 s), a high luminescence quantum yield and a high oxygen quenching efficiency. However there is an increasing attention towards pt based sensors. Pt complexes are easily excited using compact and low cost LED light source s. Furthermore the flu o roscen ce wavelengths of Pt complexes are well separated from the excitation wavelength and hence the influence of excitation light source can easily be minimized. Ru based sensors emit energy at 600 nm. Pt complex based sensors emit energy at 650nm. Watkins and others ( 1998 ) reported that Ru(dpp) 3 2 + immobilized in TEOS sol gel has sensitivity of 12 and response times 3.5 s when switching from 0% O 2 to 100% O 2 and 30 s from 100% O 2 to 0% O 2 Tang and others (2003) reported that Tris (4,7¢ diphenyl 1,10¢ phenanathroline) Ruthenium (II) chloride pentahydrate (Ru(dpp) 3 2 + ) immobilized in Octyl triEOS/TEOS sol gel has a nitrogen to oxygen intensity ratios (I N2 /I O2 ) of 16.48 where as (Yeh and others 2006) reported that oxygen sensitive dyes Platinum tetrakis pentafluorophenylporphine (PtTFPP) and platinum octaethylporphine (PtOEP) immobilized in same matrix Octyl triEOS/TEOS sol gel has I N2 /I O2 of 22 and 47 respectively This shows that modification of existing oxygen sensitive dyes with Pt complexes has resulted in increased sensitivity and photostability. 3. Modification of the support matrices: In general, the support matrix of an optical sensor not only immobilize s the dye, but also helps in oxygen to penetrate into thin film to react with the sensiti ve dye. Different matrixes yield different oxygen diffusion rates, and hence have a direct influence on the quenching efficiency of the indicator by the oxygen. Ru(dpp) 3 2 + immobilized in support matrices TEOS

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30 sol gel, Octyl triEOS/TEOS sol gel and n propyl TriMOS/TFP TriMOS have sensitivities of 12, 16.48 and 35 respectively. Yeh and others (2006) reported that Flu o rophores PtTF PP and PtOEP immobilized in Octyl triEOS/TEOS sol gel have sensitivities of 22 and 47 respectively. Chu and Lo (2007) reported that PtTFPP and PtOEP immobilized in n propyl TriMOS/TFP TriMOS have sensitivities of 68.7 and 82.5 respectively. From the above studies it is understood that the change in the support matrices has improved the sensitivity and response time of the sensors. Figure 1 2. C hemical structure of PtTFPP Figure 1 3. C hemical structure of PtOEP

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31 Table 1 2. Luminescence properties of oxygen sensitive dyes i mmobilized in sol gel matrixes. Oxygen sensitive dye Support matrix Response time I N2 /I O2 Ru(dpp) 3 2 + TEO S sol gel 0% O 2 to 100% O 2 : 3.5s; 100% O 2 to 0% O 2 : 30s 12 Ru(dpp) 3 2 + Octyl triEOS/TEOS sol gel None 16.48 Ru(dpp) 3 2 + n propyl TriMOS/TFP TriMOS Response times were<5s 35 PtTFPP Octyl triEOS/TEOS sol gel 0% O 2 to 100% O 2 : 0.6s; 100% O 2 to 0% O 2 : 5s 22 PtOEP Octyl triEOS/TEOS sol gel 0% O 2 to 100% O 2 : 0.7s; 100% O 2 to 0% O 2 : 14s 47 PtTFPP n propyl TriMOS/TFP TriMOS 0% O 2 to 100% O 2 : 3.7s; 100% O 2 to 0% O 2 : 5.3s 68.7 PtOEP n propyl TriMOS/TFP TriMOS 0% O 2 to 100% O 2 : 3.7s; 100% O 2 to 0% O 2 : 5.9s 82.5 Advantages Fiber optic sensors have several advantages compared to other measurement techniques. They have rapid response time in the range of 0.2 1 s. The main advantage of these probes is they do not consume oxygen but the conventional electrochemical probes do consume oxygen These are sensitive to low levels of oxygen. Ease of miniaturization and are good for continuous measurements. Limitations The main disadvantage of the fiber optic probes is they are sensitive to surrounding light. In order to ove rcome this overcoating of the flu o rophore has been done. However, the overcoating resulted in slow response time, poor reproducibility and fouling. Measurement of DO using these probes is position dependent. A small change

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32 in the position of the fiber or the probe can result in increase or decrease in intensity (counts). Photobleaching of the l umiphore has also been reported. Table 1 3. Comparison of different methods used to measure DO Method Advantages Disadvantages Electrochemical probe Variety of sh apes, sizes and consumables. Some probes have build on thermistor for temperature compensation (Lewis 2006) Oxygen consumption. Electrolyte solution should be free of bubbles to avoid interferences. Membrane f ouling, stagnation or rupture. Optical probe Ease of miniaturization Do not consume oxygen Sensitive to low levels of oxygen Fast response time: 0.2 1 s (Choi and Xiao 2000) Continuous measurements. Sensitive to ambient light. Photo bleaching of the fluorophore Position dependence of optic fiber Winkler titration Official reference method (ASTM D 888 92) H ighly dependent on analyst experience. Contamination with atmospheric O2 during titration. Chemical and color interferences Colorimetric methods Official methods (ASTM D 5543 94). Kit available. Able to measure ppb of O2. Not for continuous measurement s Gap in Knowledge Numerous studies on the effect of dissolved oxygen on the quality of fruit juices and also on the kinetics of dissolved oxygen consumption and ascorbic acid degradation However, contradictory data is reported on the kinetics of oxygen

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33 consumption and ascorbic acid degradation with kinetic r ate as first order, zero order and second order. There is a need to develop new methods of removing oxygen from juices without removing volatiles and affecting the overall quality of juices. Previous studies on deoxygenation of fruit juices were done by direct addition of enzymes. However, the direct addition of enzymes makes the process uneconomical and in cases violates standards of identity Enzyme immobilization offers an alternative to this meth od To GOx Cat system for deoxygenation in pineapple juice. Objectives The specific objectives of this research were To better understand the kinetics of dissolved oxyg en consumption and ascorbic acid degradation in ascorbic acid solutions and pineapple juices. To develop an Immobilized enzyme reactor using GOx Cat to remove dissolved oxygen from model solutions and fruit juices.

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34 C HAPTER 2 KINETICS OF DISSOLVE D OXYGEN CONSUMPTION IN ASCORBIC ACID SOLUTIONS AND PINEAP PLE JUICES Introduction Dissolved oxygen plays an important role in the quality of fruit juices. It causes the oxidation of ascorbic acid as well as changes in flavor and color during storage. L As corbic acid (L AA), degradation occurs by two consecutive or parallel pathways aerobic and anaerobic During the aerobic pathway, ascorbic acid in aqueous solution is easily oxidizes to 2, 3 diketogulonic acid in aqueous solution. Under anaerobic condition s, L AA is degraded to furfural and rate is slower than aerobic degradation Various studies on model solutions and citrus juices reported ascorbic acid degradation as first order, zero order and second order kinetics. Numerous studies on the kinetics of d issolved oxygen consumption and ascorbic acid degradation reported contradictory data with kinetic rate as first order, half order and second order. The oxidation of L AA to DHA produces color and flavor changes. DHA contains an dicarbonyl structure tha t participates in strecker degradation Well known examples of strecker aldehydes are methional and acetaldehyde. Oxygen is not always directly responsible for browning in fruit juices. In PPO catalyzed reactions oxygen is indirectly responsible for browni ng. The indirect effect is by way of ascorbic acid oxidation to dehydroascorbic acid with further formation of decomposition products such as furfural that has brown color. There is a misassumption that oxygen is always directly responsible for ascorbic ac id degradation. L AA degradation due to DO occurs only during initial stages of storage period from hours to days depending on the storage temperature, light intensity and package permeability. Correlation of rates of DO consumption and L AA degradation in pure solutions with those of fruit juices help in

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35 better understanding of AA retention in complex juice matrix. Initial studies on oxygen uptake in ascorbic acid solutions described the rate of reaction as first order but there is not agreement among repo rts in the literature when ascorbic acid concentration is also considered The objective of this study wa s t o better understand the kinetics of dissolved oxygen consumption and ascorbic acid degradation in ascorbic acid solutions and pineapple juices M ate rials and M ethods Pineapple variety Del Monte Gold extra sweet from Costa Rica was purchased from the local grocery store. The extraction of the pineapple juice was accomplished using a Norw alk juice press (La Jolla, CA). Immediately a fter the extraction t he juice is filled in sterile amber vials for further analysis of dissolved oxygen and ascorbic acid. Ascorbic acid solutions 2 8 mM and 2 8 mM were prepared in deionized, ultra filtered water. The concentration 2.8 mM was chosen to replicate the concentra tion in orange and pineapple juices When the probe has an overcoat we observed a slow res ponse so initially we chose high concentration (28 mM) of AA Probe over coating is explained in C hapter 1. Sa mples both AA solutions and fruit juices were kept in st erile amber vials wit hout headspace for a period of up to two weeks in temperature controlled water baths or incubators at 5.0, 13.0, 21.5, 30.5 or 40.0 0.1 C Dissolved oxygen and ascorbic acid concentrations were measured at selected time intervals I n order to ensure sterility, a ll experiments were done in autoclaved/sterilized glass amber vials Glass amber vials were chosen to protect the sample from light that can accelerate oxidative reactions as suggested by (Mack and others 1976) All the juice extraction equipment beakers and filter cloth w ere au toclaved to ensure sterility.

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36 Immediately after extraction the juice was filled in autoclaved amber glass vials under laminar hood. DO M easurement DO measurement was performed using a fiber optic FOSPOR oxygen probe with out overcoating connected to fiber optic spectrometer model SD 2 000 from Ocean Optics, (Dunedin, FL). Because the probe does not have an overcoat samples were protected from surrounding light during the measurement of DO to prevent interferences of light with fluorescence Figure 2 1 D issolved oxygen measurement setup. AA M easurement Measurement of AA was performed using capillary e lectrophoresis every two days for a period of two weeks. The CE system model P/ACE MDQ with DAD and the data acquisition and analysis software Karat 32 versi on 5.0 was from Beckman Coulter (Fullerton, CA). The capillary was bare fused silica from Polymicro Technologies

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37 (Phoenix, AZ) 50 mm i nternal d iameter 56 cm total length (48 cm to the detector). The r unning buffer was 35 mM sodium borate with 5% acetonitr ile. Results and D iscussion Kinetics of DO C onsumption in 28 mM and 2.8 mM AA S olutions and Pineapple J uices Half first and second order kinetics were used to see which kinetic order fits well for the consumption of dissolved oxygen in both model solution s and fruit juices. First order model fit better initial consumption of dissolved oxygen in ascorbic acid solutions as shown in Figures 2 2, 2 3 and 2 4 and pineapple juices (R 2 =0.999 for first order, R 2 =0.95 for second order and R 2 =0.85 for half order at 21.5 C). Mack and others (1976) als o reported that kinetics of dissolved consumption in liquid foods as first order during the first 24h of storage Figure 2 2. First order kinetics of 2.8 mM AA solutions at 21.5 C.

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38 Figure 2 3. Second order kinetics of 2.8 mM AA solutions at 21.5 C Fi gure 2 4. Half order kinetics of 2.8 mM AA solutions at 21.5 C First order reaction rate constants ranged between 1.03 x 10 5 to 2.97 x 10 4 s 1 for 5 C and 40 C, respectively for 28 mM AA solutions and 9.58 x 10 6 to 1.08 x 10 4 s 1 for 13 C and 40 C respectively for 2.8 mM AA solutions and between 8.89 x 10 6 to 4.75 x 10 5 s 1 for 13 C and 40 C, respectively for pineapple juices as shown in Table 2 1 and Table 2 2 The rate constants for 28 mM AA solution are slightly higher

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39 compared to 2.8 mM A A solution. The concentration of Ascorbic acid is al most 500 times and 50 times greater than oxygen concentration in 28 and 2.8 mM AA solutions. Higher concentration were used to obtain faster kinetic data and to ensure that we were kinetically in excess o f one the substrates and therefore, we are truly measuring the kinetics of dissolved oxygen and not the kinetics of AA. The rates were higher for 2.8 mM AA solutions compared to pineapple juices The types of reactions involved in fruit juices other than a scorbic acid degradation are difficult to predict without additional investigations. The influence of temperature on the first order kinetics of AA solutions and pineapple juices is explained by Arrhenius equation. The first order rate constant of dissolve d oxygen consumption in 2 .8 mM AA solutions, 28 mM AA solutions and pineapple juices increased exponentially with temperature following Arrhenius behavior (Figure 2 5, Figure 2 7 and Figure 2 9). The activation energy ( E a ) was calculated for both AA soluti ons and pineapple juices. This was determined with linear regression of the logarithm of the rate constant vs. the reciprocal of temperature (Figure 2 6, Figure 2 8 and Figure 2 10 ) The calculated activation energy values are 67.7 4.2 67. 12 6.72 and 48.62 2.6 1 1 for 28 mM, 2.8 mM AA solutions and pineapple juices respectively. The decrease in activation energy for pineapple juice compared to AA solutions suggests that the pineapple juice is less sensitive to temperature compared to AA solu tions. Mack and others (1976) reported activation energy in liquid infant formula foods at temperature range of 7.2 to 23.9 C as 12.42 to 47.76 1 1 Dhuique Mayer and others (2007) reported ascorbic acid degradation fitted well with first order model in orange and tangerine juice mixture and they calculated the

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40 ac tivation energy as 35.9 1 1 at temperature range of 50 to 100 C. Ahrne and others (1997) reported activation energy of liquid packed food during storage of up to 5 months at 4, 8, 20, 30, 40 and 50 C as 46 kJ mol 1 1 The model was applied to orange juice aseptically packaged in Tetra Brik Aseptic cartons. Manso and others (2001a) reported activation energy of SSOJ during sto rage in the absence of light at temperature range of 20 to 45 C as 38.6 1 1 Garcia Torres and others (2009) reviewed different studies about ascorbic acid degradation in model solutions and citrus juices. 8 of the 17 studies they reviewed reported L AA o xidation as first order reaction under different initial DO concentrations. Ea ranged from 35.9 to 143.5 1 over a temperature range of 20 and 124 C. Table 2 1. First order rate constants for dissolved oxygen (DO) consumption in 28 mM and 2.8 mM as corbic acid solutions E rror is reported as standard deviation T emp (K) k DO (s 1 ) 28 mM k DO (s 1 ) 2.8 mM 278.15 1.03 x 10 5 5.7 x 10 8 286.15 2.26 x 10 5 1.6 x 10 7 9.58 x 10 6 2.76 x 10 6 294.65 6.17 x 10 5 8.7 x 10 7 2.90 x 10 5 8 .27 x 10 6 303.65 9.75 x 10 5 1.2 x 10 6 6.71 x 10 5 5.66 x 10 6 313.15 2.97 10 4 3.21 x 10 6 1.08 x 10 4 1.05 x 10 5

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41 Figure 2 5 Effect of temperature on first order rate constants of dissolved oxygen consumption in 28 mM ascorbic acid solutions E rror is reported as standard deviation Figure 2 6 Arrhenius plot for rate constant of dissolved oxygen consumption in 28 mM ascorbic acid solutions

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42 Figure 2 7 Effect of temperature on first order rate constants of dissolved oxygen consumption in 2 8 mM ascorbic acid solutions E rror is reported as standard deviation Figure 2 8 Arrhenius plot for rate constant of dissolved oxygen consumption in 2 8 mM ascorbic acid solutions

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43 Table 2 2 First order rate constants for dissol ved oxygen (DO) consumption in pineapple juice s E rror is reported as standard deviation Temp (K) k DO (s 1 ) 286.15 8.89 x 10 6 6.82 x 10 7 294.65 1.30 x 10 5 2.96 x 10 6 303.65 3.11 x 10 5 3 13.15 4.75 x 10 5 7.25 x 10 6 Figure 2 9 Ef fect of temperature on first order rate constants of dissolved oxygen consumption in pineapple juice s E rror is reported as standard deviation

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44 Figure 2 10 Arrhenius plot for rate constant of dissolved oxygen consumption in pineapple juice s Kinetics of AA in 2.8 mM AA Solutions and Pineapple J uices Measurement of AA was performed every two days i n both 0.05% (2.8 mM) AA solutions and pineapple juices. In 2.8 mM AA solutions t here is a loss of 2 3.62 % and 25.28% AA was observed at 13 C and 21.5 C after 10 days storage period. There is a 18.7 % and 25.2% decrease in AA found after 8 days of storage at 30.5 C and 40 C. We did not observe any significant difference in decrease in Ascorbic acid between 30.5 C and 40 C after 8 days of storage

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45 Figure 2 11 Concentration of AA in 0.05% AA solutions at four different temperatures ( E rror is reported as standard deviation Measurement of Ascorbic acid was done at four different temperatures for every two day s for a period of 10 days. AA concentration decreased by 27.9% at 13 C after 10 days, 33.5 % and 31.9% decrease after 6 days of storage at 30.5 C and 40 C respectively We would expect temperature to have significant impact on the ascorbic acid degradation Although AA concentration decreased at all temperatures, temperature did not show a significant difference in decrease in the rate of AA degradation at 30.5 C and 40 C after 6 days of storage These results suggest that the activation energy is very smal l. Therefore, despite reporting rate constants at four different temperatures, our experimental results do not allow reasonable calculation of the activation energy. Also, in view of the much faster reaction of oxygen, it is evident that AA degradation in this experiment was anaerobic. Longer experiments and wider temperature ranges need to be studied to accurately determine activation energy.

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46 Figure 2 12 Co ncentration of AA in pineapple juices at four different temperatures ( 13 C, 30.5 C, 40 C) Error is reported as standard deviation Conclusions Kinetics of dissolved oxygen consumption fitted well with first order kinetics in both AA solutions and pineapple juices. The first order rate constants for DO consump tion for 28 mM AA solutions are slightly higher compared to 2.8 mM AA solutions. The first order rate constants for DO consumption in 0.05% AA solutions are higher than pineapple juices at all the four temperatures R ate of DO consumption in juice matrix i s slower than aqueous ascorbic acid solutions. This suggests that the several other reactions occur simultaneously in fruit juices. The types of reactions involved in fruit juices other than ascorbic acid degradation are difficult to predict without additi onal investigations. T he kinetics of dissolved oxygen consumption as a function of temperature followed Arrhenius relationship. The activation energies of dissolved oxygen consumption are 67.7 4.2 67. 12 6.72 and 48.62 2. 6 1 1 for 28 mM, 2. 8 mM AA solutions and pineapple juice respectively There is no s ignificant difference between 28 mM and 2.8 mM AA solutions. However, lower activation energy

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47 for pineapple juice compared to AA solutions was observed. This suggests that the pineapple juice is less sensitive to temperature compared to AA solutions. Temperature did not show a significant difference in decrease in the rate of AA degradation between 13 C and 40 C Lon g er storage times and wider temperature ranges might help in observing tempera ture effects and calculating activation energy.

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48 C HAPTER 3 THIN FILM ENZYME REA CTOR FOR DEOXYGENATI ON OF MODEL SOLUTIONS AND PINEAPPLE JUICE Introduction Dissolved oxygen (DO) plays an important role in the quality of juices. It accelerat es oxidation of vitamin C and causes non enzymatic browning during storage leading to the formation of off flavors (Trammell and others 1986; Rassis and Saguy 1995) Degradation of ascorbic acid is one of the major causes of color and quality changes during storage. To prevent these deleterious effects, fruit juices are often deaerated prior to pasteurization Deoxygenation is done by vacuum flash deaeration, gas sparging, oxygen scavengers, membrane deaerators and adding antioxidants or using enzymes. Vacuum deaeration is the most commonly used method in the citrus industry. However, in this method along with oxygen essential oil is also removed (Braddock 1999) Gas sparging consists of displacing the DO with another gas such as nitrogen or carbon dioxide. Early studies on me thods of deaeration indicate that in orange juice 99% of DO was removed using nitrogen sparging compared to 77% DO removed by vacuum deaeration (Jo slyn 1961) but significant amount of volatiles are also removed in this process. Jordan and others (2003a) reported that th e greatest losses in concentration of volatiles occurred during industrial deaeration. Enzymatic deaeration of juices using GOx Cat in solution has been reported by (Sagi and Mannheim 1990; Parpinello and others 2002) in orange juice, apple and pea r purees. The advantage of this system is glucose is present in large excess in juices with respect to DO so there is no need to ad d any substrate. Detailed literature review on enzymatic deaeration and other deaeration methods was explained in Chapter 1.

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49 However, the direct addition of enzymes makes the process uneconomical and in cases violates standards of identity. Enzyme immobili zation offers an alternative to this method. Enzyme immobilization can be done by adsorption, covalent bonding, encapsula tion, entrapment in gels, cross linking, hydrophobic interaction and polymer entrapment Unlike conventional immobilization methods, th e electrochemical formation of polymer layers of controlled thickness gives reproducible and non manual p rocedure for sensor fabrication. the usage of immobilized GOx Cat system for deoxygena tion in pineapple juice. The objective of this study was to develop and test a laboratory prototype deaeration reactor immobilizing GOx Cat in electrochemically generated poly o phenylenediamine thin films deposited on the interior wall of a platinized pl atinum tubes. Materials and Methods Glucose oxidase (EC 1.1.3.4 type X S from Aspergillus niger ), Catalase (EC: 1.11.1.6) o phenylenediamine free base, chloroplatinic acid hexahydrate, lead acetate trihydrate, hydrogen peroxide and potassium chloride we re purchased from Sigma Aldrich Chemical Co. ( St.Louis, MO ) Platinum wire (0.4mm diameter) was purchased from Fischer S cientific. Pt metal tube 250 mm length (1.60 mm o.d, 1.30mm i.d) was purchased from American Elements, (Los Angeles, CA ) All other reage nts and solvents were reagent grade. Solutions were prepared in deionized ultrafilterd water. Platinization, electro polymerization and cyclic voltammetry experiments were performed with a potentiostat/galvanostat, EG&G 263A (Perkin Elmer Instruments, Oak Ridge, TN), interfaced to a personal computer, through a GPIB board (National Instruments, Austin, TX).

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50 All potentials are reported with respect to Ag/AgCl, 3.0 M KCl reference electrode model EE008 from cypress systems inc. (Lawrence, KS) A Pt wire was us ed as the counter electrode. A syringe pump model 210 from Kd Scientific (New Hope, PA) was used for the injection of model solutions and juices. The syringe pump was interfaced to and controlled with a PC through a serial RS 232 port. Reactor C leaning R eactor was polished with 5 m alumina slurry (Buehler, Lake Bluff, IL) using miniature brushes purchased from Tanis Inc. (Waukesha, WI), rinsed with deionized water and immersed in an ultrasound bath for 5 min to remove any adherent alumina particle. For r euse, prior to polishing with alumina and after sonication, Pt tubes were immersed in concentrated H 2 SO 4 at 21C for at least 1 h and rinsed with deionized water. Reactor P latinization Platinization was carried at 100 mV vs Ag/AgCl in 4 mM H 2 PtCl 6 1 mM P b(C 2 H 3 O 2 ) (used as crystal growth promoter), 0.1 M KCl oxygen free solution for 60 min at room temperature 21C (Reyes De Corcuera and others 2005) Solutions were deoxygenated by vigorously sparging with ultrapure nitrogen ga s. A Pt wire inserted in the capsule was used as the counter electrode. To prevent counter electrode from touching the working electrode, thin glass beads were fused on the wire. Platinization of the internal wall of the reactor was performed to increase t he surface area. Reactor P olymerization Potentiostatic electropolymerization was carried out in 5 mM o phynelenediamine, 0.2 M acetate buffer (pH 5.2) and 10 g L 1 25 g L 1 and 50 g L 1 GOx solutions at 65 0 mV vs. Ag/AgCl for 30 min at 21C. Before applyi ng the polymerization potential,

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51 monomer and enzyme were allowed to diffuse for 5 min into the porous pt deposit. The concentration of catalase was adjusted from 45 L to 112.5 L to ensure complete decomposition of H 2 O 2 The reactors were stored in 0.1 M phosphate buffer pH 7.0 until further testing. Figure 3 1. Enzyme immobilization setup. Model O range J uice S olution A model orange juice solution was prepared 2.5% glucose, 2.5% fructose, 5.0% sucrose, 0.8% citric acid in distilled water (Peleg and others 1992) The pH of the above solution was adjusted to 3.5, 4.7, 5.9, 7.0 using 4 N NaO H for furt her testing. Pineapple Juice P reparation from the local grocery store. The extraction of the pineapple juice was done using a Norwalk juice press (La Jolla, CA). Soluble solids we re measured using Leica Auto ABBE refractometer. Both acidity and pH of the juice was measured using Schott Titroline easy automatic titrator.

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52 Testing the R eactor Initial tests with the reactors were carried out using 166 mM glucose solutions in 0.1 M pho sphate buffer pH 7.0. This concentration was chosen to replicate the D glucose concentration in orange juice (3.0%) 166 mM g lucose solutions model orange juice solutions and pineapple juice were pumped using a syringe pump at three different flow rates 0 .075, 0.05 and 0.025 mL min 1 Operation parameters enzyme concent ration, length and flow rate were adjusted to remove the highest % of DO Figure 3 2. Setup for testing the reactor Electrochemical determination of current was done using potentiostat at 700 mV The current is a measure of the rate of production of H 2 O 2 by the enzymatic reaction which is the principle of operation of amperometric glucose biosensors (Reyes De Corcuera and others 2005) Measurement of Dissolve d O xygen DO measurement was done using a fiber optic FOSPOR oxygen probe without over coating connected to USB 4000 spectrometer from Ocean Optics, (Dunedin, FL).

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5 3 Calibration of the probe is the important step in the measurement of DO and was done using S tern Volmer equation ( C hapter 2) In the model solutions the standard 0% oxygen concentration was obtained by using 5% sodium sulf i te solution in water in five minutes whereas in the pineapple juice the 0% concentration was achieved using 3% Glucose oxidas e in 10 minutes The other standard used was air saturated sample. The concentration of air saturated sample is dependent on temperature and pressure. At 22 C and at a pressure of 101.325 kP a the concentration of dissolved oxygen in saturated water sample is 275 M or 8.8 ppm The S tern V olmer constant is temperature dependent so all the measurements are done at constant temperature. Because the probe did not have an overcoat samples were protected from light during the measurement of DO to prevent interfe rences of light with fluorescence. Initially we used the probe with overcoating to avoid interferences from environmental light. However, due to slow response time, poor reproducibility and fouling we switched back to probes without overcoating. Detailed r eview on meas urement of DO was presented in C hapter 1. Results and Discussion Effect of Enzyme C oncentration The amount of dissolved oxygen removed increased with the enzyme concentration used in the immobilization solution as shown in Table 3 1 Reactors 3 cm long with 10 g L 1 GOx removed 41.1 % DO. In contrast, reactors 3 cm long with 25 g L 1 and 50 g L 1 GOx re moved 87.7% and 86.4% of DO respectively. There was no significant difference in DO removal by reactors with 25 g L 1 and 50 g L 1 w hich is most probably due to subst rate diffusion becoming limiting or electrochemically generated films with higher GOx concentration have reduced permeability and increased catalytic

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54 activity. E xtended operation of reactors with high concentration of enzyme lead to fi lm detachment. Table 3 1. Performance of 3 cm reactor s at selected GOx concentrations in model orange juice solution determined as [DO] at the exit of the reactor. Flow rate was 0. 025 mL min 1 error is reported as standard deviation [Enzyme] ( g L 1 ) Initial [ DO] (M) Final [DO] (M) DO removed (%) 10 276.6 27.2 163.5 26.3 41.1 3.74 25 264.7 10.1 32.8 10.9 87.7 3.65 50 27 1 .4 11.6 36.9 6.5 86.5 1.82 C urrent response also al most doubled as GOx concentration increased from 10 to 25 g L 1 in glucose solutions up to 300 mM. H owever, there was little change in current when increasing [GOx] from 25 g L 1 to 50 g L 1 as shown in F igure 3 3 This further supports that at higher GOx concentrations the rate of diffusion of substrates and products becomes limiting No replication was done for each concentration in th is experiment but the relative increase in current response between 10 g L 1 and 25 g L 1 or 50 g L 1 is consistent throug hout the glucose concentration range.

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55 Figure 3 3 Effect of GOx concentration ( 10 g/L, 25 g/L, 50 g/L) and glucose concentration on the amperometric response of 3 cm GOx reactors at a flow rate of 0.5 mL min 1 Effect of Glucose C oncentration Cur rent response vs glucose concentration showed a maximum at approximately 75 mM There is no significant difference in the current response by the reacto r at glucose concentrations 75 mM to 200 mM (Figure 3 4) A slight decrease in current response was obs erved after 75 mM which might be due to substrate inhibition as previously reported for GOx biosensors (Shin and Kim 1995; Reyes De Corcuera and others 2005) The concentration of glucose in orange and pineapple juices is around 166mM suggesting that the glucose co ncentration has little effect on the performance of the reactor.

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56 Figure 3 4 Effect of glucose conc entration on the amperometric response of a 3 cm, 25 g L 1 GOx reactor at a flow rate of 0.1 mL min 1 Error is reported as standard deviation. Effec t of R e tention T ime Reactor l ength I ncreasi ng the length of reactor to 12 cm significantly increased DO removal as indicated in Figure 3 5 However, for 12 cm or 18 cm reactors oxygen consumption leveled off due to the small concentration of DO The conce n tration of DO decreased from 270 M to 21.5 M using a 12 cm reactor. Commercial storage of deaerated orange juice is typically 15.63 mol ( 0.5 ppm ) indicating that the prototype reactor is capable of meeting the desired DO concentration.

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57 Figure 3 5 E ffect of reactor length on the performance of ( 25 g L 1 GOx 2X cat) r eactors in model orange juice solution at a flow rate of 0.025 mL min 1 Error is reported as one standard deviation. Flow r ate Retention time was also increased by adjusting flow rate for 3 cm, 12 cm long reactors. Retention times of 31.8 s and 95.5 s resulted in 22.6 and 46.3% DO removal respectively for 3 cm reactors. Retention times of 127.4 s and 382.4 s resulted in 63.6 and 91.6% DO removal respectively for 12 cm reactors as indic ated in Table 3. With retention time of 382 s, (12 cm reactor at a flow rate of 0.025 mL min 1 ) 90% removal of DO was achieved which corresponds to 23.1 M ( 0.74 ppm ) T he Reynolds number for the 12 cm reactor at these three different flow rates is below 1 This number is very small and it is difficult to operate at industrial level with these parameters. However, by adjusting retention time, tube length and diameter to a reten tion time of 382 s it is possible to remove more than 90% of DO The industrial c onditions of 4.54 kg /min could be achieved by stacking the reactors and running them in parallel Reactors can be stacked to achieve scale up.

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58 Table 3 2. Effect of flow rate on the performance of 25 g L 1 GOx r eactor in 166 mM glucose solutions Error is reported as one standard deviation. Length (cm) Retention time (s) DO removed (%) Flow rate (mL min 1 ) Initial [DO] (M) Final [DO] (M) 3 31.8 22.6 6.7 0.075 279 .0 11.3 216.5 27.6 3 63.6 33.1 6.1 0.05 279.5 2.1 187 15.6 3 95.5 46.3 10.3 0.025 284.3 3.8 153 31.2 12 127.4 63.6 0.075 250 .0 91 .0 12 254.7 76 .0 0.05 242 .0 58 .0 12 382.4 91.6 0.6 0.025 274 .0 0.7 23 .0 1.4 Effect of pH The effect of pH on the performance of the reactor was very small as shown in T able 3 3 There was no significant difference between effect of pH on the performance of reactor in the range of 3.5 to 7.0 Glucose oxidase from Aspergillus niger has optimum activity at 5.5 to 6.0. From pH 4.0 to 7.0 GOx has at least 90% of its activity (Kalisz and others 1990) Catalase from Micrococcus sp., has a working pH of 3 to 9 and the working optimum pH is 5.5. Kothapalli and others (2007) reported that there was no significant difference between GOx immobilized in low density polyethylene using UV polymerization and free GOx at Previous reports in the literature indicate that GOx Cat system is dependent on pH. All these reports are based on solution. i.e. direct addition of the enzymes. Mistry and Min (1992) reported that GOx Cat complex sh owed a maximum activity at pH 6.0 and 30 C in model salad dressing. Pickering and others (1998) reported that in Riesling grape using

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59 GOx Cat concentration of 2 g L 1 the gluconic acid produced at pH 6.0 (80 g L 1 ) was almost 3 times the gluconic acid produced at pH 3.1 (30 g L 1 ). Parpinello and others (2002) reported that GOX100 (glucose oxidase catalase system Gist Brocades, Lille, France ) showed maximum activity at pH 5.5 and 6.5 corresponding to 50 and 12 units/mg and at pH 4.0 the activity decreased to 15 and 5 units/mg for glucose oxidase and catalase respectively. One unit of glucose oxidase activity refers to 1 mol of gluconic acid formed per min at 35 C and pH 5.1. In all the above cases the enzymes were directly added to the samples. Our results with immobilized GOx C at differ from these previous reports on soluble enzymes indicating that immobilization widened the pH operational range of GOx Cat. Table 3 3. Eff ect of pH on the performance of 3 cm (GOx Cat) reactor in model orange juice solutions at a flow rate of 0. 025 mL min 1 Error is reported as one standard deviation. pH Initial [DO] ( M ) Final [DO] ( M ) DO removed (%) 3.5 2 77.7 16.8 66.2 3.78 75 .9 4.7 256.9 12.9 70.0 26.7 72 8 5.9 288.9 20.8 54.1 8.2 81.2 7 .0 2 79.3 12.6 63.1 5.5 77.4 Tes ting with Model S olutions an d Fruit J uices In both model solutions and pineapple juice approximately 90% DO was removed using a 12 cm reactor with retention time 6.4 min. Sagi and Mannheim (1990) reported that GOx Cat enzyme preparation of 17 un its L 1 reduced oxygen concentration to less than 1 ppm in 30 minutes at room temperature in orange juice. Parpinello and others

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60 (2002) reported that 2 units L 1 of GOx Cat removed 99% of DO in 120s from apple and pear purees. Ough (1975) reported that GOx Cat effectively removed DO from white and rose table wines. Hydrogen peroxide was reduced by su lfur dioxide rather than catalase. In all the above cases the enzyme complex was directly added to the samples which increases process operation costs and it is not feasible for orange juice NFC because it would violate the standards of identity. Table 3 4 Performance of 12 cm (25 g L 1 GOx 2x Cat) reactor with model orange juice solution, glucose solution and pineapple juice Error is reporte d as one standard deviation Solution Initial [DO] (M) Final [DO] (M) DO remov e d (%) A cidity (%) pH 150 mM gl ucose 265 .0 21.5 91.9 0.0 7.0 Model orange juice 274.7 1 .2 29.6 16.6 89.2 6.0 0.8 3.5 Pineapple juice 281.3 7.8 23.5 2.5 91.7 0.7 0.59 0.03 3.0 GOx Cat immobilized in poly o phenylenediamine successfully removed more than 90% DO in both model solutions and fruit juices. Conclusions Reactors with 10 g L 1 a nd 25 g L 1 GOx showed better operational stability compared to 50 g L 1 whe re film detachment was observed. Increasing t he retention time from 127.4 s to 382.4 s has increased the DO re moval from 63.6 % to 91.6% in a 12 cm reactor. Varying the pH from 3.5 to 7.0 had very little impact on the perf ormance of reactor In both model solutions and pineapple juice approximately 90% DO was

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61 removed using a 12 cm reactor at a flow rate of 0.025 mL min 1 with a retention time of 6.4 min O ver all C onclusions Kinetics of dissolved oxygen consumption fitted well with first order kinetics in both AA solutions and pineapple juices. The initial rate of DO consumption is faster in AA solutions compared to pineapple juices. The kinetics of DO consumption as a function of temperature followed Arrhenius relationship. The activation energies of dissolved oxygen consumption are 67.7 4.2 67. 12 6.72 and 48.62 2. 6 1 1 for 28 mM, 2.8 mM AA solutions and pineapple juices respectively Temperature did not show a significant difference in decrease in the rate of AA degradation between 13 C and 40 C Longer storage studies might help in determining effect of temperature and activation energy calculation Reactors with 25 g L 1 GOx showed better operational stability and performance compared to 10 and 50 g L 1 Although the enzyme activity depends on pH we did not observe any effect of pH on the performance of reactor. In principle, we would expect the pe rcentage of oxygen removed to be half compared with no catalase. But, we did not observe any effect of catalase (2 times GOx concentration) on the performance of reactor. However, when i ncreasing th e catalase concentration from 2 times 5 times the activi ty of GOx we did not observe any decrease in oxygen concentration. GOx Cat i mmobilized enzyme reactor removed more than 90% of DO in both model solutions and pineapple juices with a retention time of 6.4 min. F uture W ork With the current FOSPOR or FOXY pro be we were not able to reproducibly measure DO in orange juice as these probes degraded in that juice Similar study with

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62 both DO consumption (with a different type of sensor) and ascorbic acid degradation in orange juice would help in better understanding of AA behavior in real juice as o range is very rich in AA The current enzymatic reactor is successful in removing more than 90% of DO in both model solutions and pineapple juice s Further analysis has to be done on the quality parameters of enzymaticall y deoxygenated juice (flavor, color) Immobilization of enzymes in electrochemically generated polymer thin films resulted in stable and reproducible formation of layers. This is one of the most successful methods in the preparation of biosensors. Developm ent of thick and stable films using other polymers and immobilization method s might help in improving the operational performance and stability of the reactor W e were not able to detect the effect of catalase concentration on the performance of reactor. T here is also a need to better understand the spatial arrangement of enzyme molecules especially when a high concentration of catalase is used as this would help in successful immobilization of both GOx and catalase.

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63 LIST OF REFERENCES Agullo E, Rodriguez MS, Ramos V, Albertengo L. 2003. Present and future role of chitin and chitosan in food. Macromolecular Bioscience 3(10):521 30. Ahrne LM, Oliveira FAR, Manso MC, Drumond MC, Oste R, Gekas V. 1997. Modelling of dissolved oxygen concentration during storage of packaged liquid foods. Journal of Food Engineering 34(2):213 24. Baiano A, Marchitelli V, Tamagnone P, Nobile MAd. 2004. Use of active packaging for increasing ascorbic acid retention in food beverages. JFS 69(9):E502 E8. Baticz O, Tomoskozi S. 2002. Determination of total cholesterol content in food by flow injection analysis with immobilized cholesterol oxidase enzyme reactor. Nahrung Food 46(1):46 50. Braddock R. 1999. Handbook of Citrus by products and processing techn ology. New York: John Wiley & sons Campbell A, Uttamchandani D. 2004. Optical dissolved oxygen lifetime sensor ba sed on sol gel immobilisation. L ee Proceedings Science Measurement and Technology 151(4):291 7. Castellari M, Matricardi L, Arfelli G, Carpi G, Galassi S. 2000. Effects of high hydrostatic pressure processing and of glucose oxidase catalase addition on the color stability and sensorial score of grape juice. Food Science and Technology International 6(1):17 23. Choi MMF, Xiao D. 2000. Single stand ard calibration for an optical oxygen sensor based on luminescence quenching of a ruthenium complex. Analytica Chimica Acta 403:57 65. Chu CS, Lo YL. 2007. High performance fiber optic oxygen sensors based on fluorinated xerogels doped with Pt(II) complexe s. Sensors and Actuators B Chemical 124(2):376 82. Cole CA, Genetell.Ej. 1970. D ecarbonation and deaeration of water by use of selective hollow fiber s Environmental Science & Technology 4(6):514 &. Coughlin RW, Charles M, Julkowski K. 1978. Experimental r esults from a pilot plant for converting acid whey to potentially useful food products. AIChE Symposium Series 74(172):40 6. Dhuique Mayer C, Tbatou M, Carail M, Caris Veyrat C, Dornier M, Amiot MJ. 2007. Thermal degradation of antioxidant micronutrients i n Citrus juice: Kinetics and newly fo rmed compounds. Journal of Agricultural and Food Chemistry 55(10):4209 16.

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64 Falade KO, Babalola SO, Akinyemi SOS, Ogunlade AA. 2004. Degradation of quality attributes of sweetened Julie and Ogbomoso mango juices during s torage. European Food Research and Technology 218(5):456 9. Garcia Torres R, Ponagandla NR, Rouseff RL, Goodrich Schneider RM, Reyes de Corcuera JI. 2009. Effects of dissolved oxygen in fruit juices and methods of removal. Comprehensive Reviews in Food Sci ence and Food Safety 8(4):409 23. Gill CB, Menneer ID. 1997. Advances in gas control technology in the brewery. The Brewer:76 84. Grosova Z, Rosenberg M, Rebros M. 2008. Perspectives and applications of immobilised beta galactosidase in food industry a r eview. Czech Journal of Food Sciences 26(1):1 14. Illanes A, Wilson L, Raiman L. 1999. Design of immobilized enzyme reactors for the continuous production of fructose syrup from whey permeate. Bioprocess Engineering 21(6):509 15. Jiahui Shao Huifeng Liu, Yiliang He .2008. Boiler feed water deoxygenation using hollow fiber membrane contactor. Desalination 234 (1 3):370 77 Jiang DS, Chen X, Liu E, Huang J. 2002. Preparation and properties of sensing membrane for fiber optic oxygen sensor. Journal of Wuhan Un iversity of Technology Materials Science Edition 17(2):51 3. Johnson JR, Braddock RJ, Chen CS. 1995. Kinetics of ascorbic acid loss and nonenzymatic b rowning in orange juice serum: E xperimental rate constants. JFS 60(3):502 5. Jordan MJ, Goodner KL, Laenci na J. 2003a. Deaeration and pasteurization effects on the orange juice aromatic fraction. Lebensmittel Wissenschaft Und Technologie Food Science and Technology 36(4):391 6. Jordan MJ, Goodnerb KL, Laencinaa J. 2003b. Deaeration and pasteurization effects o n the orange juice aromatic fraction. Lebensm Wiss U Technol 36:391 6. Joslyn M. 1961. Physiological and enzymological aspects of juice production. DK T, editor. Westport: Conn: Avi Pub Co Kalisz HM, Hecht HJ, Schomburg D, Schmid RD. 1990. C rystallization and preliminary x ray diffraction studies of a deglycosylated glucose oxidase from aspergillus nige r Journal of Molecular Biology 213(2):207 9. Kennedy JF, Rivera ZS, Lloyd LL, Warner FP, Jumel K. 1992. L ascorbic acid stability in aseptically processed orange juice in TetraBrik cartons and the effect of oxygen. Food Chemistry 45:327 31.

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65 Klimant Ingo, Wolfbeis Otto S. 1995. Oxygen Sensitive Lumine scent Materials Based on Silicone Soluble Ruthenium Diimine Complexes Analytical Chemistry 67(18): 3160 66. Kothapalli A, Hayes K, Sadler G, Morgan M. 2007. Comparison of kinetic profile of free and immobilized glucose oxidase, immobilized on low density polyethylene using UV polymerization. Journal of Food Science 72(9):C478 C82. Lee SK, Okura I. 1997. Optical sensor for oxygen using a porphyrin doped sol gel glass. Analyst 122(1):81 4. Lewis ME. 2006. National Field Manual. Chapter 6.2 Dissolved oxygen. In: (USGS) USGS, editor: Office of Water Quality. Mack TE, Heldman DR, Singh RP. 1976. K inetics of oxygen uptake in liquid foo ds Journal of Food Science 41(2):309 12. Manso MC, Oliveira FAR, Oliveira JC, Frias JM. 2001a. Modelling ascorbic acid thermal de gradation and browning in orange juice under aerobic conditions. International Journal of Food Science and Technology 36(3):303 12. Manso MC, Oliveira FAR, Oliveira JC, Fras JM. 2001b. Modelling ascorbic acid thermal degradation and browning in orange jui ce under aerobic conditions Int J Food Sci Technol 36(3):303 12. McDonagh C, MacCraith BD, McEvoy AK. 1998. Tailoring of sol gel films for optical sensing of oxygen in gas and aqueous phase. Anal Chem 70:45 50. Mistry B, Min DB. 1992. R eduction of dissolve d oxygen in model salad dressing by glucose oxidase catalase dependent on p H and temperature Journal of Food Science 57(1):196 9. Ogurtsov VI, Papkovsky DB. 1998. Selection of modulation frequency of excitation for luminescence lifetime based oxygen senso rs. Sensors and Actuators B Chemical 51(1 3):377 81. Ough CS. 1975. F urther investigations with glucose oxidase catalase enzyme systems for use with win e American Journal of Enology and Viticulture 26(1):30 6. Ovsejevi K, Grazu V, Batista Viera F. 1998. b eta galactosidase from Kluyveromyces lactis immobilized on to thiolsulfinate/thiolsulfonate supports for lactose hydrolysis in milk and dairy by products. Biotechnology Techniques 12(2):143 8. Parpinello GP, Chinnici F, Versari A, Riponi C. 2002. Prelimina ry study on glucose oxidase catalase enzyme system to control the browning of apple and pear purees. Lebensmittel Wissenschaft Und Technologie Food Science and Technology 35(3):239 43.

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66 Peleg H, Naim M, Zehavi U, Rouseff RL, Nagy S. 1992. Pathways of 4 Viny lguaiacol Formation from Ferulic Acid in Model Solutions of Orange Juice. Journal of Agricultural and Food Chemistry 40(5):764 7. Pickering GJ, Heatherbell DA, Barnes MF. 1998. Optimising glucose conversion in the production of reduced alcohol wine using g lucose oxidase. Food Research International 31(10):685 92. Rassis D, Saguy IS. 1995. Oxygen effect on nonenzymatic browning and vitamin c in commercial citrus juices and concentrate Food Science and Technology Lebensmittel Wissenschaft & Technologie 28(3) :285 90. Reyes De Corcuera JI, Cavalieri RP, Powers JR, Tang JM, Kang DH. 2005. Enzyme electropolymer based amperometric biosensors: An innovative platform for time temperature integrators. Journal of Agricultural and Food Chemistry 53(23):8866 73. Ringblo m U. 2004. The orange book. 2nd ed. Lund, Sweden: Tetra pak Roig MG, Bello JF, Velasco FG, Decelis CD, Cachaza JM. 1987. A pplications of immobilized enzymes Biochemical Education 15(4):198 208. Roy I, Gupta MN. 2003. Lactose hydrolysis by Lactozym (TM) im mobilized on cellulose beads in batch and fluidized bed modes. Process Biochemistry 39(3):325 32. Roy I, Sardar M, Gupta MN. 2000. Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds. Enzyme and Microbial Techno logy 27(1 2):53 65. Sagi I, Mannheim CH. 1990. T he effect of enzymatic oxygen removal on quality of unpasteurized and pasteurized orange juice Journal of Food Processing and Preservation 14(4):253 66. Sankaran K, Godbole SS, Dsouza SF. 1989. P reparation o f spray dried, sugar free egg powder using glucose oxidase and catalase coimmobilized on cotton clot h Enzyme and Microbial Technology 11(9):617 9. Shin DH, Cheigh HS, Lee DS. 2002. The use of Na2CO3 based CO 2 absorbent systems to alleviate pressure buildu p and volume expansion of kimchi packages. Journal of Food Engineering 53(3):229 35. Shin MC, Kim HS. 1995. E ffects of enzyme concentration and film thickness on the analytical performance of a polypyrrole glucose oxidase biosenso r Analytical Letters 28(6 ):1017 31. Singh RP, Heldman DR, Kirk JR. 1976. K inetics of quality degradation ascorbic acid oxidation in infant formula during storage Journal of Food Science 41(2):304 8.

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BIOGRAPHICAL SKETCH Narsi Reddy Ponagandla was born in 1984, to Bhaskar Reddy and Annapurna in Kodad, India. He attended the Osmania University, Hyderabad, India from 2003 to 2007. He earned his Bachelor of Technology in Fo od Processing Technology in May 2007. He joined the University of Florida oo d science under Dr. Reyes De Corcuera