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Photochemically Induced Flavor Changes in Orange Juice Exposed to Light in Glass and Polyethylene Terephthalate at 4 C

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PHOTOCHEMICALLY INDUCED FLAVOR CHANGES IN ORANGE JUICE EXPOSED TO LIGHT IN GLASS AND POLYETHYLENE TEREPHTHALATE AT 4C By KRISTIN ANN NELSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Kristin Ann Nelson

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I would like to thank my husband Jared, for his support and encouragement these past two years. He has been there to offer advi ce when things did not go as planned and to cheer me on when things did. I would also li ke to thank my parents who have been there since day one. No matter where my travels ta ke me, they have always been ready with bags packed to rush to my aid when they were needed. Without my family, I would not be where I am today and therefore I dedicate this project to them.

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iv ACKNOWLEDGMENTS I would like to first thank my professor, Dr. Russell Rouseff, for all of his help on this project. His expertise in multiple fields was a great asset in setting up the experiment and analyzing the resu lts. Dr. Rouseff went above and beyond by providing hands on assistance and knowledge in constructi ng equipment for this experiment. I would also like to express my gratit ude to my committee members Dr. Renee Goodrich, Dr. Ronald Schmidt, and Dr. Kath ryn Williams for their guidance. Their insight and suggestions we re a great aid in desi gning this experiment. Also, I would like to thank the Department of Citrus and the University of Florida for their financial support of this project. Next, I would like to thank everyone at the Citrus Res earch and Education Center for their help and for allowing me to use th e facility for my experimentation and data collection. Big thanks are owed to Jack Smoot, and to my fellow lab workers, Wendy Bell, Kanjana Mahattanatawee, and Filomena Va lim for assisting me with my experiment and for helping me learn to use the laboratory instruments. Special thanks are given to April Elston for performing GC-O work for th is experiment and for taking the time to answer all my lab questions. Also, thanks go to Dr. Mickey Parish and Lorrie Friedrich for their help performing microbiology test s, Gary Coats for his help using the spectrophotometer, Yehong Xu for her help w ith ascorbic acid measurements, and Dr. Bruce Welt for help measuring dissolved oxygen.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Orange Juice Volatiles and Oxidation Reactions.........................................................3 Packaging Materials and Interactions...........................................................................4 Scalping.................................................................................................................4 Leaching................................................................................................................6 Permeation through Package.................................................................................6 Light and Oxygen Effects.............................................................................................6 Browning...............................................................................................................6 Ascorbic Acid Loss...............................................................................................8 Photochemical Reactions.......................................................................................9 Extracting and Concentra ting Flavor Volatiles..........................................................10 Sample Extraction...............................................................................................10 Sample Concentration.........................................................................................11 Gas Chromatography-Olfactom etry History and Methods.........................................12 Purpose.......................................................................................................................1 3 3 MATERIALS.............................................................................................................14 Light Chamber............................................................................................................14 Orange Juice...............................................................................................................15 Storage Containers......................................................................................................16 4 METHODS.................................................................................................................17 Initial Juice Measurements.........................................................................................17

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vi Storage Conditions......................................................................................................18 Storage Studies...........................................................................................................18 Sample Preparation.....................................................................................................19 Sensory and Analytical Tests......................................................................................19 Sensory Analysis.................................................................................................19 Juice Color Measurements..................................................................................20 Ascorbic Acid Measurement...............................................................................20 Gas Chromatograph-Flame Ioniza tion Detector / Olfactometer.........................21 Gas Chromatography – Mass Spectrometry........................................................21 Microbiological Analyses....................................................................................22 Statistical Analysis......................................................................................................22 5 RESULTS AND DISCUSSION.................................................................................23 Orange Juice Properties..............................................................................................23 Sensory Flavor Changes.............................................................................................23 Color Changes............................................................................................................25 Ascorbic Acid Changes..............................................................................................28 Aroma Active Compounds (GC-O Studies)...............................................................29 Qualitative Differences...............................................................................................35 Quantitative Differences.............................................................................................37 Sulfur Smelling Aroma Compounds...................................................................48 Accelerated Study................................................................................................50 Microbiological Evaluation........................................................................................51 6 CONCLUSIONS........................................................................................................53 APPENDIX A STATISTICAL TEST FOR “L” SIGNIFICANCE....................................................55 B STATISTICAL TEST FOR “A” SIGNIFICANCE...................................................56 C STATISTICAL TEST FOR “B” SIGNIFICANCE....................................................57 D STATISTICAL TEST FOR MYRCENE SIGNIFICANT DIFFERENCE................59 E STATISTICAL TEST FOR CARV ONE SIGNICANT DIFERENCE......................61 G GC-FID RESULTS FOR JUIC ES STORED IN PLASTIC.......................................63 H GC-FID RESULTS FOR JUIC ES STORED IN GLASS..........................................65 LIST OF REFERENCES...................................................................................................66 BIOGRAPHICAL SKETCH.............................................................................................71

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vii LIST OF TABLES Table page 1 Initial Orange Juice Properties.................................................................................23 2 Sensory Analysis of Juice after Twelve Weeks.......................................................24 3 Ascorbic Acid Loss During Storage.........................................................................29 4 Aroma Active Compounds Identified in Initial Valencia Orange Juice..................31 5 Aroma Active Compounds in Juice Stor ed in Plastic for Twelve Weeks................32 6 Aroma Active Compounds in Juice St ored in Glass for Twelve Weeks.................33 7 pcymene Comparison for Juice Stored Twelve Weeks..........................................43 8 Comparison of PVG after Twelve Weeks Storage Using GC-MS..........................47 9 GC-FID Responses for -terpineol In Juices St ored for Twelve Weeks.................48

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viii LIST OF FIGURES Figure page 1 Diagram of Light Chamber......................................................................................14 2 Picture of Light Chamber in Cold Storage...............................................................14 3 Wavelength Spectrum for Philips Cool White Fluorescent Lights..........................15 5 "L" Values for Juices after Twelve Week Storage Study........................................26 6 “a” Values for Juices after Twelve Week Storage Study.........................................27 7 “b” Values for Juices after Twelve Week Storage Study.........................................28 8 Comparison of De tector Responses.........................................................................34 9 Aroma Active Compounds in Juices Stored in PET for Twelve Weeks..................35 10 Aroma Active Compounds in Juices Stored in Glass for Twelve Weeks................36 11 Decrease in Myrcene after Twelve Week Storage Study.........................................37 12 Increase in Carvone after Twelve Week Storage Study...........................................39 13 Increase in 1,8-Cineole af ter Twelve Week Storage Study......................................41 14 p-cymene Peak Area Measurement on GC-MS.......................................................42 15 Increase in Vanillin Duri ng Twelve Week Storage Study.......................................44 16 Increase in Furaneol Duri ng Twelve Week Storage Study......................................45 17 4-Vinyl Guaiacol Peak Area Measurement on GC-MS...........................................47 1 Increase in Sulfur Compound (LRI 1116) During Twelve Week Study..................49 19 Formation of Sulfur Compound (LRI 806) after Accelerated Storage Study..........51 20 Increase in Sulfur Compound (LRI 900) after Accelerated Storage Study..............52 21 Significant Difference Between L Values................................................................55

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ix 22 Significant Difference in "a" Values........................................................................56 23 Significant Difference in "b" values.........................................................................57 24 Significant Difference in Amount of Myrcene........................................................59 25 No Difference in Amount of Myrcene.....................................................................60 26 Significant Difference in Amount of Carvone.........................................................61 27 No Difference in Amount of Carvone......................................................................62

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHOTOCHEMICALLY INDUCED FLAVOR CHANGES IN ORANGE JUICE EXPOSED TO LIGHT IN GLASS AND POLYETHYLENE TEREPHTHALATE AT 4C By Kristin Ann Nelson May 2005 Chair: Russell L. Rouseff Major Department: Food Science and Human Nutrition Pasteurized Valencia orange juice wa s stored in glass and polyethylene terephthalate containers and e xposed to fluorescent light at 4C for twelve weeks. The flavor, color and ascorbic acid concentrations of juices exposed to light were appreciably different than control samples covered with al uminum foil. Light exposed juices became darker, as indicated by signi ficant (p<0.01) decreases in “L” values. Light exposed orange juices lost 21% and 68% more ascorbic acid than unexposed controls when stored in plastic and glass cont ainers respectively. Using a defined 15 point flavor quality scal e, a trained sensory panel judged the light exposed juices to be of lower quality than controls. Juices exposed to light for twelve weeks had an average rating of 3.8 whereas control juices had an average rating of 6.8. In addition to overall flavor eval uations, individual aroma components were evaluated using GC-olfactometry (GC-O). Light exposed samples contained less -

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xi myrcene, and more carvone, 1,8-cineole, pcymene, vanillin, and Furaneol. Extracted ion chromatogram GC-MS data indicated that li ght exposed juices ha d on average 85% more pcymene than those that were not expose d. Vanillin, Furaneol, and some sulfur compounds typically form in juice due to thermal degradation a nd thermally induced non-enzymatic browning. However, all juices were stored at 4C which is well below the minimum temperatures needed to produce th ermal degradation by classical chemical means, suggesting these compounds were pr oducts of photochemical reactions. The thermally induced off-flavors -terpineol and 4-vinylguaiaco l did not increase in light exposed samples, indicating that these two de gradation products were not catalyzed by light-exposure. Two tentatively iden tified sulfur compounds were obs erved in juice samples that were exposed to light. The compound 4-mercapto-4-methylpentan-1-ol had an onionlike, moldy, or soured aroma and 3-mercapto-hexen-1-ol had a moldy or soured aroma. In addition, two sulfur smelling compounds (R .I. of 806 and 900) were produced only in light exposed juices during an accelerated st orage study. The appearance of these offaromas in only light exposed juices partia lly explains why the overall orange juice character was degraded.

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1 CHAPTER 1 INTRODUCTION Citrus is one of the most important agricultural crops in Florida with annual orange juice sales topping three billi on dollars in 2004. However, in recent years there has been a steady decline in orange juice consumption and industry profits ( 1 ). Manufacturers have found that using different packaging ma y increase sales. A recent trend is to employ clear containers in order to attract consumers with the fresh, bright color of citrus juices ( 2 ). Glass has been used for this purpose; however, this material is expensive, heavy, and prone to breakage. An alternat ive is to utilize clear plastic such as polyethylene terephthalate (PET) The plastic is lightweight robust, and inexpensive. Limitations include low oxygen a nd light barrier properties. There have been numerous reports concerni ng the influence of light, and container oxygen permeability on color changes and Vitamin C losses ( 3-5 ). Solomon et al. ( 3 ) reported that light had no effect on ascorbic acid content and an in significant effect on browning on orange juices stored for 52 da ys at 8C whereas Ahmed and coworkers ( 5 ) reported a 20% loss of ascorbic acid in only si x days at a similar storage temperature. Sensory panels have also compared light e xposed orange juices with controls and found significant differences. Alt hough overall flavor changes have been reported, the individual flavor compounds responsible for these changes have yet to be identified. Light and oxygen studies conducted using orange oil and lemon oil aqueous emulsions at room temperature and slightly elevated temper atures have shown that changes in flavor compounds are due to specific chemical and photochemical reaction products ( 6-9 ).

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2 Since orange juice has many compounds in co mmon with orange and lemon oils, it is hypothesized that similar photochemical reactions might occur in orange juice. However the rate at which these products would form at 4C is uncertain.

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3 CHAPTER 2 LITERATURE REVIEW Orange Juice Volatiles and Oxidation Reactions The aroma of orange juice consists of a combination of volatile compounds in specific proportions. The classes that ma ke up orange juice flavor are terpenes, aldehydes, esters, alcohols and sulfur compounds. Terpenes make up the largest percentage of orange volatiles, with the ma in terpenes being d-limonene, myrcene, and valencene. Although most terpenes do not play a direct role in ora nge juice flavor, they may work as carriers for othe r oil-soluble volatiles. The aldehydes that are thought to make the largest contribution to flavor are acet aldehyde, citral, octanal, nonanal, decanal, and sinensal. According to Shaw ( 10 ) ethyl butanoate, ethyl 2-methylbutanoate, ethyl propionate, methyl butanoate, and ethyl 3hydroxyhexanoate are the major esters in orange juice and are responsible for the fruit y, “top note” aroma in fresh juice. Shaw ( 10 ) has also indicated that alcohol s such as ethanol, E-2-hexeno l, Z-3-hexenol, linalool, and -terpineol are present in orange juice, but few make a signif icant contribution to flavor. Finally, it has been suggested th at various sulfur compounds th at are present at very low concentrations in the juice make a large contri bution to the overall flavor. Some of these compounds are hydrogen sulfide, methan ethiol, and dimethyl sulfide. ( 10 ) “Off-flavor” is defined as a fl avor that is not natural or normally present in fresh foods resulting from deterioration or contam ination. Off-flavors in orange juice are primarily formed due to reactions with oxyge n. Oxidation reactions decrease ascorbic

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4 acid levels, and induce terpene oxidation. Aerobic microbiological growth can also produce off-flavors in orange juice ( 11 ). These situations occur when oxygen is di ssolved in the produ ct, through contact with oxygen in the headspace and from oxygen diffusion through the container material. Decomposition of residual hydrogen peroxi de can also produce oxygen and oxidation reactions in those cases where it has been us ed as a package sanitizer. Dissolved oxygen in the initial juice can be decreased by deaeration, and oxygen in the headspace can be reduced by filling the container completely or by flushing it with nitrogen. However, the only way to reduce oxygen permeation through the package is by changing the barrier properties of the container. Packaging Materials and Interactions Orange juice is packed in a wide vari ety of materials including metal cans, paperboard cartons, plastic co ntainers, and glass bottles. Although these materials are designed to protect the juice, p ackaging materials can also aff ect the juice’s flavor in one of at least three ways. The three primary flavor altering processes associated with packaging are flavor scalping, flavor leach ing, and permeation of compounds through the package. Scalping Scalping refers to the absorption of one or more compounds from the orange juice into the packaging material. This proce ss has been observed primarily in plastic containers. Even though it is generally accepte d that volatile composition is altered as a result of scalping, there were conflicting reports as to whethe r the loss of certain volatile compounds influenced the juice’s taste or aroma.

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5 In 1987, Kwapong and Hotchkiss studied citrus e ssential oil solutions stored in low density polyethylene (LDPE) a nd two polyethylene ionomers ( 12 ). Orange oil components were not sorbed equally. Benzalde hyde and ethyl butyrate were sorbed to at approximately equal levels with Ke values ranging from 2-7, where Ke = C(plastic)/C (aq solution). Neral and geranial were moderately sorbed with Ke values ranging from 15 to 23 and 22 to 40 respectively. Limonene was h eavily sorbed especially in LDPE with a Ke value of 4700. Ten untrained panelists detect ed significant differences (p<0.05) in the aroma of the citrus samples using the triangl e test. Also in 1987, Manheim, Miltz, and Letzter compared orange and gr apefruit juices stored in lami nated cartons and glass jars at 35C for 10-12 weeks ( 13 ). They found a 25% loss of limonene in both orange and grapefruit juice stored in cart ons within 14 days of storage. A panel of twelve to fifteen experienced tasters detected a significant diffe rence (p<0.05) in the juices flavor. In 1992, Marin and colleagues observed an 80% lo ss of orange juice limonene into LDPE within 24 hours at 25C( 14 ). They used gas chromatography-olfactometry (GC-O) to determine that limonene has relatively low ar oma activity and thus contributes little if any to overall orange juice flavor and aroma. Also in 1992, Pieper and colleagues compared preference scores for orange juices stored at 4C for 24 weeks in glass and LDPE cartons ( 15 ). They found that although the plas tic cartons absorbed 50% of the limonene, no significant difference was observe d between the hedonic scores of any of the juices. Ethyl butyrate (which was thought to be an important aroma component) was not absorbed to a measurable extent by th e plastic container. In 1997, Sadler and colleagues examined sorption of orange juice volatiles into LDPE, polyethylene terephthalate (PET), polyamide (PA), and ethylene (co-)vinyl alcohol ( 16 ). The juice

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6 was maintained at 4.5C while the polymer stri ps were exposed for three weeks. Juices were stored in sterile Erlenmey er flasks in contact with the different polymer strips at a surface to volume ratio that was twice that which is used commercially. No significant flavor difference was detected in between a ny polymer treated and control juices using triangle tests with 15-22 e xperienced panelists. Leaching Leaching refers to the migration of com pounds from the container into the orange juice. This can be caused by residual m onomers, plasticizers, processing aids, and solvents from printing inks and adhesives ( 17 ). In the orange juic e industry, this problem includes off-flavors caused by juice stored in metal cans. In 2000, Takahashi and colleagues concluded that plated tin inside the can reacted with dissolved oxygen to cause unwanted reactions in fresh mandarin orange juice ( 18 ). Permeation through Package Permeation relates to movement of flavor compounds through the package. This includes flavor compounds leaving the juic e, and unwanted flavors entering the juice from outside the container. The greatest problem with permeation in orange juice is oxygen being transferred into the container and negatively impacting the juice inside. Light and Oxygen Effects When packaging materials do not provide an adequate barrier to light and oxygen, the juice’s quality can be affected. The most common problems involve browning of the juice, loss of ascorbic acid, and change s in the overall flavor of the juice. Browning Non-enzymatic browning, or the Maillard reaction, occurs when reducing sugars such as sucrose, glucose, and fructose r eact with proteins, peptides, amino acids or

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7 amines ( 19 ). The reaction is favored by higher te mperatures, lower water activities, and during extended storage. Non-enzymatic brow ning is an undesirable reaction that occurs in orange juice when heated during pasteuriza tion or storage. The reaction causes a loss of essential amino acids and leads to th e formation of brown pigments known as melanoidins that cause juice colors to dark en. Compounds such as furaneol, norfuraneol, furfural, 5-hydroxymethylfurfural, and sotol one are produced and c ontribute to flavor changes in the juice ( 20 ). The greatest driving force of these reactions is increased temperature, and the presen ce of these compounds has been correlated to elevated temperature storage. Several studies have investigated the e ffects of light and oxygen on browning in orange juice. In 1995, Solomon et al. conduc ted research on pasteu rized orange juice stored for fifty-two days at 8C ( 3 ). The juice was stored in glass containers with glass, polyethylene and paper closures. They found that browning was signi ficantly (p<0.001) correlated with the amount of dissolved oxygen in the ju ice which occurred to the greatest extent in paper capped bottles becau se they had the greatest oxygen permeability. However, the difference in the extent of juice browning due to light-exposure was found to be insignificant (p<0.05). In 1986, Trammell and colleagues conducte d an experiment on single-strength orange juice with varyin g dissolved oxygen levels ( 21 ). Juice was stored for five months at 22C. It was again found that greater am ounts of oxygen in the juice led to increased browning. However, a sensory evaluation did not detect changes in the juice flavor (p<0.05).

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8 Ascorbic Acid Loss The majority of research has investigated the effect of light and oxygen on ascorbic acid, also known as Vitamin C, in the juice because of its nutritional value. In 1976, Ahmed and colleagues investigated the effect s of fluorescent light on flavor changes and ascorbic acid loss in reconstituted orange juice and orange drinks ( 5 ). The juices were divided into plastic, glass, and paperboard containers and were placed in a light chamber at 6C for 6 days. It was found that, when e xposed to light, the ora nge juice lost 20% of its ascorbic acid and the orange drink lost 40-90%. A trai ned taste panel of 10-12 women performed a hedonic scale rating that indicate d the juice in the paperboard containers tasted significantly better (p<0.05) than juice in the lig ht exposed plastic and glass bottles. In 1992, Kennedy and colleagues studied co mmercial single-strength orange juice in TetraBrik cartons ( 22 ). The juices were stored at 4, 20, 37, 76, and 105C for sixty days. They found that juices with lower initial dissolved oxyge n (1.70ppm compared to 4.30ppm) had a slower rate of ascorbic acid loss (6.5mg/L*day compared to 25.5mg/L*day). It was also found that the temp erature the juice was stored at plays the greatest role in deterioration in that the hi gher the temperature, the more ascorbic acid was lost. Sattar and colleagues performed similar experiments in 1989 using pasteurized orange drink in clear, green and amber glass bottles as well as in a wax laminated paper (TetraPak) carton ( 4 ). The containers were stored at room temperature for thirty-two days. Ascorbic acid losses were 60.6%, 54.6%, 51.0%, and 45.5% in clear glass, green glass, TetraPak laminated paper, and amber glass respectively. This shows that greater light-exposure resulted in signifi cantly greater (p<0.05) ascorbic acid loss. Also, the loss

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9 in TetraPak cartons was greater than in amber bottles because of the higher oxygen permeability. A ten member panel performed hedonic ratings based on color, taste, and flavor and reported higher preference ratings for juices stored in amber bottles. Photochemical Reactions Several previous studies have investigat ed the changes in flavor compounds in lemon oil as a result of exposure to oxygen and light. In 1988, Schieberle and Grosch studied lemon oil in an aque ous citric acid emulsion ( 7 ). The samples were left for thirty days at 37C. The team found that neral, geranial, and linalool decreased, with a corresponding increase in pmethylacetophenone, pcresol, fenchyl alcohol, pcymene, and 1-terpinen-4-ol. They believe this ch ange in composition is responsible for the deterioration of lemon oil flavor over time. In 1997, Iwanami and colleagues also investig ated the effects of ultraviolet light on lemon oil ( 6 ). The team exposed a mixture of lem on oil, a phosphate buffer, and ethanol to UV-light ( 400nm) for four days at 30C. They found that citral (a combination of neral and geranial), limon ene, terpinolene, and nonanal decreased, while the decomposition product, pcymene, increased. The finding that citral was the most unstable component and that pcymene was produced mirrored the results of Schieberle and Grosch. In 1991, Ziegler and colleagues studied the changes in flavor compounds in orange oil after exposure to ultra-violet light ( 9 ). The orange oil was dissolved in ethanol, acidified with citric acid and homogenized to form an emulsion. The emulsions were exposed to ultraviolet light for fifty minutes at 20C. Ziegler found a significant increase in carvone, isopulegol, isomer s of carveol, limonene oxide, and linalool oxide with a corresponding decrease in nera l, geranial, and cintronellal. In addition, several new

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10 compounds formed during the study including pmentha-1,8,dien-4-ol, -cyclocitral, photocitral A, iso(iso)pulegol, carvonecam phor, methone, isomenthone, isomers of pmenth-1(7),8-dien-2-ol and isopiperitenol. Extracting and Concentrating Flavor Volatiles Volatiles in orange juice are in very low concentrations and are part of a complex matrix of insoluble and nonvolatile compounds that cannot be injected into a gas chromatograph. For these reasons the volatiles in the orange juice must be isolated and concentrated before analysis can occur. Sample Extraction Two common extraction techniques are h eadspace analysis (either static or dynamic) and liquid-liquid extraction. Thes e techniques have various advantages and limitations and the method performed depends on the compounds of interest. Regardless of the isolation technique, the goal remains to make the extraction representative of the original sample. Liquid-liquid extraction relies on the differences in polar ity to extract the desired compounds from the overall sample. The solv ent chosen for the extraction determines which compounds will be isolated. Compounds that are non-polar will be extracted to a greater extent by a non-polar so lvent such as pentane. Th e juice and extracting solvent are thoroughly mixed, and centrifuged to sepa rate aqueous and organic layers. The organic layer is retained, while the aqueous layer may be discarded or can be extracted with the same solvent or another solvent rep eatedly. However, the more solvent that is used for extraction, the further the sample must be concentrated before analysis, and thus the more volatiles may be lost.

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11 One advantage of this extraction method is that the solvent comes in direct contact with the juice and extracts the volatiles fr om the juice matrix, whereas in headspace analysis the volatiles must partition from the pulp into the aqueous phase, come to equilibrium with the headspace, and finally adhere to the fiber coating. Liquid-liquid extraction allows for a more accurate quantif ication of a wider range of volatiles, as competition for headspace and fiber coating is e liminated. One drawback is that it also isolates some non-volatile material such as ca rotenoids and lipids that can degrade in the GC injector and form artifacts. It is important to note that not all ch emical compounds have the same extraction efficiencies, and thus are not in the same proportions in the extract as they were in the original juice. This can be overcome by adding internal standa rds to the sample mixture. Internal standards should be chosen such that the standard and the compound of interest have similar structures a nd physical properties. Idea lly, for GC-MS analysis, a deuterated isomer of the desired compound should be used for comparison, since it will have the same extraction efficiencies and eva poration loses. Knowing the concentration of the internal standard can allow for b ack calculation to find the concentration of compounds in the original sample. Sample Concentration Concentration can be achieved by a vari ety of methods. The nitrogen blowdown method is commonly used. In this method, a stream of nitrogen gas is gently blown across the surface of the extrac t. This action increases the speed at which the solvent evaporates. The nitrogen blowdown method must be performed slowly so that the volatiles of interest are not lost with the solven t. Also, it is important to use a solvent that has much lower boiling point from that of th e volatiles to reduce loss. After sample

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12 preparation, the juice extract can then be injected into an anal ytical tool such as the gas chromatograph. Gas Chromatography-Olfactometry History and Methods Gas chromatography is a method that em ploys a capillary column of varying materials in order to separate chemical com pounds based on their polar ity and affinity for the column material. The temperature inside the column is steadily increased, causing the compounds to elute from the column at di fferent retention times. Various instruments such as a flame ionization detector (FID) or a mass spectrometer (MS) can be used for compound detection. In 1964, Fuller, Steltenkamp, and Tisse rand first reported the use of an olfactometer ( 23 ). This device consisted of a sniff port attached to the GC, parallel to the detector. A human assessor c ould sit at the sniff port and observe the eluting aromas. This new technique helped researchers determine compound identities and also to establish which compounds were aroma active. Based on the intensity of the aromas, researchers could also determine how much each compound contributed to the overall aroma profile. This was a significant advan cement in gas chromatography, in that some highly aroma active compounds that occurred at very low concentratio ns in orange juice and were previously overlooked, now received greater focus. There are three main types of GC-O an alysis: Dilution Analysis, Time-Intensity Analysis, and Detection Frequenc y. In Dilution Analysis, a sa mple is repeatedly reduced in concentration and analyzed on the GC-O. It is noted wh ich compounds are detected at each dilution level. This information can be used to determine each chemical compound’s threshold. The threshold is then divided by the concentration of that compound in the original sample in order to determine the compound’s aroma activity.

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13 The higher the activity, the more that co mpound contributes to the sample’s overall aroma. The two types of Dilution Anal ysis are Aroma Extract Dilution Analysis (AEDA) which measures the peak height ( 24 ), and Combined Hedonic Aroma Response Measurement (CHARM) which measures the peak area ( 25 ). Time-Intensity Analysis consists of only running the sample once on the GC-O with no subsequent dilutions. During the GC run, the aroma intensity of each compound is noted by sliding a lever across a sectioned bar. The bar contains va rious intensity descriptors such as slight, moderate, and strong, and is attached to a comp uter that records the subject’s inputs. The amount a compound contributes to the sample’s overall aroma is then based on the height or area (OSME analysis) over whic h the subject moved the lever ( 26 ). The third method, Detection Frequency, consists of measuring wh at percentage of asse ssors detects a given aroma at various concentrations and is measured by Surface of Nasal Impact Frequency (SNIF) ( 27 ). In this study time-in tensity GC-O was employed. Purpose The purpose of this study was to determ ine the effect of fluorescent light on ascorbic acid, browning and flavor of orange juices stored in glass and PET. Since both chemical and photochemical reactions occur during a storage study, this study will be conducted at temperatures just above freezi ng (4C) to minimize chemical reactions. Therefore if any major changes occur, they would be due primarily to photochemical reactions. This study will also have practic al significance as these conditions are more similar to retail or market place conditions th an any previous study. This will also be the first study to examine the indivi dual volatile components in lig ht exposed orange juice.

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14 CHAPTER 3 MATERIALS Light Chamber The light chamber was built to maximi ze the light-exposure the bottles would receive. A diagram and picture of the light chamber can be seen in Figures 1 and 2 respectively. In Figure 2, the si de door is open for viewing. Figure 1: Diagram of Light Chamber Figure 2: Picture of Light Chamber in Cold Storage A steel pipe was secured to the center of two circular tables. The pipe rotated freely on its axis to allow the tables to be turned daily. This design ensured that each

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15 container received an equal amount of light-e xposure during storage. The chamber was 32”x 32”x 26”, supported by four 2”x 4” studs and consisted of 5/8”plywood on the top, bottom, and two support sides, and 3/8” plywood on the two opening doors. It was equipped with eight 20W Phillips cool fluorescent light bulbs (Royal Philips Electronics USA, Somerset, New Jersey) that provided an average intensity of 1750 lux as measured on a Lambda Instruments LI-185 light meter (Lambda Instruments Corporation, Lincoln, Nebraska). These bulbs were chosen in order to simulate the wavele ngth of light that the juice would normally experience in a supe rmarket setting. The manufacturer’s specification of the light’s spectrum can be seen in Figure 3. Figure 3: Wavelength Spectrum for Philips Cool White Fluorescent Lights The lights were installed vertically to ensure that the top and bottom platforms would receive equal lighting a nd to reduce shadows. Eight reflective mirrors were placed on the sides of the chamber to reflect and in crease the light intensit y. Finally, two fans were installed in the sides of the chambe r in order to increa se airflow through the chamber and keep temperatures evenly distributed. Orange Juice The orange juice was from late season Va lencias, was not concentrated, and did not have pulp or flavors added. The juice was pa steurized at 212F fo r 10 seconds and then cooled to refrigeration temper ature at a local orange juice processing plant before being

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16 filled into five-gallon aseptic Scholle bags. These were trans ported in coolers in order to assure that the juice did not undergo temp erature abuse. The juice was transferred aseptically into sterilized plastic and glass bot tles and placed into the storage chamber. The total time the juice spent between leaving the processing facility and being placed into the storage chamber was about one hour. Storage Containers The bottles used in this experiment were eight-ounce “B oston round” bottles obtained from Lerman Container Corporati on (Lerman Container Co., Naugatuck, CT). The container materials were polyethylene terephthalate (PET) and glass and were similar dimensions. A graph of the light transmission characteristics for the two materials can be seen below in Figure 4. Bo th materials had similar light transmission characteristics and most of the light emitted by the fluorescent lights was in the wavelength range that the materials transmitte d. Each bottle held 250 mL of juice. 0 20 40 60 80 100 200 400 600 800Wavelength (nm)Percent Transmission Glass Plastic Light emission from Fluorescent Lights Figure 4: Light Transmission Throu gh Glass and Plastic Containers

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17 CHAPTER 4 METHODS Initial Juice Measurements Several tests were performed in order to document starting conditions. The Brix was determined as a measurement of the soluble solids in a juice. One drop of the juice was placed onto a digital refractometer. This device related the juice’s refractive index to Brix ( 28 ). The percent oil in the juice was m easured using the Scott oil test ( 29 ). This test consisted of a titration based on the chemi cal reaction between d-limonene (the main component in orange oil) and bromine. The sample was prepared by mixing it with alcohol and heating it until the alcohol and oi l evaporated. The vapor was condensed and collected for analysis. Bromine was added dro pwise until it completely reacted with all the unsaturated compounds in the oil. Know ing the concentration of the bromine and the amount added to reach the stoichiometric endpoint allowed for the calculation of dlimonene content. This is normally expre ssed as % by volume in 11.8 Brix juice. The total titratable acidity of the sample was determined using a titration procedure ( 28 ). Sodium hydroxide was added to the acid ic juice until a pH of 8.2 was achieved. This pH is used in industry because that is the endpoint of phenolphthalein indicator, which was used before electronic titration de vices were available. Using this endpoint allows for comparison of new values to those determined using the old method. Knowing the amount of juice, and the amount and concentration of sodium hydroxide used allowed for the calculation of the acidity of the orange juice.

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18 Storage Conditions The juice was divided with half going into plastic bottles and the other half into glass bottles. For each container type, there were three replications performed. For each replication there was several bottles filled su ch that a new bottle could be open, analyzed, and frozen on each of the test dates. A second group of identical bottles were also placed in the light chamber. However, this group was wrapped in aluminum foil in order to insure that the juice was not exposed to light during storage. This combination of container type and aluminum foil allowe d for comparison of compounds in juices exposed to light in plastic, light in glass, no light in plastic, and no light in glass. The light chamber was kept in cold storage at 4C. A temperat ure probe on the inside of the storage chamber recorded any temperature deviations. Storage Studies There were two storage studi es conducted during this e xperiment. The first study consisted of storing the juices in the fluorescent light chamber for twelve weeks. In this experiment, juices were tested on the first da y, as well as after 4 weeks, 8 weeks, and 12 weeks. This experiment was designed to monitor changes in juice over an extended amount of time. This should minimize thermally induced chemical reactions and maximize the possibility of photochemical r eactions. The study also had the added advantage in that it would be very similar to what juices might experience under the best of conditions in the market place. The s econd study was an accelerated storage study. At the beginning of the experiment oxygen was bubbled through each of the juices in order to increase the dissolved oxygen content and t hus any oxidation reactions that may occur over time. The juices were then stored for two weeks and samp led after Week 1 and

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19 Week 2. In this experiment all juices were stored in glass bottles and the bottles wrapped in foil were considered “control”. Sample Preparation Orange juice samples were prepared for analysis using liquid-liquid extraction. Twenty-five milliliters of juice were mixed thoroughly with 10 mL of n-pentane solvent in a 50 ml glass syringe. The mixture was then placed into a centrifuge for 10 minutes to allow the aqueous and organic layers to sepa rate. The organic t op layer was drawn off and retained, while the aqueous bottom layer was placed back into the syringe and extracted with 10 mL of ethyl ether. The same process was repeated and this organic layer was combined with the first. A small amount of sodium sulfate was added to the extract to remove any remaining aqueous mate rial. The extract was drawn from the salt and placed into a clean vial. Two internal standards were added to the solution. Fifty microliters of a 2000 ppm solution of ethyl valerate was added to mimic conditions experienced by low molecular weight com pounds, and 50 L of a 2000 ppm solution of 4-heptadecanone was added to mimic those co mpounds with higher molecular weights. A nitrogen blowdown method was em ployed in order to slowly concentrate the sample to 0.1 mL. Sensory and Analytical Tests Sensory Analysis Sensory analysis was performed by five trai ned panelists. The panelists were all members of a group trained to rate orange juice for a study conducted by Elston, Rouseff, and (publication pending). Juices were rated on a fifteen point overall flavor quality scale that reflected attributes such as aroma st rength, orange juice character, peel oil, fatty/metallic/green, fruity/floral, cooke d/heated/processed, sweetness, sourness,

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20 bitterness. In this scale juices ranked between 10 and 15 were considered superior quality, juices ranked between 5 to 10 were good quality juices a nd juices ranked 5 and below were considered to be of poor quality. These quality scores were not a reflection of hedonic rating or preference. The juice was presented at around 17C in an open room under white lighting. Juice Color Measurements The juice was measured on the first day of the experiment as well as after twelve weeks on a Gretag MacBeth Color-EYE 3000 spectrophotometer (Gretag MacBeth, Regensdorf, Switzerland). This device em its a flash of light from a pulsed xenon arc lamp and measures the light re flection from the juice. Samp les were evaluated using the International Commission of Illuminatio n’s *L, *a, and *b standard color space specification as outlined by Lee and Chen in 1998 ( 30 ). The value “L” measures relative lightness or darkness of the juices where L= 0 would correspond to black (total absence of reflected light) and L=100 would correspond to white (total refl ection of incident light). The value “a” is a measure of green to red, with negative numbers indicating more green, a value of 0 being neutral, and positive numbers indicating more red. The value “b” is a measure of blue to yellow, with ne gative values indicating more blue, a value of 0 being neutral, and positive numbers indicating more yellow. Ascorbic Acid Measurement Ascorbic acid measurements were perf ormed using capillary electrophoresis by Yehong Xu at the Florida Department of Citrus using published methods ( 31 ). The sample was prepared by placing 4 ml of jui ce into a capilla ry electrophoresis tube and adding 12 ml of 0.1% ethylenediamine tetraac etic acid (EDTA) solution and 100 l of ferulic acid as an internal standard. The sample was filtered. Injection volume was set at

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21 10 l. The run time was 30 minutes and th e running buffer was 35mM sodium borate and 5 % acetonitrile at a pH of 9.3. The column was uncoated fused silica capillary with dimensions 50 m x 70 cm with a temperat ure of 23-25C. Voltage applied was 21 kv, and scanning was performed between 200 and 360 nm using a Photodiode Array (PDA) detector. Gas Chromatograph-Flame Ionizati on Detector / Olfactometer The gas chromatograph used in this st udy was a HP 5890A (Agilent, Palo Alto, CA) with a Datu (Geneva, NY) high volume olfactometer and described in detail by Bazemore and coworkers ( 32 ). There were two columns used during analysis. The first was a thirty-meter ZB-5 column (Zebron, To rrance, CA) with a 0.32 mm inner diameter, and 0.50 m film thickness. The GC was r un in splitless mode with an injector temperature of 220C, a detect or temperature of 250C, an initial oven temperature of 40C with a 7C/min ramp up to a final ove n temperature of 265C for a 5 minute hold time. The other column was a 30 meter DB-Wax column (J&W Scientific, Folsom, CA). The column’s inner diameter, film thickness, in jection temperature, detector temperature, initial oven temperature, and temperature ramp were the same as previously stated. However, the DB-Wax column utilized a final oven temperature of 240C. All injection volumes were 0.2l. Chromperfect Spirit 5 version 5.0.0 software was used to record data and integrate the resulting chromatograms. Gas Chromatography – Mass Spectrometry The GC-MS used was a Finnigan GCQ Plus system (Finnigan, San Jose, CA) with a DB-5 column (J&W Scientific, Folsom, CA). The column was 60 m long, had an internal diameter of 0.25 mm., and a film thickness 0.25 m. Helium (99.999% purity) was used as the carrier gas. Samples were injected using the AI/AS 3000 autosampler in

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22 the splitless mode with the injector temperature at 200oC. Oven temperature was 40C, and was increased at a rate of 7oC/min to 275C and held for 5 min. Column head pressure was maintained at 14.5 psi. Transf er line and ion source temperatures were 275C and 200C, respectively. The mass spectrometer detector scanned at m/z 40-300. The ionization energy was set at 70 eV. Xcali bur version 1.3 software was used to record and integrate mass spectrometer chromatograms and spectra. Microbiological Analyses On the first day and after one month, microbi al tests were performed to insure that off-flavors were not the results of microbio logical activity. Samples from each of the four material and light-exposure combinations were plated and in cubated for 48 hours at 30C. Potato Dextrose Agar (Difco Laborator ies, Detroit, MI) with 10% tartaric acid was used to test for yeasts and molds, while Oran ge Serum Agar (Difco) was used to test for bacteria and yeast ( 33 ). Statistical Analysis Statistical analysis was performed using Minitab Statistical Software version 13.32. Tests for statistical significance were calculated using an independent two-sample t-test at a 99% confidence interval. A hypothesis of equality was assigned and Minitab was used to calculate t and p values. If the p value was less than the alpha value of 0.01, then the hypothesis was rejected and the two samples were not equal.

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23 CHAPTER 5 RESULTS AND DISCUSSION At the end of the twelve week and the accelerated storage stud ies, several data trends were observed. The results listed below are a combination of tr ends seen in both experiments. The averages and standard de viations are based on tr iplicate analysis from a singe juice container per condition. Although it may be more precise to have multiple juice containers for each condition, the size of the storage chamber and scope of this experiment limited the amount of containers us ed. Container to cont ainer variations are usually due to problems along container seems and closures as seen in metal cans and paperboard cartons. Since all containers us ed in this experiment were blow molded plastic and glass with no seams, the container to container variation should be low, and the results from each sample should be an accurate representation of each storage condition. Orange Juice Properties Initial juice properties ca n be seen in Table 1. Table 1: Initial Orange Juice Properties PropertyValue Brix12.8 Percent Total Acid0.724 Brix/acid ratio17.7 Percent Oil0.0246 Sensory Flavor Changes After twelve weeks, the juices were rem oved from the storage chamber and frozen until sensory evaluations. Juice was also frozen on the first day of the experiment to be

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24 used as a control. Shown in Table 2 ar e the cumulative sensory comments for both orthonasal aroma and flavor impressions for each juice from five trained panelists. Panelists also evaluated each juice for overall fl avor quality based on a 15 point scale. Table 2: Sensory Analysis of Juice after Twelve Weeks Conditions Aroma Flavor Quality Rating Initial Juice Good OJ character, cooked, apricot, citrusy, slight oxidized note Processed, cooked, good sweet/sour balance, good overall flavor, very peely, somewhat musty, oxidized flavor, 8.8 1.3 Light-Plastic No OJ character, peppery, cooked vegetable, solventy, painty, sweet caramel notes, fermented Almost no OJ character, apricot, candy sweetness, burning backend, processed, cotton candy flavor, lack of fruity notes 3.6 1.5 No light-Plastic Sulfury, over mature, fermented, weak OJ character, slightly peppery, fatty aroma, metallic More OJ character, good sweet/sour balance, furaneol sweet, orange peel, bitter, fatty/musty offflavor, no fruity notes 3.8 0.4 Light-Glass Terpeney, peely, week OJ character, slightly peppery, sweet/processed, painty off-flavor Almost no OJ character, artificial candy sweet, sulfury vegetable, acidic, pineapple, bitter, cardboard-like, pronounced off-flavor 4.0 1.6 No light-Glass Musty, peely, terpeney, slightly fermented, peppery, bleach, some OJ character, no offflavor Floral, candy sweet, some OJ character, little sour, slightly cleaner like 6.4 1.1 There was a considerable loss of flavor quality after 12 weeks storage at 4C as noted from the difference in average flavor quality score of th e control juice (8.8) compared to even the highest rated stored ju ice (6.4, no light – glass) Since all juices

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25 (except control) were stored for the same time (12 weeks) and at the same temperature (4C), any flavor changes must be due to c ontainer properties or e xposure to light. Since juices exposed to light in either PET or gla ss had similar ratings, it appears that container material made no difference in overall flavor score. However, it a ppears that container oxygen permeability can influence juices protected from the light. Juices in plastic and not exposed to light had about the same flavor quality scores as light exposed juices. However, orange juice stored in glass and not exposed light had higher flavor quality scores than the similar juice stored in P ET, suggesting that oxygen permeability also influences flavor even at 4C. In general, juices exposed to light exhibited diminished orange juice character and ra ted lower than those that we re not exposed. Therefore exposure to light appears to be a major factor in juice quality. Color Changes The color of the juices exposed to li ght during the twelve week storage study appeared darker than those unexposed. Juice color was evaluated instrumentally using a spectrophotometer to determine their respecti ve L, a, and b values. The relative lightness, redness and yellowness of the stored juices and control are shown in Figures 57. Error bars represent one standard devi ation above and below the average value. Since L measures relative lightness or darkness of the juices, it can be readily seen from Figure 5 that juices expos ed to light have darkened. The light exposed juice had a significantly lower L value (p<0.01) than the or iginal juice (see Appe ndix A for statistical calculations). Those that were not exposed to light showed less change from the original juice. Non-enzymatic browning is typically re sponsible for darkening citrus juices during elevated temperature storage in the abse nce of light. A colored compound has been identified that is thought to be formed by the condensation of the norfuraneol with the

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26 aldehyde group of furfural at elevated temperatures ( 20 ). The darkening of the juices in this study which occurred at 4C indicates that browning can be induced by light as well as elevated storage temperature. 50 54 58 Starting MaterialLight, In GlassNo Light, In GlassLight, In PlasticNo Light, In Plastic Figure 5: "L" Values for Juices after Twelve Week Storage Study

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27 -5 -4 -3 -2 -1 0 Starting MaterialLight, In GlassNo Light, In GlassLight, In PlasticNo Light, In Plastic Figure 6: “a” Values for Juices after Twelve Week Storage Study Since “a” is a measure of green to red, with negative numbers indicating more green, a value of 0 being neutral, and positive numbers indicating more red, it was found that juices that were exposed to light had significantly less (p<0.05) of a green color than the original juice (see Appendix B).

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28 25 30 35 40 Starting MaterialLight, In GlassNo Light, In GlassLight, In PlasticNo Light, In Plastic Figure 7: “b” Values for Juices after Twelve Week Storage Study Since “b” values are a measure of blue to yellow, with negative values indicating more blue, a value of 0 being neutral, and positive numbers indicating more yellow, it was found that those juices that were exposed to light had significantly less of a yellow color than those that were protected (s ee Appendix C). These three measurements confirm that those juices exposed to light did become darker in color and more brown than light yellow or orange. Ascorbic Acid Changes During the twelve month study, ascorbic acids measurements were taken periodically in order to mon itor any losses. The results of these measurements can be seen below in Table 3.

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29 Table 3: Ascorbic Acid Loss During Storage Concentration Ascorbic Acid (g/ml) Initial 320 Light, In Plastic 36 No Light, In Plastic 87 Light, In Glass 45 No Light, In Glass 160 It is apparent that those samples that were exposed to light lost more vitamin C than those samples that were not exposed. Juice in plastic bottles lost 21.5% more vitamin C when exposed to light as compared to juice that was not exposed to light, and juice in glass lost 68.0% more. Also, samples that were stored in plastic bottles lost more ascorbic acid than thos e stored in glass. The results found in this study correspond with those reported in earlier studies concerning ascorbic acid loss and sensory ratings The increased loss of ascorbic acid in juices exposed to light and corresponding decrease in consumer acceptance (through hedonic ratings) is consistent with the fi ndings of both Ahmed and colleagues in 1976 ( 5 )and Sattar and colleagues in 1989 ( 4 ). Also the increased loss of ascorbic acid in juices stored in plastic an d thus allowing more oxygen permeation is consistent with findings by Kennedy and colleagues ( 22 ). Aroma Active Compounds (GC-O Studies) Not all volatiles in orange juice are arom a active. Aroma activity for each volatile must be established using human assessors Instruments detect only those volatiles present in highest concentration. Humans respond only to volatiles with particular functional groups and molecular shape. Ta ble 4 contains a list of the aroma active

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30 compounds that were identifie d in the initial juice by comparing GC-olfactometry descriptors and retention index from bot h DB-5 and Wax columns with published standards ( 34 ). After storage in the light chamber for twelve weeks, some compounds decreased, some increased, and some new compounds form ed. A comparison of the GC-O relative responses (peak area of compound divided by the peak area of the internal standard ethyl valerate) of samples exposed to light and not exposed to light during storage in plastic containers can be seen in Table 5. Likewise, a comparis on of GC-O relative responses for samples stored in glass containers af ter twelve weeks can be seen in Table 6. It is also important to note that some aroma active compounds occur at such low concentrations that they are below the detec tion limits of the flame ionization detector. Alternatively, some compounds th at occur in large concentrat ions in orange juice have very low aroma activity and thus are not dete cted through the olfactometer. An example of this can be seen in Figure 8.

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31 Table 4: Aroma Active Compounds Identifie d in Initial Valencia Orange Juice LRI on DB-5 Identification Se nsory Description LRI on Wax 805 ethyl butanoate grassy, fruity 1041 825 sulfur smelling compound skunk 851 z-3-hexen-1-ol fruity 1383 859 sulfur smelling compound rotten fruit 867 1-hexanol sour fruit 1352 871 2-methyl-3-furanthiol cooked grain 900 ethyl valerate (I.S.) fruity 1141 907 methional baked potato 1468 924 2-acetyl-1-pyrroline graham cracker 935 -pinene pine tree 1024 942 4-mercapto-4methylpentan-2-one chicken, moldy 1376 982 1-octen-3-one mushroom 1313 986 b-pinene musty, soil 1106 992 -myrcene green 1171 1003 octanal fruity, lemon 1309 1006 ethyl hexanoate lemon 1035 pcymene minty, fresh 1225 1041 limonene licorice, minty 1211 1046 4-mercapto-4methylpentan-2-ol fruity 1545 1065 (E)-2-octenal fruity, green 1071 Furaneol caramel 2038 1085 unknown coffee, burnt, processed 1099 linalool lemon 1553 1100 nonanal citrus, floral 1394 1105 fenchol lemony, citrus 1116 unknown sweet, popcorn 1134 2,6-nonadienal roses, green, cucumber 1601 1207 decanal lemon, sour, woody 1518 1233 neral lemon, sweet, floral 1252 carvone minty 1754 1320 eugenol balsamic, cloves 2176 1412 vanillin vanilla, sweet 2565 1455 wine lactone dill, crayons 2254 1494 b-ionone raspberry 1955 1564 dodecanoic acid musty 2500 1706 b-sinensal marine, old house 2243 1755 a-sinenesal marine, dusty 2420 1822 nookatone green, spicy, fruity co-elutes with vanillin

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32 Table 5: Aroma Active Compounds in Juice Stored in Plastic for Twelve Weeks Light Exposed No Lightexposure DB-5 LRI Descriptor Identity 0.79 0.54 805 grassy ethyl butanoate 0.00 0.46 825 skunk butyric acid 0.98 0.39 851 fruity z-3-hexen-1-ol 0.00 0.27 856 rotten unknown sulfur 0.00 0.69 867 sour fruit 1-hexanol 1.07 0.84 871 cooked grain 2-methyl-3-furanthiol 1.00 1.00 900 fruity ethyl valerate 0.79 0.64 907 baked potato methional 0.00 0.32 924 graham cracker 1.33 0.68 935 pine tree a-pinene 0.45 0.57 982 mushroom 1-octen-3-one 0.68 0.46 986 green,soil b-myrcene 0.81 0.46 1006 lemon ethyl hexanoate 1.04 0.44 1035 licorice,minty 1,8-cineole 0.63 0.89 1041 minty limonene 1.25 0.40 1046 moldy 4-mercapto-4methylpentan-2-ol 1.16 0.74 1071 caramel furaneol 0.50 0.51 1085 fruity,sweet tetramethyl-pyrazine 0.00 0.41 1099 lemon,burnt linalool 1.52 0.56 1100 sweet,citrus nonanal 0.77 0.00 1105 green 0.00 0.48 1116 citrus,floral unknown 0.92 0.00 1116 moldy 3-mercapto-hexen-1-ol* 0.56 0.58 1121 sweet,popcorn 0.62 0.43 1134 sweet ethyl 3-hydroxyhexanoate 0.93 0.00 1207 lemon,sour unknown 0.58 0.39 1233 lemon neral 0.84 0.50 1252 minty carvone 0.90 0.00 1285 cloves,burnt 1.31 0.83 1320 balsamic 4-vinyl guaiacol 0.87 0.38 1350 metallic 1.25 1.29 1383 very metallic 1.57 1.04 1412 vanilla vanillin 0.73 0.58 1455 dill wine lactone 0.82 0.77 1494 berries b-ionone 1.31 0.67 1564 marine b-sinensal 0.00 0.54 1584 burnt,spicy 0.96 0.43 1706 marine,old house a-sinenesal 0.00 0.47 1755 musty,dusty 0.00 0.19 1822 green nookatone

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33 Table 6: Aroma Active Compounds in Jui ce Stored in Glass for Twelve Weeks Light Exposed No Lightexposure DB-5 LRI Descriptor Identity 0.52 0.73 805 grassy ethyl butanoate 0.62 0.54 825 skunk butyric acid 0.41 0.39 851 fruity z-3-hexen-1-ol 0.68 0.39 856 wet dog unknown sulfur 0.36 0.57 867 fruity 1-hexanol 0.57 0.60 871 cooked grain 2-methyl-3-furanthiol 1.00 1.00 900 fruity ethyl valerate 0.81 0.64 907 baked potato methional 0.00 0.00 924 graham cracker 0.52 0.55 935 pine a-pinene 0.39 0.96 942 chicken 4-mercapto-4-methylpentan-2one 0.42 0.41 982 mushroom 1-octen-3-one 0.75 0.51 986 green,dirt b-myrcene 0.35 0.00 1003 fruity,lemon octanal 0.67 0.00 1006 lemon ethyl hexanoate 0.55 0.38 1035 licorice,minty 1,8-cineole 0.40 0.49 1041 minty limonene 0.83 0.00 1046 moldy,soured 4-mercapto-4-methylpentan-2ol 0.25 0.00 1065 fruity,green (E)-2-octenal 0.87 0.46 1071 caramel furaneol 0.87 0.62 1085 coffee tetramethyl-pyrazine 0.50 0.51 1099 burnt,lemon linalool 0.94 0.00 1100 sweet nonanal 0.00 0.59 1116 lemon,citrus unknown 0.47 0.00 1116 sweet,moldy 3-mercapto-hexen-1-ol* 0.91 0.57 1121 onion 0.59 0.34 1134 sweet ethyl 3-hydroxyhexanoate 0.54 0.28 1207 floral,sour unknown 0.36 0.35 1233 lemon,woody neral 0.49 0.37 1252 minty carvone 0.91 0.64 1285 burnt 0.71 0.63 1320 cloves 4-vinyl guaiacol 0.93 0.83 1383 metallic 0.74 0.48 1412 vanilla vanillin 0.76 0.79 1455 dill,old house wine lactone 0.40 0.80 1494 berries b-ionone 0.00 0.53 1564 marine b-sinensal 0.60 0.38 1706 marine a-sinenesal 0.46 0.48 1755 peppery 0.00 0.29 1822 green nookatone

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34 Figure 8: Comparison of Detector Responses The compound valencene has a large peak on the FID, but is not present on the GCO. Whereas the compound b-ionone has a larg e peak on the GC-O but is not present on the GC-FID. For this reason it is important to look for changes in the juice using both detection methods. However, the GC-O data will indicate which FID peaks are associated with aroma activity and which are not.

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35 Qualitative Differences In Figures 9 and 10, the average relative intensities of the aroma active components in light exposed and control juices in PET and glass are compared. The average aroma intensities are indicated by the bar height and are compared “head to tail” or “fishbone” by inverting the control juice data. ethyl butanoate butyric acid z-3-hexen-1-ol unknown (rotten) 1-hexanol methional a-pinene ethyl hexanoate 1,8-cineole limonene tetramethyl-pyrazine nonanal ethyl 3-hydroxyhexanoate carvone PVG unknown (metallic) vanillin wine lactone b-ionone b-sinensal unknown (spicy) a-sinensal nookatone(metallic)unknown (musty) unknown (cloves)4-mercapto-4-methylpentan-2-olunknown (graham cracker)unknown (green)furaneol3-mercapto-hexen-1-ol* neral unknown (popcorn) unknown (citrus,floral) b-myrcene 1-octen-3-one2-methyl-3-furanthiolethyl valerate linalool unknown (lemon,sour)-2.0 -1.0 0.0 1.0 2.0Relative Responses (GC-O)Light Exposed Juice No Light Exposure Juice Figure 9: Aroma Active Compounds in Juices St ored in PET for Twelve Weeks. Note ethyl valerate was an aroma active internal standard. In the case of plastic, 31 aroma activ e components were observed in juices exposed to light and 36 aroma active compone nts were noted in the control juices. Twenty six aroma components were common to both juices. From a qualitative point of view, the primary effect of light was the lo ss of several aroma components. The loss of the aroma components unbalanced the orange flavor. A few negative aroma components

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36 were formed from light-exposure, but since th e overall sensory quality of these juices were similar, it appears that that thei r flavor reducing impact was minimal. ethyl butanoate butyric acid z-3-hexen-1-ol unknown (wet dog) 1-hexanol 2-methyl-3-furanthiol ethyl valerate methional b-myrcene nonanal unknown (onion) carvone unknown (burnt) PVG unknown (metallic) vanillin wine lactone b-ionone b-sinensal a-sinensal unknown (pepper) nookatone 3-mercapto-hexen-1-ol*4-SH-4-methylpentan-2-onefuraneol (E)-2-octenal 1,8-cineole ethyl hexanoateunknown (floral)1-octen-3-one neral ethyl 3-hydroxyhexanoatetetramethyl-pyrazinelinalool 4-mercapto-4-methylpentan-2-ol limonene a-pinene-2.4 -1.4 -0.4 0.6 1.6Relative Responses (GC-O)Light Exposed Juice No Light Exposure Juice Figure 10: Aroma Active Compounds in Juices St ored in Glass for Twelve Weeks. Note ethyl valerate was an aroma active internal standard. In Figure 11 the aroma components for light exposed and contro l juices are also compared. The number of aroma active compon ents in both juice types were almost identical and the vast majority (30 aroma co mponents) were common to both juice types. However, the major aroma difference appear s to be due to the production of the extremely potent sulfur component, 4-mercapto -4-methyl-2-pentanol in the light exposed juice.

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37 Quantitative Differences At the end of storage the aroma active co mpound myrcene was found to decrease in concentration when exposed to light. The d ecrease in myrcene after twelve weeks of storage as measured on the GC -FID is shown in Figure 11. 0.7 0.9 1.1 -40481216WeekRelative Response (GC-FID) Figure 11: Decrease in Myrcene af ter Twelve Week Storage Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass co ntainers exposed to light, and represents juice in glass cont ainers not exposed to light The amount of myrcene is shown as a meas ure of “relative re sponse”, the area of the GC-FID peak for myrcene divided by the ar ea of the GC-FID peak of the internal standard, ethyl valerate. Myr cene decreased significantly (p <0.01) in those samples that were exposed to light, whereas the myrcene le vels in protected samples did not change (see Appendix D). After twelve weeks th e amount of myrcene decreased by 13.3% in juices in plastic and exposed to light, 1.7% in plastic and not expos ed to light, 11.9% in

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38 glass and exposed to light, and 6.1% in glass and not exposed to light. It should be noted that the sample that was stored in plastic and not exposed to light had the least amount of myrcene loss. Therefore the loss of this compound is mainly dictated by exposure to light and not by sorption into the plastic container. The compound -myrcene degrades in an acidic environment such as orange juice to form geraniol and its isomer nerol ( 35 ). This is perhaps why -myrcene decreased initially and then leveled off. The increased amounts of myrcene lost in those samples that were exposed to light can be explained by light acting as a catalyst that increased the rate of reaction in those juices. Normally, changes occurring in juice are produced by chemical reactions induced by heat. However, in the case of this expe riment, all samples were kept at the same temperature, such that reactions that occurred to a greater extent after light-exposure must have been catalyzed by the energy from light. An example of the difference between heat and light catalyzed reactions can be seen below. Energy of Activation from Heat: A + B AB Energy of Activation from Light: A + B AB The compound that showed the most drama tic increase during all three experiments was carvone. Figure 12 shows the increase in carvone after tw elve weeks of storage as measured by the GC-FID. It is apparent that the amount of car vone increased significantly (p<0.01) in samples that were exposed to light, whereas th ere was little change in those samples that were not exposed (see Appendix E). After tw elve weeks of storage, carvone increased by h v

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39 240.1% in plastic and exposed to light, by 66.6% in plastic not exposed to light, 368.0% in glass and exposed to light, and 27. 7% in glass not exposed to light. 0.0 0.1 0.2 0.3 -404812WeekRelative Response (GC-FID) Figure 12: Increase in Carvone af ter Twelve Week Storage Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass co ntainers exposed to light, and represents juice in glass cont ainers not exposed to light Carvone is an oxidation product of limonene ( 36 ). This oxidation of limonene is perhaps why juices that were stored in plastic with no light and thus had a greater chance of oxygen exposure had a greater amount of car vone formation than those juices that were stored in glass. Also, increases in carvone, which has a minty aroma, and the subsequent decrease in limonene, which has very little aroma activity, could be a contributing factor to the overa ll change in aroma and flavor of the orange juice after storage.

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40 Studies by Ziegler and colleagues conduc ted on orange oil also documented a significant increase in carvone ( 9 ). The changes in carvone in the orange juice used in this experiment occurred to a lesser extent than those changes in the orange oil. This is most likely due to the matrix the flavor co mpounds are suspended in. The compounds in the orange oil are in close proximity to one a nother and therefore have a greater chance of reacting with one another and with light induced oxidation. The compounds in the orange juice are separated by large quan tities of water that decrease compound interaction and oxidation reacti ons. The insoluble material (c loud) in the juice may also work as a reflective material that blocks so me of the entering light from contacting the flavor compounds. This difference between juice and oil may also explain why compounds that changed in orange and lemon oils (such as neral and geranial) did not show significant changes in this experiment. The minty smelling compound 1,8-cineole also increased during storage. This compound co-elutes on a DB-5 column with limonene. Since the concentration of limonene is relatively large in orange juice, the resulting GC-FID peak is also large and therefore the peak for 1,8-cineole could not be quantified. Instead, the peak was quantified using results from the GC-O which was able to separate the two compounds. Figure 13 shows the increase in this oxidation product during storage. Although initially, 1,8-cineole in creased in juice not exposed to light, it leveled off after four weeks of storage. At this time, juice exposed to light showed an increase in this compound, with a greater increase in the juic e that was stored in a plastic bottle and therefore had a greater possibili ty of oxygen content. After twelve weeks of storage, 1,8cineole increased by 173.5% in plastic and exposed to light, by 15.6% in plastic not

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41 exposed to light, by 44.7% in glass and e xposed to light, and by 0.7% in glass not exposed to light. 0.0 0.4 0.8 1.2 -404812WeekRelative Response (GC-O) Figure 13: Increase in 1,8-Cineole after Twelve Week Storage Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass containers exposed to light, and represents juice in glass co ntainers not exposed to light The volatile 1,8-cineole is a decomposition product of limonene. It has been reported in past literature that 1,8-cineole only formed when stored at 23C and not at 6C ( 37 ). The orange juice in this study was stor ed at 4C, and thus the increase of 1,8cineole may be light induced as well as heat induced. The volatile compound pcymene was found to increase in samples that had been exposed to light. Since pcymene has a similar retention time as limonene on the DB-5 column (LRI=1026 and LRI=1031 respectively) the two compounds coeluted and could not be quantified by GC-FID or GC-O. Howe ver, the polar wax column in the GC-MS

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42 allowed for separation of the tw o compounds. The peak areas of pcymene were measured based on an extracted ion chromat ogram at m/z of 119. An example of how the peak area was integrated and the library standard mass spectra for pcymene can be seen in Figure 14. 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9Time (min) 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100Relative Abundance 17.65 17.74 17.42 17.85 RT: 17.42TIC EIC m/z= 119 40 80 120 160 0 50 100 51 65 77 91 103 119 134 Relative Abundance Figure 14: p-cymene Peak Area Measurement on GC-MS The peak area of the internal standa rd was measured using the total ion chromatograph for all samples. The internal standard used in this comparison was 4heptadecanone. The increase in pcymene in those samples that were exposed to light can be seen in Table 7.

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43 Table 7: pcymene Comparison for Juice Stored Twelve Weeks Sample Peak Area Cymene Peak Area IS2 Ratio Light, In Plastic 9.66x10^4 3.54x10^7 2.73 No Light, In Plastic 4.94x10^4 4.24x10^7 1.16 Light, In Glass 6.97x10^4 3.10x10^7 2.25 No Light, In Glass 2.04x10^4 1.33x10^7 1.53 The samples that were exposed to light have on average 85% more pcymene than those that were not exposed. Howe ver, there was little difference in pcymene content from juices stored in plastic or glass containers either light exposed or not, suggesting that difference in oxygen permeability of storag e container materials was not a factor in pcymene formation. The compound pcymene is formed from the acid catalyzed decomposition of citral ( 38 ). These results of increased pcymene after exposure to fluorescent light mirror the findings of both Schieberle and Grosch and Iwanami and colleagues for pcymene in lemon oil ( 6;8 ). The compounds vanillin and 4-hydroxy-2,5dimethyl-3(2H)-furanone (Furaneol) also showed a slight increase during storage. These compounds occurred at levels below the detection limits of the GC-FID and GC -MS and therefore the results reported are based on GC-O data. Figures 15 and 16 below s how the increase in vanillin and Furaneol respectively.

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44 0.0 0.6 1.2 1.8 2.4 -40481216WeekRelative Response (GC-O) Figure 15: Increase in Vanillin Du ring Twelve Week Storage Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass co ntainers exposed to light, and represents juice in glass cont ainers not exposed to light There was an increase in vanillin in juice samples that were exposed to light. It is also evident that samples stored in plastic bot tles had a greater incr ease of vanillin than those that were stored in glass. This may be caused by a greater amount of oxygen being present in the juice stored in plastic bottles At the end of twelve weeks, vanillin increased by 171.4% in plastic and exposed to light, by 80.0% in plastic not exposed to light, by 28.3% in glass and exposed to lig ht, and decreased by 17.4% in glass not exposed to light. Vanillin has been reported to form from the thermal degradation of ferulic acid in citrus juices ( 39 ). In this experiment, all of the jui ces were kept at the same temperature, so thermal degradation should have occurred equally amongst all samples. However, the

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45 juices that were exposed to light formed more vanillin that those that were protected. Therefore, light may have provided the energy source needed for ferulic acid decomposition in place of heat. It has also be en noted that vanillin can further degrade to form phenols and cresols ( 40 ). This could explain why th e amount of increase in vanillin levels off after four weeks and actually decrea ses in the case of juices stored in glass bottles. Furaneol increased significantly in samples that were exposed to light during this study and increased to a le sser extent in those samples left unexposed. 0.0 0.7 1.4 -40481216WeekRelative Response (GC-O) Figure 16: Increase in Furaneol Du ring Twelve Week Storage Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass containers exposed to light, and represents juice in glass contai ners not exposed to light. Also, juices that were stored in plastic containers again had a greater increase in Furaneol than juices stored in glass bottles. Furaneol is an undesirable product that is

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46 formed through non-enzymatic browni ng during orange juice storage ( 41 ). It has been documented as occurring in juices that have been subjected to temperature abuse. Again, since all of the juices were under the same temperature condi tions throughout storage, the increased amount of Furaneol in juice exposed to light i ndicates that light may be a possible energy source that catalyzes the reaction. A compound that has been used as a marker for temperature abuse in orange juice is 4-vinyl guaiacol ( 42 ). Like vanillin, this compound forms from the thermal degradation of ferulic acid ( 43 ). Because extraction efficienci es are low, it is difficult to quantify 4-vinyl guaiacol (PVG) using GC-FID and GC-O. However, quantification can be achieved by measuring the peak area of an extracted ion chromatogram at m/z of 150. An example of the integration and the library standard mass spectra for PVG can be seen in Figure 17. This peak area can be divided by the peak ar ea of an internal st andard in order to determine the relative response. The internal standard used for this comparison was 4heptadecanone because of its similar rete ntion time to PVG on a wax column (RT=33.89 and 34.89min respectively). It can be seen in Table 8 that the amount of PVG was essentially the same after twelve weeks of st orage regardless of container type and lightexposure. Therefore, PVG, probably the most important off flavor formed from thermal abuse is not responsible for the flavor differe nces in the light expos ed samples at 4C.

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47 34.6 34.7 34.8 34.9 35.0 35.1 35.2 35.3Time (min) 0 20 40 60 80 100Relative Abundance 0 20 40 60 80 100Relative Abundance 34.71 34.89 RT: 34.89 RT: 34.67 RT: 35.04 RT: 35.18 RT: 35.26TIC EIC m/z= 150 40 80 120 160 200 0 50 100 51 677789107123135 150 OH O M+ Figure 17: 4-Vinyl Guaiacol Peak Area Measurement on GC-MS Table 8: Comparison of PVG after Twelve Weeks Storage Using GC-MS Sample Peak Area PVG Peak Area IS2 Ratio Light, In Plastic 8.44x10^4 3.54x10^7 2.38 No Light, In Plastic 6.38x10^4 4.24x10^7 2.56 Light, In Glass 6.38x10^4 3.10x10^7 2.06 No Light, In Glass 8.29x10^4 1.33x10^7 2.47 Another compound that has been used as a marker for temperature abuse in orange juice is -terpineol. This compound forms from acid catalyzed hydration of limonene and linalool in the presence of wa ter at elevated temperatures ( 44 ). It is present in higher concentrations, and is therefore easier to identify and quantify than PVG ( 45 ). It can be seen in Table 9 that the amount of -terpineol was the same after twelve weeks of storage regardless of container mate rial and light-exposure.

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48 Table 9: GC-FID Responses for -terpineol In Juices St ored for Twelve Weeks Samples Relative Response of terpineol Initial Juice 0.126 Light, In Plastic 0.449 No Light, In Plastic 0.497 Light, In Glass 0.464 No Light, In Glass 0.477 Although the samples increased in -terpineol during the twelve weeks of storage, the formation was equivalent regardless of c ontainer material or light-exposure. Again this shows that changes in other compounds su ch as vanillin and Furaneol were caused by exposure to light and not due to thermally induced reactions. Two additional compounds that are used as a marker for temperature abuse in orange juice are furfural a nd 5-hydroxymethyl furfural ( 46 ). However, due to their low extraction efficiencies, they were not identified or quantified using either GC or GC-MS. Their thresholds were too high to be aroma active and they were not detected using GCO. Sulfur Smelling Aroma Compounds As seen in Figures 9 and 10, a total of five sulfur containing/smelling aroma compounds were observed in the juices after twelve weeks storage. Methional and 2methyl-3-furnathiol levels we re essentially the same in light exposed and control samples. Methional is a Strecker aldehyde formed from the decomposition of the sulfurcontaining amino acid, methionine. The unknow n sulfur compounds were higher in light exposed sample when stored in glass, but sli ght lower when stored in plastic, and did not seem to make a major contribution to the ove rall flavor. However, two thiol (mercapto) compounds (4-mercapto-4-methylpe ntan-2-ol and 3-mercapto-h exen-1-ol) were formed

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49 only in light exposed juices in both gla ss and PET. The aroma of 4-mercapto-4methylpentan-2-ol was described as moldy or soured, and 3-mercapto-hexen-1-ol was described as onions, moldy, or soured. It shoul d be noted that the id entity of 3-mercaptohexen-1-ol must be consider ed tentative as it has been identified only on the basis of retention time matching. Ther efore, the appearance of these profoundly negative aroma compounds in only the light exposed juices woul d explain in part th e diminished flavor quality of these juices. As shown in Fi gure 18, 3-mercapto-hexen-1-ol increased during the twelve week storage study only in samples exposed to light. Also, the concentration increased at an earlier date a nd to a greater extent in jui ce that was stored in plastic bottles as compared to juice th at was stored in glass. 0.0 0.2 0.4 0.6 0.8 1.0 -40481216WeekRelative Response (GC-O) Figure 1: Increase in Sulf ur Compound (LRI 1116) Du ring Twelve Week Study. represents juice in plastic containers exposed to light, represents juice in plastic containers not exposed to light, represents juice in glass containers exposed to light, and represents juice in glass containers not exposed to light

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50 Finally, the sulfur smelling compounds that formed or increased were probably caused by the degradation of known sulfur compounds such as the amino acid methionine or the vitamin thiamine which are bo th present in fresh orange juice ( 47 ). The appearance of these skunky, ch icken-like, or onion-like arom as in only light exposed juices partially explains why the overall orange juice character was degraded. Accelerated Study At the beginning of the accelerated storage study, oxygen was bubbled through all juice samples. Control juices were wrappe d in aluminum foil. After two weeks, two unidentified sulfur (skunklike) smelling compounds increased. These compounds occurred at very low concentrations in the juice and thus were only detected on the GCO. The linear retention indices were 806 a nd 900 on the DB-5 column. The peak at LRI 900 was observed just before the internal sta ndard, ethyl valerate, eluted. The sulfur smelling compound (LRI 806) that increased in the accelerated study is not the same as the early eluting unknown sulfur (LRI 856) observed in the twelve week study. The sulfur smelling compound at R.I. 806 had a skunk like aroma, whereas the compound at R.I. 856 smelled like wet dog. As seen in Figure 19, the skunky aroma was at higher concentrations in the sample exposed to light. The sulfur compound at R.I. 900 also ha d a skunk like aroma and formed during the accelerated storage study. As seen in Figure 20, this compound was not present in the original juice, and only formed in the juice that was exposed to light after two weeks. The increase or formation of sulfur smelling compounds after light-exposure has been documented in previous literature. It has been found that the amino acid methionine degrades during ultrav iolet light-exposure in the pres ence of riboflavin and oxygen to

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51 form methional. Methional further degrades to form methanethiol, dimethyl sulfide, and dimethyl disulfide ( 41 ). 0.0000 0.1000 0.2000 0.3000 0.4000 0.5000 Week 0Week 1, In LightWeek 2, In LightWeek 2, No LightRelative Response (GC-O) Figure 19: Formation of Sulfur Compound (L RI 806) after Accelerated Storage Study Microbiological Evaluation At the beginning of the study and af ter one month, microbial counts were performed. There was one colony formed on se veral of the orange serum agar plates before and after the study, however, this colony was identified as a bacillus strain that does not affect juice flavor. Therefore, e ssentially no microbial growth was observed in the orange juice before or dur ing the experiment. Therefore any changes in juice flavor were not caused by microbial contamination.

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52 0.0000 0.1000 0.2000 0.3000 Week 0Week 1, In LightWeek 2, In LightWeek 2, No LightRelative Response (GC-O) Figure 20: Increase in Sulfur Compound (LRI 900) after Accelerated Storage Study

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53 CHAPTER 6 CONCLUSIONS The decrease in -myrcene, the increase in carvone, pcymene, 1,8-cineole, vanillin, and Furaneol, and the formation and increase of various sulfur smelling compounds helps explain the overall changes in aroma and flavor of orange juice that has been exposed to light. It is these change s that upset the usual balance of flavor compounds in fresh orange juice. The increase in vanillin, Furaneol, and su lfur smelling compounds in samples that were not subjected to increased temperature abuse indicates that li ght may also play a role in non-enzymatic browning and thermal degradation. Changes in volatiles were not significant during the first month of storage. Therefore, orange juice exposed to light in a retail setting where th ere is a high turnover rate would not be affected to a measurable extent and no loss of ove rall quality should be detected. However, juices that are sold at a lower turnover rate are at a higher risk for developing these off-flavors and ove rall deterioration of quality. It was shown in this experiment that in general juices that are stored in glass retained more orange juice character and had less photoxidation r eactions occurring. This is probably due to less oxygen being ab le to permeate the container, and fewer oxidation reactions occurring in the juice. Re tailers can therefore gua rd against some of these reactions by using glass containers or multilayer pl astic materials with higher oxygen barrier properties. New packaging technology which utilizes higher oxygen

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54 barrier materials or oxygen scavenging capabi lity and shields the ju ice from light would protect orange juice quality and ensure consumer satisfaction.

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55 APPENDIX A STATISTICAL TEST FOR “L” SIGNIFICANCE Two-sample T for Initial vs L-P N Mean StDev SE Mean Initial 8 55.437 0.199 0.070 L-P 2 52.0135 0.0841 0.059 Difference = mu Initial mu L-P Estimate for difference: 3.4234 95% CI for difference: (3.1678, 3.6789) T-Test of difference = 0 (vs not =): T-Value = 37.19 PValue = 0.000 DF = 4 L-P Initial 56 55 54 53 52 Boxplots of Initial and L-P(means are indicated by solid circles) Figure 21: Significant Difference Between L Values

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56 APPENDIX B STATISTICAL TEST FOR “A” SIGNIFICANCE Two-sample T for Initial vs L-P N Mean StDev SE Mean Initial 8 -4.1694 0.0754 0.027 L-P 2 -0.6200 0.0283 0.020 Difference = mu Initial mu L-P Estimate for difference: -3.5494 95% CI for difference: (-3.6351, -3.4637) T-Test of difference = 0 (vs not =): T-Value = -106.50 PValue = 0.000 DF = 5 L-P Initial -0.5 -1.5 -2.5 -3.5 -4.5 Boxplots of Initial and L-P(means are indicated by solid circles) Figure 22: Significant Difference in "a" Values Since the p value is 0.000, which is less th an the alpha value of 0.01, we reject the null hypothesis. The “a” value of the initial ju ice is not equal to th e “a” value of juice exposed to light in plastic.

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57 APPENDIX C STATISTICAL TEST FOR “B” SIGNIFICANCE Two-sample T for Initial vs L-P N Mean StDev SE Mean Initial 8 33.760 0.308 0.11 L-P 2 28.129 0.210 0.15 Difference = mu Initial mu L-P Estimate for difference: 5.631 95% CI for difference: (4.839, 6.424) T-Test of difference = 0 (vs not =): T-Value = 30.58 PValue = 0.001 DF = 2 L-P Initial 34 33 32 31 30 29 28 Boxplots of Initial and L-P(means are indicated by solid circles) Figure 23: Significant Difference in "b" values Since the p value is 0.001, which is less th an the alpha value of 0.01, we reject the null hypothesis. The “b” value of the initial ju ice is not equal to th e “b” value of juice exposed to light in plastic.

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58 The Appendices in the Guide for Preparing Theses and Dissertations provided by the Graduate School’s Editorial Office give numerous examples regarding the proper construction of an appendix. In most cases, the appendices will vary from dissertation to dissertation and may vary within a disse rtation, depending on the content of the individual appendix. Obey the ge neral guidelines given in the Guide for Preparing Theses and Dissertations Although the margins have been set thr oughout this document correctly, please pay close attention to the possibility of picture frames overlapping the margin. The base style to use is Normal The remainder of this text is extraneous We included it in this version of the template so that appendix A can have page numbers on all its pages. The text that follows was copied directly from the Guide for Preparing Theses & Dissertations. Candidates in the English department w ho author a collection of poems, short stories, or a novel for a thesis degree shoul d consult the Editorial Office and not other theses as a guide to format. Typing, spacing, margin, heading,, numbering, and formatting requirements in this guide apply to all theses. If a thesis consists of a co llection of poems that are not grouped under headings, the first page of each poem has a 2-inch top margin. Each poe m title is centered and in all capital letters. The first page of each poem is numbered bottom center with the rest of the pages of the poem numbered in the top margin. The poems may be doubleor singlespaced but must conform to the other margin and formatting requirements in this guide.

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59 APPENDIX D STATISTICAL TEST FOR MYRCENE SIGNIFICANT DIFFERENCE Two-sample T for Initial vs L-P N Mean StDev SE Mean Initial 2 1.03376 0.00165 0.0012 L-P 3 0.8629 0.0264 0.015 Difference = mu Initial mu L-P Estimate for difference: 0.1709 95% CI for difference: (0.1051, 0.2367) T-Test of difference = 0 (vs not =): T-Value = 11.18 PValue = 0.008 DF = 2 L-P Initial 1.05 0.95 0.85 Boxplots of Initial and L-P(means are indicated by solid circles) Figure 24: Significant Difference in Amount of Myrcene Since the p value is 0.008, which is less th an the alpha value of 0.01, we reject the null hypothesis. The amount of myrcene in the initial juice is not equal to the amount in the juice exposed to light in plastic.

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60 Two-sample T for Initial vs D-P N Mean StDev SE Mean Initial 2 1.03376 0.00165 0.0012 D-P 3 0.9782 0.0331 0.019 Difference = mu Initial mu D-P Estimate for difference: 0.0556 95% CI for difference: (-0.0268, 0.1380) T-Test of difference = 0 (vs not =): T-Value = 2.90 PValue = 0.101 DF = 2 D-P Initial 1.04 1.03 1.02 1.01 1.00 0.99 0.98 0.97 0.96 0.95 0.94 Boxplots of Initial and D-P(means are indicated by solid circles) Figure 25: No Difference in Amount of Myrcene Since the p value is 0.101, which is greater than the alpha value of 0.01, we fail to reject the null hypothesis. The amount of myrcene in the in itial juice is equal to the amount in the juice protected from light in plastic.

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61 APPENDIX E STATISTICAL TEST FOR CARV ONE SIGNICANT DIFERENCE Two-sample T for Initial vs L-P N Mean StDev SE Mean Initial 2 0.04826 0.00196 0.0014 L-P 3 0.1750 0.0207 0.012 Difference = mu Initial mu L-P Estimate for difference: -0.1267 95% CI for difference: (-0.1784, -0.0750) T-Test of difference = 0 (vs not =): T-Value = -10.55 PValue = 0.009 DF = 2 L-P Initial 0.20 0.15 0.10 0.05 Boxplots of Initial and L-P(means are indicated by solid circles) Figure 26: Significant Differe nce in Amount of Carvone Since the p value is 0.009, which is less th an the alpha value of 0.01, we reject the null hypothesis. The amount of carvone in the initial juice is not e qual to the amount in the juice exposed to light in plastic.

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62 Two-sample T for Initial vs D-P N Mean StDev SE Mean Initial 2 0.04826 0.00196 0.0014 D-P 3 0.0857 0.0187 0.011 Difference = mu Initial mu D-P Estimate for difference: -0.0374 95% CI for difference: (-0.0843, 0.0095) T-Test of difference = 0 (vs not =): T-Value = -3.43 PValue = 0.076 DF = 2 D-P Initial 0.11 0.10 0.09 0.08 0.07 0.06 0.05 Boxplots of Initial and D-P(means are indicated by solid circles) Figure 27: No Difference in Amount of Carvone Since the p value is 0.076, which is greater than the alpha value of 0.01, we fail to reject the null hypothesis. The amount of car vone in the initial ju ice is equal to the amount in the juice protected from light in plastic.

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APPENDIX F GC-FID RESULTS FOR JUIC ES STORED IN PLASTIC

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64 Week 0Week 4Week 8Week 12Week 0Week 4Week 8Week 12Identities Ave AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve TimeDB-5 LRI 0.2640.2650.0930.3660.2640.5040.2550.3332.54731 0.0600.0540.0890.0600.0890.0510.1243.27777 0.1290.0660.2740.2300.1290.1190.2680.2333.68802ethyl butanoate 1.0001.0000.4001.0001.0001.0000.4001.0005.55901ethyl valerate 0.3060.2600.2850.2720.3060.2940.3050.2776.29936a-pinene 0.9950.8140.8920.8630.9951.0031.0520.9787.54993b-myrcene 0.1850.0500.1040.1850.1440.1417.801005octanal 0.0380.0390.0170.0380.0360.0190.0348.0010143-carene 54.07644.19747.99847.17354.07651.38753.61050.6538.641042limonene 0.1400.2430.1010.1500.1400.1040.1090.2629.351073p-cresol 0.5530.4610.5130.6150.5530.5490.5910.67410.031103linalool 0.0380.0440.0390.0730.0380.0460.0330.07310.541126trans-rose oxide 0.2280.2290.2470.3270.2280.2580.2650.35610.641130ethyl 3-hydroxyhexanoa t 0.1110.1110.2040.1060.1350.1230.21611.821183 0.1260.2090.2890.4490.1260.2500.3660.49712.111197a-terpineol 0.1700.0930.0970.1730.1700.1280.1100.08312.331207 0.0330.0400.0490.0890.0330.0360.0390.05412.721225nerol 0.0280.0220.0130.0380.0280.0300.0310.05512.861231neral 0.0550.0690.1190.1750.0550.0620.0680.08613.251250carvone 0.0270.0180.0960.0270.0400.05814.731321eugenol 0.0450.0550.0690.1370.0450.0530.0570.10715.271347E-2-undecenal 0.0320.0260.0380.0640.0320.0330.0300.07116.511410b-demascenone 0.0620.0530.0650.0840.0620.0650.0730.09816.931432 0.0520.0470.0600.0740.0520.0620.0700.09517.491461wine lactone 0.0750.0670.0770.0980.0750.0810.0890.12917.991488 0.2630.2300.2660.3270.2630.2690.3170.43318.151497 3.1922.7883.3114.1043.1923.3393.7615.04018.371508b-ionone 0.1240.1460.1490.1820.1240.1380.1500.31718.531517 0.2170.2140.2440.3140.2170.2370.2830.38618.811533 0.0320.0270.0170.0690.0320.0350.0380.08319.411565dodecanoic acid 0.0300.0430.0480.0820.0300.0510.0520.12421.261672 0.0310.0230.0210.0340.0310.0230.04121.731699b-sinensal 0.0710.0630.0940.1530.0710.1280.1130.21522.761761a-sinensal 0.2330.1960.2560.3930.2330.2320.3000.52923.781825nootkatone 0.0210.0160.0460.0210.0510.07924.331859 1.3970.7401.0341.9031.3971.1340.6522.43924.651880 0.8380.8081.2722.6010.8381.9541.5783.88024.881895 0.1380.1240.1750.2540.1380.1390.1780.34629.972251 0.1860.1510.8850.1190.1860.2490.2100.22230.442286 0.0460.0411.1360.1930.0460.1370.0450.27330.742308 0.1160.0710.1040.1990.1160.2460.1090.20431.012329 0.0560.0370.0450.0560.0600.0530.05831.242346 0.1720.1400.1740.1750.1720.1800.1930.24431.362356 0.0930.0570.0670.0870.0930.1340.0850.18231.692381 Light, In PlasticNo Light, In Plastic

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65 APPENDIX G GC-FID RESULTS FOR JUIC ES STORED IN GLASS Week 0Week 4Week 8Week 12Week 0Week 4Week 8Week 12IdentitiesAve AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve AreaAve TimeDB-5 LRI 0.2230.5050.3100.6500.2230.2710.2730.8722.54731 0.0480.0980.0320.1630.0480.0520.0550.2163.27777 0.1170.1140.2230.2990.1170.0680.2070.1833.68802ethyl butanoate1.0001.0000.4001.0001.0001.0000.4001.0005.55901ethyl valerate0.2880.2710.2960.2800.2880.2890.3080.2686.29936a-pinene0.9950.8730.9360.8770.9950.9631.0160.9357.54993b-myrcene0.1670.1180.0610.1677.801005octanal0.0360.0330.0360.0310.0348.0010143-carene50.48947.11851.76248.31250.48950.58352.60447.3308.641042limonene0.2060.0940.1890.1020.2060.2310.1840.1289.351073p-cresol0.4920.4860.5470.6320.4920.4960.5270.57110.031103linalool0.0360.0600.0580.1550.0360.0370.0340.15210.541126trans-rose oxide0.2240.2440.2870.3650.2240.2530.2690.28410.641130ethyl 3-hydroxyhexanoate0.0920.1140.1640.2210.0920.1440.1540.25211.821183 0.1080.2220.2890.4630.1080.2380.3470.47712.111197a-terpineol0.1410.1210.0900.1530.1410.0380.0570.09012.331207 0.0290.0450.0610.1050.0290.0290.03012.721225nerol0.0230.0270.0230.0310.03112.861231neral0.0480.0940.1670.2410.0480.0400.0560.06613.251250carvone0.0230.0280.0580.1000.0230.0370.04114.731321eugenol0.0430.0660.0940.1450.0430.0530.0540.07015.271347E-2-undecenal0.0230.0360.0440.0660.0230.0230.02916.511410b-demascenone0.0520.0630.0740.1250.0520.0610.0720.07716.931432 0.0450.0600.0700.0900.0450.0580.0670.06917.491461wine lactone0.0640.0740.0910.1220.0640.0740.0950.09117.991488 0.2290.2670.3210.4020.2290.2510.2970.29418.151497 2.7273.1963.8344.7512.7273.0583.5373.64218.371508b-ionone0.1120.1440.1350.1900.1120.1490.1440.16018.531517 0.1970.2270.2740.3390.1970.2320.2580.26918.811533 0.0250.0390.0520.0730.0250.0400.03319.411565dodecanoic acid0.0270.0540.0570.1030.0270.0470.0490.07021.261672 0.0320.0270.0530.0320.0300.02621.731699b-sinensal0.0870.0990.1530.2370.0870.1000.1840.12622.761761a-sinensal0.2070.2570.3000.4460.2070.2290.2830.37023.781825nootkatone0.0150.0230.0820.0780.0150.0550.06724.331859 1.1961.4000.6712.3001.1961.1820.6731.89924.651880 0.6911.3581.2133.5440.6912.7683.8512.21624.881895 0.1230.1600.2120.3540.1230.1500.1810.27029.972251 0.1510.3830.2720.1760.1510.2360.3210.13930.442286 0.0270.1230.0630.3150.0270.1130.1080.19530.742308 0.0810.1930.1410.2460.0810.1880.2700.19231.012329 0.0350.0540.0370.0350.0490.0760.19031.242346 0.1260.1850.1100.2730.1260.1770.2610.24631.362356 0.0830.0930.0530.0830.0780.11831.692381 Light, In GlassNo Light, In Glass

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66 LIST OF REFERENCES (1) Brown, C.; Brown, M.; Lee, J. Y.; Sodek, V.; Gunter, D. L.; Collins, S.; Sparks, M. Citrus Reference Book ; Florida Department of Citrus Economic and Market Research Department: Gainesville, FL, 2004; 61,62. (2) Weggeman, P. Reconsidering PET Bottling Coatings. In Beverage Industry March 2001; pp 60-62. (3) Solomon, O.; Svanberg, U.; Sahlstrom, A. Effect of Oxygen and Fluorescent Light on the Quality of Orange Juice During Storage at 8-Degrees-C. Food Chemistry 1995 53 363-368. (4) Sattar, A.; Durrani, M. J.; Khan, R. N. ; Hussain, B. H. Effect of Packaging Materials and Fluorescent Light on Htst-Pasteurized Orange Drink. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung 1989 188 430-433. (5) Ahmed, A. A.; Watrous, G. H.; Hargrove G. L.; Dimick, P. S. Effects of Fluorescent Light on Flavor and Ascorb ic-Acid Content in Refrigerated Orange Juice and Drinks. Journal of Milk and Food Technology 1976 39 332-336. (6) Iwanami, Y.; Tateba, H.; Kodama, N.; Kishino, K. Changes of lemon flavor components in an aqueous solution during UV irradiation. Journal of Agricultural and Food Chemistry 1997 45 463-466. (7) Schieberle, P.; Grosch, W. Quantitative analysis of important volatile flavor compounds in fresh and stored le mon oil/citric acid emulsions. Lebensmittel-Wissenschaft und -Technologie 1988 21 158-162. (8) Schieberle, P.; Grosch, W. Identification of potent flavor compounds formed in an aqueous lemon oil/citric acid emulsion. Journal of Agricultural and Food Chemistry 1988 36 797-800.

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68 (18) Handwerk, R. L.; Coleman, R. L. Approach es to the Citrus Browning Problem: A Review. J. Agric. Food Chem. 1988 36 231-236. (19) Belitz, H. D.; Grosch, W.; Schieberle, P. Food Chemistry 3 ed.; Springer: Heidelberg, 2004; 269-283. (20) Trammell, D. J.; Dalsis, D. E.; Malone, C. T. Effect of Oxygen on Taste, Ascorbic-Acid Loss and Browning for Htst-Pasteurized, Single-Strength Orange Juice. Journal of Food Science 1986 51 1021-1023. (21) Kennedy, J. F.; Rivera, Z. S.; Lloyd, L. L.; Warner, F. P.; Jumel, K. L-Ascorbic acid stability in aseptically processed orange juice in TetraBrik cartons and the effect of oxygen. Food Chemistry 1992 45 327-331. (22) Fuller, G. H.; Steltenkamp, R.; Tisserand, G. A. The Gas Chromatograph with Human Sensor: Perfumer Model. Annals of the New York Academy of Sciences 1964 116 711-724. (23) Guth, H.; Grosch, W. Comparison of stored soya-bean and rapeseed oils by aroma extract dilution analysis. Lebensmittel Wissenschaft und Technologie 1990 23 59-65. (24) Acree, T. E.; Cunningham, D. G. A pro cedure for the sensory analysis of gas chromatographic effluents. Food Chemistry 1984 14 273-286. (25) Da Silva, M. M. A. P.; Lundahl, D. S.; McDaniel, M. R. The capability and psychophysics of osme: A new gc-olfactometry technique. In Trends in flavor research ; H. Maarse and D. G. van der Heij, Eds.; Elsevier Science Publishers: Amsterdam, 1994; pp 518. (26) Debonneville, C.; Orsier, B.; Flament, I.; Chaintreau, A. Improved hardware and software for quick gas chromatogr aphy-olfactometry using CHARM and GC-"SNIF" analysis. Anal. Chem. 2002 74 2345-2351. (27) Orange Juice Quality and Categories. In The Orange Book ; Tetra Pak Processing Systems AB: Lund, Sweden, 1998; pp 16-29.

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69 (28) Scott, W. C.; Veldhuis, M. K. Rapid Es timation of Recoverable Oil in Citrus Juices by Bromate Titration. Journal of the Association of Official Analytical Chemists 1966 49 628-633. (29) Lee, H. S.; Chen, C. S. Rates of Vita min C Loss and Discoloration in Clear Orange Juice Concentrate during St orage at Temperatures of 4-24C. J. Agric. Food Chem. 1998 46 4723-4727. (30) Cancalon, P. F. Vitamin Analysis by Capillary Electrophoresis. LC GC Europe 2003 March 2-5. (31) Bazemore, R.; Goodner, K.; Rouseff, R. Volatiles from unpasteurized and excessively heated orange juice analy zed with solid phase microextraction. J. Food Sci. 1999 64 800 -804. (32) Arias, C. R.; Burns, J. K.; Friedrich, L. M.; Goodrich, R. M.; Parish, M. E. Yeast Species Associated with Orange Juice: Evalution of Different Identification Methods. Applied and Environemental Microbiology 2002 68 1955-1961. (33) Rouseff, R. Flavor Database: LRI DB-5. http://www.crec.ifas.ufl.edu//rouseff/Website 2002/Subpages/database_f_Frameset.htm Lake Alfred, FL, March 2005 (34) Clark, J., B.C.; Chamblee, T. S. Acid-Cat alyzed Reactions of Citrus Oils and Other Terpene-Containing Flavors. In Off Flavors of Foods and Beverages ; G. Charalambous, Ed.; Elsevier Science Publishers: Amsterdam, 1992. (35) Royals, E. E.; Horne, S. E. Conversion of d-limonene to l-carvone. Journal of the American Chemical Society 1951 73 5856-5857. (36) Ebbesen, A.; Rysstad, G.; Baxter, A. Eff ect of temperature oxygen and packaging material on orange juice quality during storage. Fruit Processing 1998 8 446-455. (37) Schieberle, P.; Ehrmeier, H.; Grosc h, W. Aroma Compounds Resulting from the Acid-Catalyzed breakdown of Citral. Zeitschrift fuer LebensmittelUntersuchung und -Forschung 1988 187 35-39.

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70 (38) Naim, M.; Striem, B. J.; Kanner, J.; Pele g, H. Potential of ferulic acid as a precursor to off-flavors in stored orange juice. Journal of Food Science 1988 53 500-503. (39) Fisher, C.; Scott, T. R. Flavour Compounds. In Food Flavours Biology and Chemistry ; The Royal Society of Chemis try: Cambridge, 1997; pp 15-55. (40) Sucan, M. K. Identifying and Preventing Off-Flavors. In Food Technology 2004; pp 36-40. (41) Naim, M.; Zehavi, U.; Nagy, S.; Rouseff, R. L. Hydroxycinnamic acids as offflavor precursors in citrus fruits and their products. ACS Symposium Series 1992 506 180-191. (42) Peleg, H.; Naim, M.; Zehavi, U.; Rouseff, R. L.; Nagy, S. Pathyways of 4vinylguaiacol Formation from Ferulic Ac id in Model Solutions of Orange Juice. J. Agric. Food Chem. 1992 40 764-767. (43) Haleva-Toledo, E.; Naim, M.; Zehavi, U.; Rouseff, R. L. Formation of alphaterpineol in citrus juices, model and buffer solutions. J. Agric. Food Chem. 1999 64 838-841. (44) Naim, M.; Rouseff, R. L.; Zehavi, U.; Sc hutz, O.; Haleva Toledo, E. The use of chemical analysis and aroma similarity to evaluate the significance of offflavors in citrus products. In Flavor Analysis: Developments in Isolation and Characterizatio ; C. J. Mussinan and M. Morrello, Eds.; American Chemical Society: Washington DC, 1998; pp 303-319. (45) Rouseff, R. L.; Nagy, S.; Shaw, P. E. Impact of Food Research Development, Proceedings of the International Conference on the Impact of Food Research on New product Development, Karachi ; University of Karachi, Dep. Food Sci. & Technol.; Vol. 2, p 149-161. (46) Dreher, J. G.; Rouseff, R. L.; Naim, M. GC-olfactometric characterization of aroma volatiles from the thermal degr adation of thiamin in model orange juice. J. Agric. Food Chem. 51 3097-3102.

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71 BIOGRAPHICAL SKETCH Kristin Nelson graduated from Niceville High School in 1997. She received a Bachelor of Science degree in Chemical Engineering from the Georgia Institute of Technology in 2002, graduating with honors. Kris tin completed her Master of Science in food science and human nutrition at the Univer sity of Florida in 2005. Her research was in the field of flavor chemistry and was c onducted at the Citrus Research and Education Center in Lake Alfred, Florida. She is now part of Kerry’s graduate student management training program working in Lakeland, Florida, in the flavor division. As part of the program, Kristin trains in various areas of the company such as research and development, beverage applications, pilot plant scale-up, production, quality control, and analytical analysis.


Permanent Link: http://ufdc.ufl.edu/UFE0010289/00001

Material Information

Title: Photochemically Induced Flavor Changes in Orange Juice Exposed to Light in Glass and Polyethylene Terephthalate at 4 C
Physical Description: Mixed Material
Copyright Date: 2008

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Holding Location: University of Florida
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Permanent Link: http://ufdc.ufl.edu/UFE0010289/00001

Material Information

Title: Photochemically Induced Flavor Changes in Orange Juice Exposed to Light in Glass and Polyethylene Terephthalate at 4 C
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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PHOTOCHEMICALLY INDUCED FLAVOR CHANGES IN ORANGE JUICE
EXPOSED TO LIGHT IN GLASS AND POLYETHYLENE TEREPHTHALATE AT
40C
















By

KRISTIN ANN NELSON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Kristin Ann Nelson




























I would like to thank my husband Jared, for his support and encouragement these past
two years. He has been there to offer advice when things did not go as planned and to
cheer me on when things did. I would also like to thank my parents who have been there
since day one. No matter where my travels take me, they have always been ready with
bags packed to rush to my aid when they were needed. Without my family, I would not
be where I am today and therefore I dedicate this project to them.















ACKNOWLEDGMENTS

I would like to first thank my professor, Dr. Russell Rouseff, for all of his help on

this project. His expertise in multiple fields was a great asset in setting up the experiment

and analyzing the results. Dr. Rouseff went above and beyond by providing hands on

assistance and knowledge in constructing equipment for this experiment.

I would also like to express my gratitude to my committee members Dr. Renee

Goodrich, Dr. Ronald Schmidt, and Dr. Kathryn Williams for their guidance. Their

insight and suggestions were a great aid in designing this experiment.

Also, I would like to thank the Department of Citrus and the University of Florida

for their financial support of this project.

Next, I would like to thank everyone at the Citrus Research and Education Center

for their help and for allowing me to use the facility for my experimentation and data

collection. Big thanks are owed to Jack Smoot, and to my fellow lab workers, Wendy

Bell, Kanjana Mahattanatawee, and Filomena Valim for assisting me with my experiment

and for helping me learn to use the laboratory instruments. Special thanks are given to

April Elston for performing GC-O work for this experiment and for taking the time to

answer all my lab questions. Also, thanks go to Dr. Mickey Parish and Lorrie Friedrich

for their help performing microbiology tests, Gary Coats for his help using the

spectrophotometer, Yehong Xu for her help with ascorbic acid measurements, and Dr.

Bruce Welt for help measuring dissolved oxygen.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ....................................................... .. ........... ............ .. vii

L IST O F FIG U R E S .............. ............................ ............. ........... ... ........ viii

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 LITER A TU R E REV IEW ............................................................. ....................... 3

Orange Juice Volatiles and Oxidation Reactions ........................................ ..............3
Packaging M materials and Interactions.................................... ..................................... 4
S c alp in g .................................................................. ................................ . 4
L teaching ..................................................................... . 6
Perm eation through Package ........................................ ........................... 6
L ight and O xygen E effects ............................................................................. ...... .6
B row ning ........................................... ............................ .. 6
A scorbic A cid L oss ....................... ............................ .... ......... ............ ..
Photochemical Reactions........................... ......... .......................... 9
Extracting and Concentrating Flavor Volatiles .................................. ............... 10
Sam ple Extraction ................................................. ............ 10
Sam ple C concentration ..................... ............... .................... ..... ...... ..... 11
Gas Chromatography-Olfactometry History and Methods............... .... ..............12
P u rp o se ............................................................................ 1 3

3 MATERIALS ............................... ... ...... ... ................... 14

Light Cham ber................................................. 14
O ra n g e Ju ic e ............................................................................................................... 1 5
Storage C containers .................. ........................................ .. .......... 16

4 M ETHOD S ..................................... ................................. ........... 17

Initial Juice M easurem ents ............................................................ ............... 17



v









Storage C onditions.......... ............................................................... ........ .. 18
S to ra g e S tu d ie s ..................................................................................................... 1 8
Sam ple Preparation ................................................. ....... ............... 19
Sensory and A nalytical Tests.............................................. ............................ 19
S en so ry A n aly sis ........................................................................................... 19
Juice Color M easurem ents ............................................................................ 20
A scorbic A cid M easurem ent .......................................................... ..................20
Gas Chromatograph-Flame Ionization Detector / Olfactometer .......................21
Gas Chromatography Mass Spectrometry..................................................21
M icrobiological A nalyses......................................................... ............... 22
Statistical Analysis................... .. .................. ......... 22

5 RESULTS AND DISCU SSION ........................................... .......................... 23

O range Juice P properties ..................................................................... ...................23
Sensory F lavor C changes ..................................................................... ..................23
C olor C changes .........................................................................25
A scorbic A cid C changes ............................................................... .......................28
Aroma Active Compounds (GC-O Studies)............................................................29
Qualitative Differences ............................................. .. ...... ................. 35
Quantitative D differences ............................................................................. 37
Sulfur Smelling Aroma Compounds ....................................... ............... 48
A accelerated Study ............ ...................................... .... .... ........ 50
M icrobiological Evaluation ............................................... ............................. 51

6 CON CLU SION S ............................ ........ ... ......... ........ ..... ...... 53

APPENDIX

A STATISTICAL TEST FOR "L" SIGNIFICANCE..................................................55

B STATISTICAL TEST FOR "A" SIGNIFICANCE ................................................56

C STATISTICAL TEST FOR "B" SIGNIFICANCE......................... .....................57

D STATISTICAL TEST FOR MYRCENE SIGNIFICANT DIFFERENCE...............59

E STATISTICAL TEST FOR CARVONE SIGNICANT DIFERENCE....................61

G GC-FID RESULTS FOR JUICES STORED IN PLASTIC ..................................... 63

H GC-FID RESULTS FOR JUICES STORED IN GLASS .......................................65

L IST O F R E F E R E N C E S ........................................................................ .....................66

B IO G R A PH IC A L SK E TCH ..................................................................... ..................71
















LIST OF TABLES


Table pge

1 Initial O range Juice Properties ........................................... .......................... 23

2 Sensory Analysis of Juice after Twelve W eeks .....................................................24

3 Ascorbic Acid Loss During Storage...................................................................... 29

4 Aroma Active Compounds Identified in Initial Valencia Orange Juice ..................31

5 Aroma Active Compounds in Juice Stored in Plastic for Twelve Weeks ..............32

6 Aroma Active Compounds in Juice Stored in Glass for Twelve Weeks ................33

7 p-cymene Comparison for Juice Stored Twelve Weeks .......................................43

8 Comparison of PVG after Twelve Weeks Storage Using GC-MS ..........................47

9 GC-FID Responses for a-terpineol In Juices Stored for Twelve Weeks .................48
















LIST OF FIGURES


Figure pge

1 D iagram of L ight C ham ber .......................................................................... ..... 14

2 Picture of Light Chamber in Cold Storage..................... ................................14

3 Wavelength Spectrum for Philips Cool White Fluorescent Lights........................15

5 "L" Values for Juices after Twelve Week Storage Study .....................................26

6 "a" Values for Juices after Twelve Week Storage Study .....................................27

7 "b" Values for Juices after Twelve Week Storage Study .....................................28

8 Comparison of Detector Responses .. .. ............................. ....................... ............... 34

9 Aroma Active Compounds in Juices Stored in PET for Twelve Weeks ..............35

10 Aroma Active Compounds in Juices Stored in Glass for Twelve Weeks ..............36

11 Decrease in Myrcene after Twelve Week Storage Study......................................37

12 Increase in Carvone after Twelve Week Storage Study................ ..................39

13 Increase in 1,8-Cineole after Twelve Week Storage Study.................................41

14 p-cymene Peak Area Measurement on GC-MS ................................................. 42

15 Increase in Vanillin During Twelve Week Storage Study ....................................44

16 Increase in Furaneol During Twelve Week Storage Study ....................................45

17 4-Vinyl Guaiacol Peak Area Measurement on GC-MS .......................................47

1 Increase in Sulfur Compound (LRI 1116) During Twelve Week Study.................49

19 Formation of Sulfur Compound (LRI 806) after Accelerated Storage Study ..........51

20 Increase in Sulfur Compound (LRI 900) after Accelerated Storage Study.............. 52

21 Significant Difference Between L Values.............. ...... ...................55









22 Significant D difference in "a" V alues ............................................... .................. 56

23 Significant D difference in "b" values.................................. .......................... 57

24 Significant Difference in Amount of Myrcene ......................................................59

25 No Difference in Amount of M yrcene .................... ...................... ............... .60

26 Significant Difference in Amount of Carvone ............................... ...............61

27 No Difference in Amount of Carvone..................................... ........................62















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 Science

PHOTOCHEMICALLY INDUCED FLAVOR CHANGES IN ORANGE JUICE
EXPOSED TO LIGHT IN GLASS AND POLYETHYLENE TEREPHTHALATE AT
40C

By

Kristin Ann Nelson

May 2005

Chair: Russell L. Rouseff
Major Department: Food Science and Human Nutrition

Pasteurized Valencia orange juice was stored in glass and polyethylene

terephthalate containers and exposed to fluorescent light at 40C for twelve weeks. The

flavor, color and ascorbic acid concentrations of juices exposed to light were appreciably

different than control samples covered with aluminum foil. Light exposed juices became

darker, as indicated by significant (p<0.01) decreases in "L" values. Light exposed

orange juices lost 21% and 68% more ascorbic acid than unexposed controls when stored

in plastic and glass containers respectively.

Using a defined 15 point flavor quality scale, a trained sensory panel judged the

light exposed juices to be of lower quality than controls. Juices exposed to light for

twelve weeks had an average rating of 3.8 whereas control juices had an average rating of

6.8. In addition to overall flavor evaluations, individual aroma components were

evaluated using GC-olfactometry (GC-O). Light exposed samples contained less 3-









myrcene, and more carvone, 1,8-cineole, p-cymene, vanillin, and Furaneol. Extracted ion

chromatogram GC-MS data indicated that light exposed juices had on average 85% more

p-cymene than those that were not exposed. Vanillin, Furaneol, and some sulfur

compounds typically form in juice due to thermal degradation and thermally induced

non-enzymatic browning. However, all juices were stored at 40C which is well below the

minimum temperatures needed to produce thermal degradation by classical chemical

means, suggesting these compounds were products of photochemical reactions. The

thermally induced off-flavors a-terpineol and 4-vinylguaiacol did not increase in light

exposed samples, indicating that these two degradation products were not catalyzed by

light-exposure.

Two tentatively identified sulfur compounds were observed in juice samples that

were exposed to light. The compound 4-mercapto-4-methylpentan-1-ol had an onion-

like, moldy, or soured aroma and 3-mercapto-hexen-l-ol had a moldy or soured aroma.

In addition, two sulfur smelling compounds (R.I. of 806 and 900) were produced only in

light exposed juices during an accelerated storage study. The appearance of these off-

aromas in only light exposed juices partially explains why the overall orange juice

character was degraded.














CHAPTER 1
INTRODUCTION

Citrus is one of the most important agricultural crops in Florida with annual orange

juice sales topping three billion dollars in 2004. However, in recent years there has been

a steady decline in orange juice consumption and industry profits (1). Manufacturers

have found that using different packaging may increase sales. A recent trend is to

employ clear containers in order to attract consumers with the fresh, bright color of citrus

juices (2). Glass has been used for this purpose; however, this material is expensive,

heavy, and prone to breakage. An alternative is to utilize clear plastic such as

polyethylene terephthalate (PET). The plastic is lightweight, robust, and inexpensive.

Limitations include low oxygen and light barrier properties.

There have been numerous reports concerning the influence of light, and container

oxygen permeability on color changes and Vitamin C losses (3-5). Solomon et al. (3)

reported that light had no effect on ascorbic acid content and an insignificant effect on

browning on orange juices stored for 52 days at 80C whereas Ahmed and coworkers (5)

reported a 20% loss of ascorbic acid in only six days at a similar storage temperature.

Sensory panels have also compared light exposed orange juices with controls and found

significant differences. Although overall flavor changes have been reported, the

individual flavor compounds responsible for these changes have yet to be identified.

Light and oxygen studies conducted using orange oil and lemon oil aqueous emulsions at

room temperature and slightly elevated temperatures have shown that changes in flavor

compounds are due to specific chemical and photochemical reaction products (6-9).






2


Since orange juice has many compounds in common with orange and lemon oils, it is

hypothesized that similar photochemical reactions might occur in orange juice. However

the rate at which these products would form at 40C is uncertain.














CHAPTER 2
LITERATURE REVIEW

Orange Juice Volatiles and Oxidation Reactions

The aroma of orange juice consists of a combination of volatile compounds in

specific proportions. The classes that make up orange juice flavor are terpenes,

aldehydes, esters, alcohols and sulfur compounds. Terpenes make up the largest

percentage of orange volatiles, with the main terpenes being d-limonene, myrcene, and

valencene. Although most terpenes do not play a direct role in orange juice flavor, they

may work as carriers for other oil-soluble volatiles. The aldehydes that are thought to

make the largest contribution to flavor are acetaldehyde, citral, octanal, nonanal, decanal,

and sinensal. According to Shaw (10) ethyl butanoate, ethyl 2-methylbutanoate, ethyl

propionate, methyl butanoate, and ethyl 3-hydroxyhexanoate are the major esters in

orange juice and are responsible for the fruity, "top note" aroma in fresh juice. Shaw (10)

has also indicated that alcohols such as ethanol, E-2-hexenol, Z-3-hexenol, linalool, and

a-terpineol are present in orange juice, but few make a significant contribution to flavor.

Finally, it has been suggested that various sulfur compounds that are present at very low

concentrations in the juice make a large contribution to the overall flavor. Some of these

compounds are hydrogen sulfide, methanethiol, and dimethyl sulfide. (10)

"Off-flavor" is defined as a flavor that is not natural or normally present in fresh

foods resulting from deterioration or contamination. Off-flavors in orange juice are

primarily formed due to reactions with oxygen. Oxidation reactions decrease ascorbic









acid levels, and induce terpene oxidation. Aerobic microbiological growth can also

produce off-flavors in orange juice (11).

These situations occur when oxygen is dissolved in the product, through contact

with oxygen in the headspace and from oxygen diffusion through the container material.

Decomposition of residual hydrogen peroxide can also produce oxygen and oxidation

reactions in those cases where it has been used as a package sanitizer. Dissolved oxygen

in the initial juice can be decreased by deaeration, and oxygen in the headspace can be

reduced by filling the container completely or by flushing it with nitrogen. However, the

only way to reduce oxygen permeation through the package is by changing the barrier

properties of the container.

Packaging Materials and Interactions

Orange juice is packed in a wide variety of materials including metal cans,

paperboard cartons, plastic containers, and glass bottles. Although these materials are

designed to protect the juice, packaging materials can also affect the juice's flavor in one

of at least three ways. The three primary flavor altering processes associated with

packaging are flavor scalping, flavor leaching, and permeation of compounds through the

package.

Scalping

Scalping refers to the absorption of one or more compounds from the orange juice

into the packaging material. This process has been observed primarily in plastic

containers. Even though it is generally accepted that volatile composition is altered as a

result of scalping, there were conflicting reports as to whether the loss of certain volatile

compounds influenced the juice's taste or aroma.









In 1987, Kwapong and Hotchkiss studied citrus essential oil solutions stored in low

density polyethylene (LDPE) and two polyethylene ionomers (12). Orange oil

components were not sorbed equally. Benzaldehyde and ethyl butyrate were sorbed to at

approximately equal levels with Ke values ranging from 2-7, where Ke = C(plastic)/C (aq

solution). Neral and geranial were moderately sorbed with Ke values ranging from 15 to 23

and 22 to 40 respectively. Limonene was heavily sorbed especially in LDPE with a Ke

value of 4700. Ten untrained panelists detected significant differences (p<0.05) in the

aroma of the citrus samples using the triangle test. Also in 1987, Manheim, Miltz, and

Letzter compared orange and grapefruit juices stored in laminated cartons and glass jars

at 350C for 10-12 weeks (13). They found a 25% loss oflimonene in both orange and

grapefruit juice stored in cartons within 14 days of storage. A panel of twelve to fifteen

experienced tasters detected a significant difference (p<0.05) in the juices flavor. In

1992, Marin and colleagues observed an 80% loss of orange juice limonene into LDPE

within 24 hours at 250C(14). They used gas chromatography-olfactometry (GC-O) to

determine that limonene has relatively low aroma activity and thus contributes little if

any to overall orange juice flavor and aroma. Also in 1992, Pieper and colleagues

compared preference scores for orange juices stored at 40C for 24 weeks in glass and

LDPE cartons (15). They found that although the plastic cartons absorbed 50% of the

limonene, no significant difference was observed between the hedonic scores of any of

the juices. Ethyl butyrate (which was thought to be an important aroma component) was

not absorbed to a measurable extent by the plastic container. In 1997, Sadler and

colleagues examined sorption of orange juice volatiles into LDPE, polyethylene

terephthalate (PET), polyamide (PA), and ethylene (co-)vinyl alcohol (16). The juice









was maintained at 4.50C while the polymer strips were exposed for three weeks. Juices

were stored in sterile Erlenmeyer flasks in contact with the different polymer strips at a

surface to volume ratio that was twice that which is used commercially. No significant

flavor difference was detected in between any polymer treated and control juices using

triangle tests with 15-22 experienced panelists.

Leaching

Leaching refers to the migration of compounds from the container into the orange

juice. This can be caused by residual monomers, plasticizers, processing aids, and

solvents from printing inks and adhesives (17). In the orange juice industry, this problem

includes off-flavors caused by juice stored in metal cans. In 2000, Takahashi and

colleagues concluded that plated tin inside the can reacted with dissolved oxygen to cause

unwanted reactions in fresh mandarin orange juice (18).

Permeation through Package

Permeation relates to movement of flavor compounds through the package. This

includes flavor compounds leaving the juice, and unwanted flavors entering the juice

from outside the container. The greatest problem with permeation in orange juice is

oxygen being transferred into the container and negatively impacting the juice inside.

Light and Oxygen Effects

When packaging materials do not provide an adequate barrier to light and oxygen,

the juice's quality can be affected. The most common problems involve browning of the

juice, loss of ascorbic acid, and changes in the overall flavor of the juice.

Browning

Non-enzymatic browning, or the Maillard reaction, occurs when reducing sugars

such as sucrose, glucose, and fructose react with proteins, peptides, amino acids or









amines (19). The reaction is favored by higher temperatures, lower water activities, and

during extended storage. Non-enzymatic browning is an undesirable reaction that occurs

in orange juice when heated during pasteurization or storage. The reaction causes a loss

of essential amino acids and leads to the formation of brown pigments known as

melanoidins that cause juice colors to darken. Compounds such as furaneol, norfuraneol,

furfural, 5-hydroxymethylfurfural, and sotolone are produced and contribute to flavor

changes in the juice (20). The greatest driving force of these reactions is increased

temperature, and the presence of these compounds has been correlated to elevated

temperature storage.

Several studies have investigated the effects of light and oxygen on browning in

orange juice. In 1995, Solomon et al. conducted research on pasteurized orange juice

stored for fifty-two days at 80C (3). The juice was stored in glass containers with glass,

polyethylene and paper closures. They found that browning was significantly (p<0.001)

correlated with the amount of dissolved oxygen in the juice which occurred to the

greatest extent in paper capped bottles because they had the greatest oxygen permeability.

However, the difference in the extent of juice browning due to light-exposure was found

to be insignificant (p<0.05).

In 1986, Trammell and colleagues conducted an experiment on single-strength

orange juice with varying dissolved oxygen levels (21). Juice was stored for five months

at 220C. It was again found that greater amounts of oxygen in the juice led to increased

browning. However, a sensory evaluation did not detect changes in the juice flavor

(p<0.05).









Ascorbic Acid Loss

The majority of research has investigated the effect of light and oxygen on ascorbic

acid, also known as Vitamin C, in the juice because of its nutritional value. In 1976,

Ahmed and colleagues investigated the effects of fluorescent light on flavor changes and

ascorbic acid loss in reconstituted orange juice and orange drinks (5). The juices were

divided into plastic, glass, and paperboard containers and were placed in a light chamber

at 60C for 6 days. It was found that, when exposed to light, the orange juice lost 20% of

its ascorbic acid and the orange drink lost 40-90%. A trained taste panel of 10-12 women

performed a hedonic scale rating that indicated the juice in the paperboard containers

tasted significantly better (p<0.05) than juice in the light exposed plastic and glass

bottles.

In 1992, Kennedy and colleagues studied commercial single-strength orange juice

in TetraBrik cartons (22). The juices were stored at 4, 20, 37, 76, and 1050C for sixty

days. They found that juices with lower initial dissolved oxygen (1.70ppm compared to

4.30ppm) had a slower rate of ascorbic acid loss (6.5mg/L*day compared to

25.5mg/L*day). It was also found that the temperature the juice was stored at plays the

greatest role in deterioration in that the higher the temperature, the more ascorbic acid

was lost.

Sattar and colleagues performed similar experiments in 1989 using pasteurized

orange drink in clear, green and amber glass bottles as well as in a wax laminated paper

(TetraPak) carton (4). The containers were stored at room temperature for thirty-two

days. Ascorbic acid losses were 60.6%, 54.6%, 51.0%, and 45.5% in clear glass, green

glass, TetraPak laminated paper, and amber glass respectively. This shows that greater

light-exposure resulted in significantly greater (p<0.05) ascorbic acid loss. Also, the loss









in TetraPak cartons was greater than in amber bottles because of the higher oxygen

permeability. A ten member panel performed hedonic ratings based on color, taste, and

flavor and reported higher preference ratings for juices stored in amber bottles.

Photochemical Reactions

Several previous studies have investigated the changes in flavor compounds in

lemon oil as a result of exposure to oxygen and light. In 1988, Schieberle and Grosch

studied lemon oil in an aqueous citric acid emulsion (7). The samples were left for thirty

days at 370C. The team found that neral, geranial, and linalool decreased, with a

corresponding increase in p-methylacetophenone, p-cresol, fenchyl alcohol, p-cymene,

and 1-terpinen-4-ol. They believe this change in composition is responsible for the

deterioration of lemon oil flavor over time.

In 1997, Iwanami and colleagues also investigated the effects of ultraviolet light on

lemon oil (6). The team exposed a mixture of lemon oil, a phosphate buffer, and ethanol

to UV-light (< 400nm) for four days at 300C. They found that citral (a combination of

neral and geranial), limonene, terpinolene, and nonanal decreased, while the

decomposition product, p-cymene, increased. The finding that citral was the most

unstable component and thatp-cymene was produced mirrored the results of Schieberle

and Grosch.

In 1991, Ziegler and colleagues studied the changes in flavor compounds in orange

oil after exposure to ultra-violet light (9). The orange oil was dissolved in ethanol,

acidified with citric acid and homogenized to form an emulsion. The emulsions were

exposed to ultraviolet light for fifty minutes at 200C. Ziegler found a significant increase

in carvone, isopulegol, isomers of carveol, limonene oxide, and linalool oxide with a

corresponding decrease in neral, geranial, and cintronellal. In addition, several new









compounds formed during the study including p-mentha-1,8,dien-4-ol, a-cyclocitral,

photocitral A, iso(iso)pulegol, carvonecamphor, methone, isomenthone, isomers ofp-

menth-1(7),8-dien-2-ol and isopiperitenol.

Extracting and Concentrating Flavor Volatiles

Volatiles in orange juice are in very low concentrations and are part of a complex

matrix of insoluble and nonvolatile compounds that cannot be injected into a gas

chromatograph. For these reasons, the volatiles in the orange juice must be isolated and

concentrated before analysis can occur.

Sample Extraction

Two common extraction techniques are headspace analysis (either static or

dynamic) and liquid-liquid extraction. These techniques have various advantages and

limitations and the method performed depends on the compounds of interest. Regardless

of the isolation technique, the goal remains to make the extraction representative of the

original sample.

Liquid-liquid extraction relies on the differences in polarity to extract the desired

compounds from the overall sample. The solvent chosen for the extraction determines

which compounds will be isolated. Compounds that are non-polar will be extracted to a

greater extent by a non-polar solvent such as pentane. The juice and extracting solvent

are thoroughly mixed, and centrifuged to separate aqueous and organic layers. The

organic layer is retained, while the aqueous layer may be discarded or can be extracted

with the same solvent or another solvent repeatedly. However, the more solvent that is

used for extraction, the further the sample must be concentrated before analysis, and thus

the more volatiles may be lost.









One advantage of this extraction method is that the solvent comes in direct contact

with the juice and extracts the volatiles from the juice matrix, whereas in headspace

analysis the volatiles must partition from the pulp into the aqueous phase, come to

equilibrium with the headspace, and finally adhere to the fiber coating. Liquid-liquid

extraction allows for a more accurate quantification of a wider range of volatiles, as

competition for headspace and fiber coating is eliminated. One drawback is that it also

isolates some non-volatile material such as carotenoids and lipids that can degrade in the

GC injector and form artifacts.

It is important to note that not all chemical compounds have the same extraction

efficiencies, and thus are not in the same proportions in the extract as they were in the

original juice. This can be overcome by adding internal standards to the sample mixture.

Internal standards should be chosen such that the standard and the compound of interest

have similar structures and physical properties. Ideally, for GC-MS analysis, a

deuterated isomer of the desired compound should be used for comparison, since it will

have the same extraction efficiencies and evaporation loses. Knowing the concentration

of the internal standard can allow for back calculation to find the concentration of

compounds in the original sample.

Sample Concentration

Concentration can be achieved by a variety of methods. The nitrogen blowdown

method is commonly used. In this method, a stream of nitrogen gas is gently blown

across the surface of the extract. This action increases the speed at which the solvent

evaporates. The nitrogen blowdown method must be performed slowly so that the

volatiles of interest are not lost with the solvent. Also, it is important to use a solvent that

has much lower boiling point from that of the volatiles to reduce loss. After sample









preparation, the juice extract can then be injected into an analytical tool such as the gas

chromatograph.

Gas Chromatography-Olfactometry History and Methods

Gas chromatography is a method that employs a capillary column of varying

materials in order to separate chemical compounds based on their polarity and affinity for

the column material. The temperature inside the column is steadily increased, causing

the compounds to elute from the column at different retention times. Various instruments

such as a flame ionization detector (FID) or a mass spectrometer (MS) can be used for

compound detection.

In 1964, Fuller, Steltenkamp, and Tisserand first reported the use of an

olfactometer (23). This device consisted of a sniff port attached to the GC, parallel to the

detector. A human assessor could sit at the sniff port and observe the eluting aromas.

This new technique helped researchers determine compound identities and also to

establish which compounds were aroma active. Based on the intensity of the aromas,

researchers could also determine how much each compound contributed to the overall

aroma profile. This was a significant advancement in gas chromatography, in that some

highly aroma active compounds that occurred at very low concentrations in orange juice

and were previously overlooked, now received greater focus.

There are three main types of GC-O analysis: Dilution Analysis, Time-Intensity

Analysis, and Detection Frequency. In Dilution Analysis, a sample is repeatedly reduced

in concentration and analyzed on the GC-O. It is noted which compounds are detected at

each dilution level. This information can be used to determine each chemical

compound's threshold. The threshold is then divided by the concentration of that

compound in the original sample in order to determine the compound's aroma activity.









The higher the activity, the more that compound contributes to the sample's overall

aroma. The two types of Dilution Analysis are Aroma Extract Dilution Analysis

(AEDA) which measures the peak height (24), and Combined Hedonic Aroma Response

Measurement (CHARM) which measures the peak area (25). Time-Intensity Analysis

consists of only running the sample once on the GC-O with no subsequent dilutions.

During the GC run, the aroma intensity of each compound is noted by sliding a lever

across a sectioned bar. The bar contains various intensity descriptors such as slight,

moderate, and strong, and is attached to a computer that records the subject's inputs. The

amount a compound contributes to the sample's overall aroma is then based on the height

or area (OSME analysis) over which the subject moved the lever (26). The third method,

Detection Frequency, consists of measuring what percentage of assessors detects a given

aroma at various concentrations and is measured by Surface of Nasal Impact Frequency

(SNIF) (27). In this study time-intensity GC-O was employed.

Purpose

The purpose of this study was to determine the effect of fluorescent light on

ascorbic acid, browning and flavor of orange juices stored in glass and PET. Since both

chemical and photochemical reactions occur during a storage study, this study will be

conducted at temperatures just above freezing (40C) to minimize chemical reactions.

Therefore if any major changes occur, they would be due primarily to photochemical

reactions. This study will also have practical significance as these conditions are more

similar to retail or market place conditions than any previous study. This will also be the

first study to examine the individual volatile components in light exposed orange juice.












CHAPTER 3
MATERIALS

Light Chamber
The light chamber was built to maximize the light-exposure the bottles would

receive. A diagram and picture of the light chamber can be seen in Figures 1 and 2

respectively. In Figure 2, the side door is open for viewing.








Figure 1: Diagram of Light Chamber





'V 'f -^ -ii .1^ t J
"- I -




A ^-i


Figure 2: Picture of Light Chamber in Cold Storage

A steel pipe was secured to the center of two circular tables. The pipe rotated

freely on its axis to allow the tables to be turned daily. This design ensured that each










container received an equal amount of light-exposure during storage. The chamber was

32"x 32"x 26", supported by four 2"x 4" studs, and consisted of 5/8"plywood on the top,

bottom, and two support sides, and 3/8" plywood on the two opening doors. It was

equipped with eight 20W Phillips cool fluorescent light bulbs (Royal Philips Electronics

USA, Somerset, New Jersey) that provided an average intensity of 1750 lux as measured

on a Lambda Instruments LI-185 light meter (Lambda Instruments Corporation, Lincoln,

Nebraska). These bulbs were chosen in order to simulate the wavelength of light that the

juice would normally experience in a supermarket setting. The manufacturer's

specification of the light's spectrum can be seen in Figure 3.

0.75
Cool White

2 0.50


S0.25


3010 400 500 600 700 800 9oo00 1000
Wavelength (nm)
Figure 3: Wavelength Spectrum for Philips Cool White Fluorescent Lights

The lights were installed vertically to ensure that the top and bottom platforms

would receive equal lighting and to reduce shadows. Eight reflective mirrors were placed

on the sides of the chamber to reflect and increase the light intensity. Finally, two fans

were installed in the sides of the chamber in order to increase airflow through the

chamber and keep temperatures evenly distributed.

Orange Juice

The orange juice was from late season Valencias, was not concentrated, and did not

have pulp or flavors added. The juice was pasteurized at 2120F for 10 seconds and then

cooled to refrigeration temperature at a local orange juice processing plant before being










filled into five-gallon aseptic Scholle bags. These were transported in coolers in order

to assure that the juice did not undergo temperature abuse. The juice was transferred

aseptically into sterilized plastic and glass bottles and placed into the storage chamber.

The total time the juice spent between leaving the processing facility and being placed

into the storage chamber was about one hour.

Storage Containers

The bottles used in this experiment were eight-ounce "Boston round" bottles

obtained from Lerman Container Corporation (Lerman Container Co., Naugatuck, CT).

The container materials were polyethylene terephthalate (PET) and glass and were

similar dimensions. A graph of the light transmission characteristics for the two

materials can be seen below in Figure 4. Both materials had similar light transmission

characteristics and most of the light emitted by the fluorescent lights was in the

wavelength range that the materials transmitted. Each bottle held 250 mL of juice.

100
Glass

Ss80o Plastic


S60 Light emission from
SFluorescent Lights
I-



20
10 ,



200 400 600 800
Wavelength (nm)


Figure 4: Light Transmission Through Glass and Plastic Containers














CHAPTER 4
METHODS

Initial Juice Measurements

Several tests were performed in order to document starting conditions. The Brix

was determined as a measurement of the soluble solids in a juice. One drop of the juice

was placed onto a digital refractometer. This device related the juice's refractive index to

Brix (28).

The percent oil in the juice was measured using the Scott oil test (29). This test

consisted of a titration based on the chemical reaction between d-limonene (the main

component in orange oil) and bromine. The sample was prepared by mixing it with

alcohol and heating it until the alcohol and oil evaporated. The vapor was condensed and

collected for analysis. Bromine was added drop-wise until it completely reacted with all

the unsaturated compounds in the oil. Knowing the concentration of the bromine and the

amount added to reach the stoichiometric endpoint allowed for the calculation of d-

limonene content. This is normally expressed as % by volume in 11.8 Brix juice.

The total titratable acidity of the sample was determined using a titration procedure

(28). Sodium hydroxide was added to the acidic juice until a pH of 8.2 was achieved.

This pH is used in industry because that is the endpoint of phenolphthalein indicator,

which was used before electronic titration devices were available. Using this endpoint

allows for comparison of new values to those determined using the old method.

Knowing the amount of juice, and the amount and concentration of sodium hydroxide

used allowed for the calculation of the acidity of the orange juice.









Storage Conditions

The juice was divided with half going into plastic bottles and the other half into

glass bottles. For each container type, there were three replications performed. For each

replication there was several bottles filled such that a new bottle could be open, analyzed,

and frozen on each of the test dates. A second group of identical bottles were also

placed in the light chamber. However, this group was wrapped in aluminum foil in order

to insure that the juice was not exposed to light during storage. This combination of

container type and aluminum foil allowed for comparison of compounds in juices

exposed to light in plastic, light in glass, no light in plastic, and no light in glass. The

light chamber was kept in cold storage at 40C. A temperature probe on the inside of the

storage chamber recorded any temperature deviations.

Storage Studies

There were two storage studies conducted during this experiment. The first study

consisted of storing the juices in the fluorescent light chamber for twelve weeks. In this

experiment, juices were tested on the first day, as well as after 4 weeks, 8 weeks, and 12

weeks. This experiment was designed to monitor changes in juice over an extended

amount of time. This should minimize thermally induced chemical reactions and

maximize the possibility of photochemical reactions. The study also had the added

advantage in that it would be very similar to what juices might experience under the best

of conditions in the market place. The second study was an accelerated storage study. At

the beginning of the experiment oxygen was bubbled through each of the juices in order

to increase the dissolved oxygen content and thus any oxidation reactions that may occur

over time. The juices were then stored for two weeks and sampled after Week 1 and









Week 2. In this experiment all juices were stored in glass bottles and the bottles wrapped

in foil were considered "control".

Sample Preparation

Orange juice samples were prepared for analysis using liquid-liquid extraction.

Twenty-five milliliters of juice were mixed thoroughly with 10 mL of n-pentane solvent

in a 50 ml glass syringe. The mixture was then placed into a centrifuge for 10 minutes to

allow the aqueous and organic layers to separate. The organic top layer was drawn off

and retained, while the aqueous bottom layer was placed back into the syringe and

extracted with 10 mL of ethyl ether. The same process was repeated and this organic

layer was combined with the first. A small amount of sodium sulfate was added to the

extract to remove any remaining aqueous material. The extract was drawn from the salt

and placed into a clean vial. Two internal standards were added to the solution. Fifty

microliters of a 2000 ppm solution of ethyl valerate was added to mimic conditions

experienced by low molecular weight compounds, and 50 [tL of a 2000 ppm solution of

4-heptadecanone was added to mimic those compounds with higher molecular weights.

A nitrogen blowdown method was employed in order to slowly concentrate the sample to

0.1 mL.

Sensory and Analytical Tests

Sensory Analysis

Sensory analysis was performed by five trained panelists. The panelists were all

members of a group trained to rate orange juice for a study conducted by Elston, Rouseff,

and (publication pending). Juices were rated on a fifteen point overall flavor quality scale

that reflected attributes such as aroma strength, orange juice character, peel oil,

fatty/metallic/green, fruity/floral, cooked/heated/processed, sweetness, sourness,









bitterness. In this scale juices ranked between 10 and 15 were considered superior

quality, juices ranked between 5 to 10 were good quality juices and juices ranked 5 and

below were considered to be of poor quality. These quality scores were not a reflection

of hedonic rating or preference. The juice was presented at around 17C in an open room

under white lighting.

Juice Color Measurements

The juice was measured on the first day of the experiment as well as after twelve

weeks on a Gretag MacBeth Color-EYE 3000 spectrophotometer (Gretag MacBeth,

Regensdorf, Switzerland). This device emits a flash of light from a pulsed xenon arc

lamp and measures the light reflection from the juice. Samples were evaluated using the

International Commission of Illumination's *L, *a, and *b standard color space

specification as outlined by Lee and Chen in 1998 (30). The value "L" measures relative

lightness or darkness of the juices where L= 0 would correspond to black (total absence

of reflected light) and L=100 would correspond to white (total reflection of incident

light). The value "a" is a measure of green to red, with negative numbers indicating more

green, a value of 0 being neutral, and positive numbers indicating more red. The value

"b" is a measure of blue to yellow, with negative values indicating more blue, a value of

0 being neutral, and positive numbers indicating more yellow.

Ascorbic Acid Measurement

Ascorbic acid measurements were performed using capillary electrophoresis by

Yehong Xu at the Florida Department of Citrus using published methods (31). The

sample was prepared by placing 4 ml of juice into a capillary electrophoresis tube and

adding 12 ml of 0.1% ethylenediamine tetraacetic acid (EDTA) solution and 100 ul of

ferulic acid as an internal standard. The sample was filtered. Injection volume was set at









10 [l. The run time was 30 minutes and the running buffer was 35mM sodium borate

and 5 % acetonitrile at a pH of 9.3. The column was uncoated fused silica capillary with

dimensions 50 u[m x 70 cm with a temperature of 23-250C. Voltage applied was 21 kv,

and scanning was performed between 200 and 360 nm using a Photodiode Array (PDA)

detector.

Gas Chromatograph-Flame Ionization Detector / Olfactometer

The gas chromatograph used in this study was a HP 5890A (Agilent, Palo Alto,

CA) with a Datu (Geneva, NY) high volume olfactometer and described in detail by

Bazemore and coworkers (32). There were two columns used during analysis. The first

was a thirty-meter ZB-5 column (Zebron, Torrance, CA) with a 0.32 mm inner diameter,

and 0.50 u[m film thickness. The GC was run in splitless mode with an injector

temperature of 2200C, a detector temperature of 2500C, an initial oven temperature of

400C with a 70C/min ramp up to a final oven temperature of 2650C for a 5 minute hold

time. The other column was a 30 meter DB-Wax column (J&W Scientific, Folsom, CA).

The column's inner diameter, film thickness, injection temperature, detector temperature,

initial oven temperature, and temperature ramp were the same as previously stated.

However, the DB-Wax column utilized a final oven temperature of 2400C. All injection

volumes were 0.2[al. Chromperfect Spirit 5 version 5.0.0 software was used to record

data and integrate the resulting chromatograms.

Gas Chromatography Mass Spectrometry

The GC-MS used was a Finnigan GCQ Plus system (Finnigan, San Jose, CA) with

a DB-5 column (J&W Scientific, Folsom, CA). The column was 60 m long, had an

internal diameter of 0.25 mm., and a film thickness 0.25 Om. Helium (99.999% purity)

was used as the carrier gas. Samples were injected using the AI/AS 3000 autosampler in









the splitless mode with the injector temperature at 2000C. Oven temperature was 400C,

and was increased at a rate of 7C/min to 2750C and held for 5 min. Column head

pressure was maintained at 14.5 psi. Transfer line and ion source temperatures were

2750C and 2000C, respectively. The mass spectrometer detector scanned at m/z 40-300.

The ionization energy was set at 70 eV. Xcalibur version 1.3 software was used to record

and integrate mass spectrometer chromatograms and spectra.

Microbiological Analyses

On the first day and after one month, microbial tests were performed to insure that

off-flavors were not the results of microbiological activity. Samples from each of the

four material and light-exposure combinations were plated and incubated for 48 hours at

300C. Potato Dextrose Agar (Difco Laboratories, Detroit, MI) with 10% tartaric acid was

used to test for yeasts and molds, while Orange Serum Agar (Difco) was used to test for

bacteria and yeast (33).

Statistical Analysis

Statistical analysis was performed using Minitab Statistical Software version 13.32.

Tests for statistical significance were calculated using an independent two-sample t-test

at a 99% confidence interval. A hypothesis of equality was assigned and Minitab was

used to calculate t and p values. If the p value was less than the alpha value of 0.01, then

the hypothesis was rejected and the two samples were not equal.















CHAPTER 5
RESULTS AND DISCUSSION

At the end of the twelve week and the accelerated storage studies, several data

trends were observed. The results listed below are a combination of trends seen in both

experiments. The averages and standard deviations are based on triplicate analysis from

a singe juice container per condition. Although it may be more precise to have multiple

juice containers for each condition, the size of the storage chamber and scope of this

experiment limited the amount of containers used. Container to container variations are

usually due to problems along container seems and closures as seen in metal cans and

paperboard cartons. Since all containers used in this experiment were blow molded

plastic and glass with no seams, the container to container variation should be low, and

the results from each sample should be an accurate representation of each storage

condition.

Orange Juice Properties

Initial juice properties can be seen in Table 1.

Table 1: Initial Orange Juice Properties
Property Value
Brix 12.8
Percent Total Acid 0.724
Brix/acid ratio 17.7
Percent Oil 0.0246

Sensory Flavor Changes

After twelve weeks, the juices were removed from the storage chamber and frozen

until sensory evaluations. Juice was also frozen on the first day of the experiment to be









used as a control. Shown in Table 2 are the cumulative sensory comments for both

orthonasal aroma and flavor impressions for each juice from five trained panelists.

Panelists also evaluated each juice for overall flavor quality based on a 15 point scale.

Table 2: Sensory Analysis of Juice after Twelve Weeks
Conditions Aroma Flavor Quality Rating
Processed, cooked,
Good OJ character, good sweet/sour
Initial Juice cooked, apricot, balance, good overall 8.8 1.3
Initial Juice 8.8 1.3
citrusy, slight flavor, very peely,
oxidized note somewhat musty,
oxidized flavor,
Almost no OJ
No OJ character, A
peppery, cooked character, apricot,
peppery, cooked
candy sweetness,
vegetable, solventy, candy sweetness,
Light-Plastic t burning backed, 3.6+ 1.5
painty, sweet
arael ntes, processed, cotton candy
caramel notes,
fermented flavor, lack of fruity
notes
Sulfury, over More OJ character,
mature, fermented, good sweet/sour
No light-Plastic weak OJ character, balance, furaneol 38
No light-Plastic 3.8 0.4
slightly peppery, sweet, orange peel,
fatty aroma, bitter, fatty/musty off-
metallic flavor, no fruity notes
Almost no OJ
Terpeney, peely, character, artificial
week OJ character, candy sweet, sulfury
Light-Glass slightly peppery, vegetable, acidic, 4.0 + 1.6
sweet/processed, pineapple, bitter,
painty off-flavor cardboard-like,
pronounced off-flavor
Musty, peely,
terpeney, slightly Floral, candy sweet,
No light s fermented, peppery, some OJ character, 11
No light-Glass 6.4 1.1
bleach, some OJ little sour, slightly
character, no off- cleaner like
flavor

There was a considerable loss of flavor quality after 12 weeks storage at 40C as

noted from the difference in average flavor quality score of the control juice (8.8)

compared to even the highest rated stored juice (6.4, no light glass). Since all juices









(except control) were stored for the same time (12 weeks) and at the same temperature

(40C), any flavor changes must be due to container properties or exposure to light. Since

juices exposed to light in either PET or glass had similar ratings, it appears that container

material made no difference in overall flavor score. However, it appears that container

oxygen permeability can influence juices protected from the light. Juices in plastic and

not exposed to light had about the same flavor quality scores as light exposed juices.

However, orange juice stored in glass and not exposed light had higher flavor quality

scores than the similar juice stored in PET, suggesting that oxygen permeability also

influences flavor even at 40C. In general, juices exposed to light exhibited diminished

orange juice character and rated lower than those that were not exposed. Therefore

exposure to light appears to be a major factor in juice quality.

Color Changes

The color of the juices exposed to light during the twelve week storage study

appeared darker than those unexposed. Juice color was evaluated instrumentally using a

spectrophotometer to determine their respective L, a, and b values. The relative

lightness, redness and yellowness of the stored juices and control are shown in Figures 5-

7. Error bars represent one standard deviation above and below the average value.

Since L measures relative lightness or darkness of the juices, it can be readily seen

from Figure 5 that juices exposed to light have darkened. The light exposed juice had a

significantly lower L value (p<0.01) than the original juice (see Appendix A for statistical

calculations). Those that were not exposed to light showed less change from the original

juice. Non-enzymatic browning is typically responsible for darkening citrus juices during

elevated temperature storage in the absence of light. A colored compound has been

identified that is thought to be formed by the condensation of the norfuraneol with the










aldehyde group of furfural at elevated temperatures (20). The darkening of the juices in

this study which occurred at 40C indicates that browning can be induced by light as well

as elevated storage temperature.


58










54










50
Starting Material Light, In Glass No Light, In Glass Light, In Plastic No Light, In Plastic

Figure 5: "L" Values for Juices after Twelve Week Storage Study











Starting Material Light, In Glass No Light, In Glass Light, In Plastic No Light, In Plastic
0'




-1




-2 -




-3 -




-4 -




-5

Figure 6: "a" Values for Juices after Twelve Week Storage Study

Since "a" is a measure of green to red, with negative numbers indicating more

green, a value of 0 being neutral, and positive numbers indicating more red, it was found

that juices that were exposed to light had significantly less (p<0.05) of a green color than

the original juice (see Appendix B).










40






35






30






25
Starting Material Light, In Glass No Light, In Glass Light, In Plastic No Light, In Plastic

Figure 7: "b" Values for Juices after Twelve Week Storage Study

Since "b" values are a measure of blue to yellow, with negative values indicating

more blue, a value of 0 being neutral, and positive numbers indicating more yellow, it

was found that those juices that were exposed to light had significantly less of a yellow

color than those that were protected (see Appendix C). These three measurements

confirm that those juices exposed to light did become darker in color and more brown

than light yellow or orange.

Ascorbic Acid Changes

During the twelve month study, ascorbic acids measurements were taken

periodically in order to monitor any losses. The results of these measurements can be

seen below in Table 3.









Table 3: Ascorbic Acid Loss During Storage
Concentration
Ascorbic Acid
(ftg/ml)
Initial 320
Light, In Plastic 36
No Light, In
Plastic 87
Light, In Glass 45
No Light, In
Glass 160

It is apparent that those samples that were exposed to light lost more vitamin C than

those samples that were not exposed. Juice in plastic bottles lost 21.5% more vitamin C

when exposed to light as compared to juice that was not exposed to light, and juice in

glass lost 68.0% more. Also, samples that were stored in plastic bottles lost more

ascorbic acid than those stored in glass.

The results found in this study correspond with those reported in earlier studies

concerning ascorbic acid loss and sensory ratings. The increased loss of ascorbic acid in

juices exposed to light and corresponding decrease in consumer acceptance (through

hedonic ratings) is consistent with the findings of both Ahmed and colleagues in 1976

(5)and Sattar and colleagues in 1989 (4). Also the increased loss of ascorbic acid in

juices stored in plastic and thus allowing more oxygen permeation is consistent with

findings by Kennedy and colleagues (22).

Aroma Active Compounds (GC-O Studies)

Not all volatiles in orange juice are aroma active. Aroma activity for each volatile

must be established using human assessors. Instruments detect only those volatiles

present in highest concentration. Humans respond only to volatiles with particular

functional groups and molecular shape. Table 4 contains a list of the aroma active









compounds that were identified in the initial juice by comparing GC-olfactometry

descriptors and retention index from both DB-5 and Wax columns with published

standards (34).

After storage in the light chamber for twelve weeks, some compounds decreased,

some increased, and some new compounds formed. A comparison of the GC-O relative

responses (peak area of compound divided by the peak area of the internal standard ethyl

valerate) of samples exposed to light and not exposed to light during storage in plastic

containers can be seen in Table 5. Likewise, a comparison of GC-O relative responses

for samples stored in glass containers after twelve weeks can be seen in Table 6.

It is also important to note that some aroma active compounds occur at such low

concentrations that they are below the detection limits of the flame ionization detector.

Alternatively, some compounds that occur in large concentrations in orange juice have

very low aroma activity and thus are not detected through the olfactometer. An example

of this can be seen in Figure 8.









Table 4: Aroma Active Compounds Identified in Initial Valencia Orange Juice
LRI on DB-5 Identification Sensory Description LRI on Wax
805 ethyl butanoate grassy, fruity 1041
825 sulfur smelling compound skunk
851 z-3-hexen-1-ol fruity 1383
859 sulfur smelling compound rotten fruit
867 1-hexanol sour fruit 1352
871 2-methyl-3-furanthiol cooked grain
900 ethyl valerate (I.S.) fruity 1141
907 methional baked potato 1468
924 2-acetyl-1-pyrroline graham cracker
935 a-pinene pine tree 1024
4-mercapto-4-
942 methylpentan-2-one chicken, moldy 1376
982 1-octen-3-one mushroom 1313
986 b-pinene musty, soil 1106
992 P-myrcene green 1171
1003 octanal fruity, lemon 1309
1006 ethyl hexanoate lemon
1035 p-cymene minty, fresh 1225
1041 limonene licorice, minty 1211
4-mercapto-4-
1046 methylpentan-2-ol fruity 1545
1065 (E)-2-octenal fruity, green
1071 Furaneol caramel 2038
coffee, burnt,
1085 unknown processed
1099 linalool lemon 1553
1100 nonanal citrus, floral 1394
1105 fenchol lemony, citrus
1116 unknown sweet, popcorn
roses, green,
1134 2,6-nonadienal cucumber 1601
1207 decanal lemon, sour, woody 1518
1233 neral lemon, sweet, floral
1252 carvone minty 1754
1320 eugenol balsamic, cloves 2176
1412 vanillin vanilla, sweet 2565
1455 wine lactone dill, crayons 2254
1494 b-ionone raspberry 1955
1564 dodecanoic acid musty 2500
1706 b-sinensal marine, old house 2243
1755 a-sinenesal marine, dusty 2420
co-elutes with
1822 nookatone green, spicy, fruity vanillin









Table 5: Aroma Active Compounds in Juice Stored in Plastic for Twelve Weeks
Light No Light- DB-5
Exposed exposure LRI Descriptor Identity
0.79 0.54 805 grassy ethyl butanoate
0.00 0.46 825 skunk butyric acid
0.98 0.39 851 fruity z-3-hexen-1-ol
0.00 0.27 856 rotten unknown sulfur
0.00 0.69 867 sour fruit 1-hexanol
1.07 0.84 871 cooked grain 2-methyl-3-furanthiol
1.00 1.00 900 fruity ethyl valerate
0.79 0.64 907 baked potato methional
0.00 0.32 924 graham cracker
1.33 0.68 935 pine tree a-pinene
0.45 0.57 982 mushroom 1-octen-3-one
0.68 0.46 986 green,soil b-myrcene
0.81 0.46 1006 lemon ethyl hexanoate
1.04 0.44 1035 licorice,minty 1,8-cineole
0.63 0.89 1041 minty limonene
4-mercapto-4-
1.25 0.40 1046 moldy methylpentan-2-ol
1.16 0.74 1071 caramel furaneol
0.50 0.51 1085 fruity,sweet tetramethyl-pyrazine
0.00 0.41 1099 lemon,bumt linalool
1.52 0.56 1100 sweet,citrus nonanal
0.77 0.00 1105 green
0.00 0.48 1116 citrus,floral unknown
0.92 0.00 1116 moldy 3-mercapto-hexen- -ol*
0.56 0.58 1121 sweet,popcorn
0.62 0.43 1134 sweet ethyl 3-hydroxyhexanoate
0.93 0.00 1207 lemon,sour unknown
0.58 0.39 1233 lemon neral
0.84 0.50 1252 minty carvone
0.90 0.00 1285 cloves,burmt
1.31 0.83 1320 balsamic 4-vinyl guaiacol
0.87 0.38 1350 metallic
1.25 1.29 1383 very metallic
1.57 1.04 1412 vanilla vanillin
0.73 0.58 1455 dill wine lactone
0.82 0.77 1494 berries b-ionone
1.31 0.67 1564 marine b-sinensal
0.00 0.54 1584 burnt,spicy
0.96 0.43 1706 marine,old house a-sinenesal
0.00 0.47 1755 musty,dusty
0.00 0.19 1822 green nookatone









Table 6: Aroma Active Compounds in Juice Stored in Glass for Twelve Weeks
Light No Light- DB-5
Exposed exposure LRI Descriptor Identity
0.52 0.73 805 grassy ethyl butanoate
0.62 0.54 825 skunk butyric acid
0.41 0.39 851 fruity z-3-hexen-l-ol
0.68 0.39 856 wet dog unknown sulfur
0.36 0.57 867 fruity 1-hexanol
0.57 0.60 871 cooked grain 2-methyl-3-furanthiol
1.00 1.00 900 fruity ethyl valerate
0.81 0.64 907 baked potato methional
0.00 0.00 924 graham cracker
0.52 0.55 935 pine a-pinene
4-mercapto-4-methylpentan-2-
0.39 0.96 942 chicken one
0.42 0.41 982 mushroom 1-octen-3-one
0.75 0.51 986 green,dirt b-myrcene
0.35 0.00 1003 fruity,lemon octanal
0.67 0.00 1006 lemon ethyl hexanoate
0.55 0.38 1035 licorice,minty 1,8-cineole
0.40 0.49 1041 minty limonene
4-mercapto-4-methylpentan-2-
0.83 0.00 1046 moldy,soured ol
0.25 0.00 1065 fruity,green (E)-2-octenal
0.87 0.46 1071 caramel furaneol
0.87 0.62 1085 coffee tetramethyl-pyrazine
0.50 0.51 1099 burnt,lemon linalool
0.94 0.00 1100 sweet nonanal
0.00 0.59 1116 lemon,citrus unknown
0.47 0.00 1116 sweet,moldy 3-mercapto-hexen- 1-ol*
0.91 0.57 1121 onion
0.59 0.34 1134 sweet ethyl 3-hydroxyhexanoate
0.54 0.28 1207 floral,sour unknown
0.36 0.35 1233 lemon,woody neral
0.49 0.37 1252 minty carvone
0.91 0.64 1285 burnt
0.71 0.63 1320 cloves 4-vinyl guaiacol
0.93 0.83 1383 metallic
0.74 0.48 1412 vanilla vanillin
0.76 0.79 1455 dill,old house wine lactone
0.40 0.80 1494 berries b-ionone
0.00 0.53 1564 marine b-sinensal
0.60 0.38 1706 marine a-sinenesal
0.46 0.48 1755 peppery
0.00 0.29 1822 green nookatone



















a) >

0
n0




C
LF C0
U- I

UC C 7, E_

Tim M2 Z C
L- 0








a




Q, E E 0 Cc

o 0 1 0 )

(9 Q- 2 > E CL)



0 CD
( ) a)C E D



7 2



11 12 13 14 15 16 17 18 19 20

Time Minutes (ZB-Wax Column)
Figure 8: Comparison of Detector Responses


The compound valencene has a large peak on the FID, but is not present on the GC-


0. Whereas the compound b-ionone has a large peak on the GC-0 but is not present on


the GC-FID. For this reason it is important to look for changes in the juice using both


detection methods. However, the GC-0 data will indicate which FID peaks are


associated with aroma activity and which are not.









Qualitative Differences

In Figures 9 and 10, the average relative intensities of the aroma active components

in light exposed and control juices in PET and glass are compared. The average aroma

intensities are indicated by the bar height and are compared "head to tail" or "fishbone"

by inverting the control juice data.

2.0


- 1.0
0
o
0)


Light Exposed Juice







2 n -lUil.l U. l-


...... ..I o El H i


= o >
.- .
-1. a) a) -0 a) o

'a oa o r o -- oo o


-.= No Light Exposure Juice
-2.0

Figure 9: Aroma Active Compounds in Juices Stored in PET for Twelve Weeks. Note
ethyl valerate was an aroma active internal standard.

In the case of plastic, 31 aroma active components were observed in juices

exposed to light and 36 aroma active components were noted in the control juices.

Twenty six aroma components were common to both juices. From a qualitative point of

view, the primary effect of light was the loss of several aroma components. The loss of

the aroma components unbalanced the orange flavor. A few negative aroma components
3:C 0W OM 2 -C: C C
1.0 X E C: >1 Cb-: ~ OCi-n ,- c

















the aroma components unbalanced the orange flavor. A few negative aroma components









were formed from light-exposure, but since the overall sensory quality of these juices

were similar, it appears that that their flavor reducing impact was minimal.


1.0


0 0.6

o,


oNo, Light Exo r Ji
[ -0.4 I II I
a0 0C0. -_ C 0
0F m a C 0 C )-
Sn F i = 0 U li xa
a o a a'o m at =i t a a o


id W m c n 0 ,
S.|, 1 2 3 2 I :




-2.4
Figure 10: Aroma Active Compounds in Juices Stored in Glass for Twelve Weeks. Note
ethyl valerate was an aroma active internal standard.

In Figure 11 the aroma components for light exposed and control juices are also

compared. The number of aroma active components in both juice types were almost


However, the major aroma difference appears to be due to the production of the

extremely potent sulfur component, 4-mercapto-4-methyl-2-pentanol in the light exposed

juice.
juice.


Light Exposed Juice




-lh lll .1 il1ll










Quantitative Differences

At the end of storage the aroma active compound myrcene was found to decrease in

concentration when exposed to light. The decrease in myrcene after twelve weeks of

storage as measured on the GC-FID is shown in Figure 11.


1.1




a .. - - -




o 0.9 \
CL
I- -










0.7
0.7 -----------------------------------------------------





0 4 8 12
Week

Figure 11: Decrease in Myrcene after Twelve Week Storage Study. o represents juice in
plastic containers exposed to light, represents juice in plastic containers not
exposed to light, A represents juice in glass containers exposed to light, and A
represents juice in glass containers not exposed to light

The amount of myrcene is shown as a measure of "relative response", the area of

the GC-FID peak for myrcene divided by the area of the GC-FID peak of the internal

standard, ethyl valerate. Myrcene decreased significantly (p<0.01) in those samples that

were exposed to light, whereas the myrcene levels in protected samples did not change

(see Appendix D). After twelve weeks the amount of myrcene decreased by 13.3% in

juices in plastic and exposed to light, 1.7% in plastic and not exposed to light, 11.9% in









glass and exposed to light, and 6.1% in glass and not exposed to light. It should be noted

that the sample that was stored in plastic and not exposed to light had the least amount of

myrcene loss. Therefore the loss of this compound is mainly dictated by exposure to

light and not by sorption into the plastic container.

The compound P-myrcene degrades in an acidic environment such as orange juice

to form geraniol and its isomer nerol (35). This is perhaps why P-myrcene decreased

initially and then leveled off. The increased amounts of myrcene lost in those samples

that were exposed to light can be explained by light acting as a catalyst that increased the

rate of reaction in those juices.

Normally, changes occurring in juice are produced by chemical reactions induced

by heat. However, in the case of this experiment, all samples were kept at the same

temperature, such that reactions that occurred to a greater extent after light-exposure must

have been catalyzed by the energy from light. An example of the difference between heat

and light catalyzed reactions can be seen below.

Energy of Activation from Heat:

A+B B AB

Energy of Activation from Light:
hv
A+B AB

The compound that showed the most dramatic increase during all three experiments

was carvone. Figure 12 shows the increase in carvone after twelve weeks of storage as

measured by the GC-FID.

It is apparent that the amount of carvone increased significantly (p<0.01) in

samples that were exposed to light, whereas there was little change in those samples that

were not exposed (see Appendix E). After twelve weeks of storage, carvone increased by









240.1% in plastic and exposed to light, by 66.6% in plastic not exposed to light, 368.0%

in glass and exposed to light, and 27.7% in glass not exposed to light.


0.3











0 4 8 12
0.2 -














Week

Figure 12: Increase in Carvone after Twelve Week Storage Study. 0 represents juice in
plastic containers exposed to light, m represents juice in plastic containers not
exposed to light, A represents juice in glass containers exposed to light, and A
represents juice in glass containers not exposed to light

Carvone is an oxidation product oflimonene (36). This oxidation oflimonene is

perhaps why juices that were stored in plastic with no light, and thus had a greater chance

of oxygen exposure had a greater amount of carvone formation than those juices that

were stored in glass. Also, increases in carvone, which has a minty aroma, and the

subsequent decrease in limonene, which has very little aroma activity, could be a

contributing factor to the overall change in aroma and flavor of the orange juice after

storage.
s. .^ --S -. -. -'- -n


























storage.









Studies by Ziegler and colleagues conducted on orange oil also documented a

significant increase in carvone (9). The changes in carvone in the orange juice used in

this experiment occurred to a lesser extent than those changes in the orange oil. This is

most likely due to the matrix the flavor compounds are suspended in. The compounds in

the orange oil are in close proximity to one another and therefore have a greater chance of

reacting with one another and with light induced oxidation. The compounds in the

orange juice are separated by large quantities of water that decrease compound

interaction and oxidation reactions. The insoluble material (cloud) in the juice may also

work as a reflective material that blocks some of the entering light from contacting the

flavor compounds. This difference between juice and oil may also explain why

compounds that changed in orange and lemon oils (such as neral and geranial) did not

show significant changes in this experiment.

The minty smelling compound 1,8-cineole also increased during storage. This

compound co-elutes on a DB-5 column with limonene. Since the concentration of

limonene is relatively large in orange juice, the resulting GC-FID peak is also large and

therefore the peak for 1,8-cineole could not be quantified. Instead, the peak was

quantified using results from the GC-O which was able to separate the two compounds.

Figure 13 shows the increase in this oxidation product during storage.

Although initially, 1,8-cineole increased in juice not exposed to light, it leveled off

after four weeks of storage. At this time, juice exposed to light showed an increase in

this compound, with a greater increase in the juice that was stored in a plastic bottle and

therefore had a greater possibility of oxygen content. After twelve weeks of storage, 1,8-

cineole increased by 173.5% in plastic and exposed to light, by 15.6% in plastic not










exposed to light, by 44.7% in glass and exposed to light, and by 0.7% in glass not

exposed to light.


1.2






S0.8





S0.4 -






0.0
0. ---------------------------------




0 4 8 12
Week

Figure 13: Increase in 1,8-Cineole after Twelve Week Storage Study. 0 represents juice
in plastic containers exposed to light, represents juice in plastic containers
not exposed to light, A represents juice in glass containers exposed to light,
and A represents juice in glass containers not exposed to light

The volatile 1,8-cineole is a decomposition product of limonene. It has been

reported in past literature that 1,8-cineole only formed when stored at 230C and not at

6C (37). The orange juice in this study was stored at 40C, and thus the increase of 1,8-

cineole may be light induced as well as heat induced.

The volatile compound p-cymene was found to increase in samples that had been

exposed to light. Since p-cymene has a similar retention time as limonene on the DB-5

column (LRI=1026 and LRI=1031 respectively), the two compounds coeluted and could

not be quantified by GC-FID or GC-O. However, the polar wax column in the GC-MS







42


allowed for separation of the two compounds. The peak areas ofp-cymene were

measured based on an extracted ion chromatogram at m/z of 119. An example of how

the peak area was integrated and the library standard mass spectra forp-cymene can be

seen in Figure 14.







100
100 1765
Sso TIC 17.42
S70 1



20 10C 119

8 0 RT: 17.42
S100
S90 50 1
= so EIC m/z= 119 i
70-
60
a so 1 77 1134
Z 40 5
2 30 0 40 80 120 160
a0 20
10

17.1 17.2 17.3 17.4 17 5 17.6 17.7 17.8 17.9
Time (mm)




Figure 14: p-cymene Peak Area Measurement on GC-MS

The peak area of the internal standard was measured using the total ion

chromatograph for all samples. The internal standard used in this comparison was 4-

heptadecanone. The increase inp-cymene in those samples that were exposed to light

can be seen in Table 7.









Table 7: p-cymene Comparison for Juice Stored Twelve Weeks
Sample Peak Area Cymene Peak Area IS2 Ratio
Light, In Plastic 9.66x10A4 3.54x10^7 2.73
No Light, In
Plastic 4.94x10^4 4.24x10^7 1.16
Light, In Glass 6.97x10A4 3.10x10^7 2.25
No Light, In
Glass 2.04x10^4 1.33x10^7 1.53

The samples that were exposed to light have on average 85% morep-cymene than

those that were not exposed. However, there was little difference inp-cymene content

from juices stored in plastic or glass containers either light exposed or not, suggesting

that difference in oxygen permeability of storage container materials was not a factor in

p-cymene formation.

The compound p-cymene is formed from the acid catalyzed decomposition of citral

(38). These results of increased p-cymene after exposure to fluorescent light mirror the

findings of both Schieberle and Grosch and Iwanami and colleagues forp-cymene in

lemon oil (6;8).

The compounds vanillin and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (Furaneol)

also showed a slight increase during storage. These compounds occurred at levels below

the detection limits of the GC-FID and GC-MS and therefore the results reported are

based on GC-O data. Figures 15 and 16 below show the increase in vanillin and Furaneol

respectively.










2.4




S1.8



o




S0.6
0. 1.2 -








0.0 .
0 4 8 12
Week

Figure 15: Increase in Vanillin During Twelve Week Storage Study. 0 represents juice in
plastic containers exposed to light, represents juice in plastic containers not
exposed to light, A represents juice in glass containers exposed to light, and A
represents juice in glass containers not exposed to light

There was an increase in vanillin in juice samples that were exposed to light. It is

also evident that samples stored in plastic bottles had a greater increase of vanillin than

those that were stored in glass. This may be caused by a greater amount of oxygen being

present in the juice stored in plastic bottles. At the end of twelve weeks, vanillin

increased by 171.4% in plastic and exposed to light, by 80.0% in plastic not exposed to

light, by 28.3% in glass and exposed to light, and decreased by 17.4% in glass not

exposed to light.

Vanillin has been reported to form from the thermal degradation of ferulic acid in

citrus juices (39). In this experiment, all of the juices were kept at the same temperature,

so thermal degradation should have occurred equally amongst all samples. However, the









juices that were exposed to light formed more vanillin that those that were protected.

Therefore, light may have provided the energy source needed for ferulic acid

decomposition in place of heat. It has also been noted that vanillin can further degrade to

form phenols and cresols (40). This could explain why the amount of increase in vanillin

levels off after four weeks and actually decreases in the case of juices stored in glass

bottles.

Furaneol increased significantly in samples that were exposed to light during this

study and increased to a lesser extent in those samples left unexposed.

1.4






0

0.7








0.0
"-/




0 4 8 12
Week

Figure 16: Increase in Furaneol During Twelve Week Storage Study. 0 represents juice
in plastic containers exposed to light, represents juice in plastic containers
not exposed to light, A represents juice in glass containers exposed to light,
and A represents juice in glass containers not exposed to light.

Also, juices that were stored in plastic containers again had a greater increase in

Furaneol than juices stored in glass bottles. Furaneol is an undesirable product that is









formed through non-enzymatic browning during orange juice storage (41). It has been

documented as occurring in juices that have been subjected to temperature abuse. Again,

since all of the juices were under the same temperature conditions throughout storage, the

increased amount of Furaneol in juice exposed to light indicates that light may be a

possible energy source that catalyzes the reaction.

A compound that has been used as a marker for temperature abuse in orange juice

is 4-vinyl guaiacol (42). Like vanillin, this compound forms from the thermal

degradation of ferulic acid (43). Because extraction efficiencies are low, it is difficult to

quantify 4-vinyl guaiacol (PVG) using GC-FID and GC-O. However, quantification can

be achieved by measuring the peak area of an extracted ion chromatogram at m/z of 150.

An example of the integration and the library standard mass spectra for PVG can be seen

in Figure 17.

This peak area can be divided by the peak area of an internal standard in order to

determine the relative response. The internal standard used for this comparison was 4-

heptadecanone because of its similar retention time to PVG on a wax column (RT=33.89

and 34.89min respectively). It can be seen in Table 8 that the amount of PVG was

essentially the same after twelve weeks of storage regardless of container type and light-

exposure. Therefore, PVG, probably the most important off flavor formed from thermal

abuse is not responsible for the flavor differences in the light exposed samples at 40C.












3 71

TIC
34.89
\ \ !


RT: 34.89


EIC m/z = 150


J20 RT: 34.67 RT: 35.04 RT: 35.18RT: 35.26

34.6 34.7 34.8 34.9 35.0 35.1 35.2 35.3
Time (min)


Figure 17: 4-Vinyl Guaiacol Peak Area Measurement on GC-MS

Table 8: Comparison of PVG after Twelve Weeks Storage Using GC-MS


Peak Area
Sample PVG Peak Area IS2 Ratio
Light, In Plastic 8.44x10A4 3.54x10A7 2.38
No Light, In Plastic 6.38x10A4 4.24x10A7 2.56
Light, In Glass 6.38x10A4 3.10x10A7 2.06
No Light. In Glass 21.\10 4 1 33\10 7 2 47

Another compound that has been used as a marker for temperature abuse in orange

juice is a-terpineol. This compound forms from acid catalyzed hydration of limonene

and linalool in the presence of water at elevated temperatures (44). It is present in higher

concentrations, and is therefore easier to identify and quantify than PVG (45). It can be

seen in Table 9 that the amount of a-terpineol was the same after twelve weeks of storage

regardless of container material and light-exposure.









Table 9: GC-FID Responses for a-terpineol In Juices Stored for Twelve Weeks
Relative Response of a-
Samples terpineol
Initial Juice 0.126
Light, In Plastic 0.449
No Light, In
Plastic 0.497
Light, In Glass 0.464
No Light, In Glass 0.477

Although the samples increased in a-terpineol during the twelve weeks of storage,

the formation was equivalent regardless of container material or light-exposure. Again

this shows that changes in other compounds such as vanillin and Furaneol were caused by

exposure to light and not due to thermally induced reactions.

Two additional compounds that are used as a marker for temperature abuse in

orange juice are furfural and 5-hydroxymethyl furfural (46). However, due to their low

extraction efficiencies, they were not identified or quantified using either GC or GC-MS.

Their thresholds were too high to be aroma active and they were not detected using GC-

0.

Sulfur Smelling Aroma Compounds

As seen in Figures 9 and 10, a total of five sulfur containing/smelling aroma

compounds were observed in the juices after twelve weeks storage. Methional and 2-

methyl-3-fumathiol levels were essentially the same in light exposed and control

samples. Methional is a Strecker aldehyde formed from the decomposition of the sulfur-

containing amino acid, methionine. The unknown sulfur compounds were higher in light

exposed sample when stored in glass, but slight lower when stored in plastic, and did not

seem to make a major contribution to the overall flavor. However, two thiol (mercapto)

compounds (4-mercapto-4-methylpentan-2-ol and 3-mercapto-hexen-1-ol) were formed










only in light exposed juices in both glass and PET. The aroma of 4-mercapto-4-

methylpentan-2-ol was described as moldy or soured, and 3-mercapto-hexen-l-ol was

described as onions, moldy, or soured. It should be noted that the identity of 3-mercapto-

hexen-l-ol must be considered tentative as it has been identified only on the basis of

retention time matching. Therefore, the appearance of these profoundly negative aroma

compounds in only the light exposed juices would explain in part the diminished flavor

quality of these juices. As shown in Figure 18, 3-mercapto-hexen-1-ol increased during

the twelve week storage study only in samples exposed to light. Also, the concentration

increased at an earlier date and to a greater extent in juice that was stored in plastic

bottles as compared to juice that was stored in glass.


1.0



0.8



S0.6
0
0.4

0.2


0.2/



0 .0 -
0 4 8 12
Week

Figure 1: Increase in Sulfur Compound (LRI 1116) During Twelve Week Study. o
represents juice in plastic containers exposed to light, m represents juice in
plastic containers not exposed to light, A represents juice in glass containers
exposed to light, and A represents juice in glass containers not exposed to
light









Finally, the sulfur smelling compounds that formed or increased were probably

caused by the degradation of known sulfur compounds such as the amino acid methionine

or the vitamin thiamine which are both present in fresh orange juice (47). The

appearance of these skunky, chicken-like, or onion-like aromas in only light exposed

juices partially explains why the overall orange juice character was degraded.

Accelerated Study

At the beginning of the accelerated storage study, oxygen was bubbled through all

juice samples. Control juices were wrapped in aluminum foil. After two weeks, two

unidentified sulfur (skunk-like) smelling compounds increased. These compounds

occurred at very low concentrations in the juice and thus were only detected on the GC-

O. The linear retention indices were 806 and 900 on the DB-5 column. The peak at LRI

900 was observed just before the internal standard, ethyl valerate, eluted. The sulfur

smelling compound (LRI 806) that increased in the accelerated study is not the same as

the early eluting unknown sulfur (LRI 856) observed in the twelve week study. The

sulfur smelling compound at R.I. 806 had a skunk like aroma, whereas the compound at

R.I. 856 smelled like wet dog. As seen in Figure 19, the skunky aroma was at higher

concentrations in the sample exposed to light.

The sulfur compound at R.I. 900 also had a skunk like aroma and formed during

the accelerated storage study. As seen in Figure 20, this compound was not present in the

original juice, and only formed in the juice that was exposed to light after two weeks.

The increase or formation of sulfur smelling compounds after light-exposure has

been documented in previous literature. It has been found that the amino acid methionine

degrades during ultraviolet light-exposure in the presence of riboflavin and oxygen to











form methional. Methional further degrades to form methanethiol, dimethyl sulfide, and

dimethyl disulfide (41).


0.5000



0.4000



a 0.3000
0
C.

0 0.2000



0.1000



0.0000
Week 0 Week 1, In Light Week 2, In Light Week 2, No Light

Figure 19: Formation of Sulfur Compound (LRI 806) after Accelerated Storage Study

Microbiological Evaluation

At the beginning of the study and after one month, microbial counts were

performed. There was one colony formed on several of the orange serum agar plates

before and after the study, however, this colony was identified as a bacillus strain that

does not affect juice flavor. Therefore, essentially no microbial growth was observed in

the orange juice before or during the experiment. Therefore any changes in juice flavor

were not caused by microbial contamination.







52



0.3000







0 0.2000



0
0
U)
w


0.1000








0.0000 ,,
Week 0 Week 1, In Light Week 2, In Light Week 2, No Light

Figure 20: Increase in Sulfur Compound (LRI 900) after Accelerated Storage Study














CHAPTER 6
CONCLUSIONS

The decrease in P-myrcene, the increase in carvone, p-cymene, 1,8-cineole,

vanillin, and Furaneol, and the formation and increase of various sulfur smelling

compounds helps explain the overall changes in aroma and flavor of orange juice that has

been exposed to light. It is these changes that upset the usual balance of flavor

compounds in fresh orange juice.

The increase in vanillin, Furaneol, and sulfur smelling compounds in samples that

were not subjected to increased temperature abuse indicates that light may also play a

role in non-enzymatic browning and thermal degradation.

Changes in volatiles were not significant during the first month of storage.

Therefore, orange juice exposed to light in a retail setting where there is a high turnover

rate would not be affected to a measurable extent and no loss of overall quality should be

detected. However, juices that are sold at a lower turnover rate are at a higher risk for

developing these off-flavors and overall deterioration of quality.

It was shown in this experiment that in general juices that are stored in glass

retained more orange juice character and had less photoxidation reactions occurring.

This is probably due to less oxygen being able to permeate the container, and fewer

oxidation reactions occurring in the juice. Retailers can therefore guard against some of

these reactions by using glass containers or multilayer plastic materials with higher

oxygen barrier properties. New packaging technology which utilizes higher oxygen






54


barrier materials or oxygen scavenging capability and shields the juice from light would

protect orange juice quality and ensure consumer satisfaction.


















APPENDIX A
STATISTICAL TEST FOR "L" SIGNIFICANCE



Two-sample T for Initial vs L-P


N
Initial 8
L-P 2


Mean
55.437
52.0135


StDev
0.199
0.0841


SE Mean
0.070
0.059


Difference = mu Initial mu L-P
Estimate for difference: 3.4234
95% CI for difference: (3.1678, 3.6789)
T-Test of difference = 0 (vs not =): T-Value = 37.19 P-Value = 0.000 DF = 4

Boxplots of Initial and L-P
(means are indicated by solid circles)


Figure 21: Significant Difference Between L Values


t--l


















APPENDIX B
STATISTICAL TEST FOR "A" SIGNIFICANCE


Two-sample T for Initial vs L-P

N Mean StDev SE Mean
Initial 8 -4.1694 0.0754 0.027
L-P 2 -0.6200 0.0283 0.020

Difference = mu Initial mu L-P
Estimate for difference: -3.5494
95% CI for difference: (-3.6351, -3.4637)
T-Test of difference = 0 (vs not =): T-Value = -106.50 P-Value = 0.000 DF = 5

Boxplots of Initial and L-P
(means are indicated by solid circles)


-0 5-


Figure 22: Significant Difference in "a" Values

Since the p value is 0.000, which is less than the alpha value of 0.01, we reject the


null hypothesis. The "a" value of the initial juice is not equal to the "a" value of juice


exposed to light in plastic.


a

















APPENDIX C
STATISTICAL TEST FOR "B" SIGNIFICANCE


Two-sample T for Initial vs L-P


N
Initial 8
L-P 2


Mean
33.760
28.129


StDev SE Mean
0.308 0.11
0.210 0.15


Difference = mu Initial mu L-P
Estimate for difference: 5.631
95% CI for difference: (4.839, 6.424)
T-Test of difference = 0 (vs not =): T-Value


30.58 P-Value = 0.001 DF = 2


Boxplots of Initial and L-P
(means are indicated by solid circles)


Initial


Figure 23: Significant Difference in "b" values


Since the p value is 0.001, which is less than the alpha value of 0.01, we reject the


null hypothesis. The "b" value of the initial juice is not equal to the "b" value of juice


exposed to light in plastic.


I











The Appendices in the Guide for Preparing Theses and Dissertations provided by

the Graduate School's Editorial Office give numerous examples regarding the proper

construction of an appendix. In most cases, the appendices will vary from dissertation to

dissertation and may vary within a dissertation, depending on the content of the

individual appendix. Obey the general guidelines given in the Guide for Preparing

Theses and Dissertations.

Although the margins have been set throughout this document correctly, please pay

close attention to the possibility of picture frames overlapping the margin. The base style

to use is Normal.

The remainder of this text is extraneous. We included it in this version of the

template so that appendix A can have page numbers on all its pages. The text that

follows was copied directly from the Guide for Preparing Theses & Dissertations.

Candidates in the English department who author a collection of poems, short

stories, or a novel for a thesis degree should consult the Editorial Office and not other

theses as a guide to format. Typing, spacing, margin, heading,, numbering, and

formatting requirements in this guide apply to all theses.

If a thesis consists of a collection of poems that are not grouped under headings, the

first page of each poem has a 2-inch top margin. Each poem title is centered and in all

capital letters. The first page of each poem is numbered bottom center with the rest of the

pages of the poem numbered in the top margin. The poems may be double- or single-

spaced but must conform to the other margin and formatting requirements in this guide.


















APPENDIX D
STATISTICAL TEST FOR MYRCENE SIGNIFICANT DIFFERENCE



Two-sample T for Initial vs L-P

N Mean StDev SE Mean
Initial 2 1.03376 0.00165 0.0012
L-P 3 0.8629 0.0264 0.015

Difference = mu Initial mu L-P
Estimate for difference: 0.1709
95% CI for difference: (0.1051, 0.2367)
T-Test of difference = 0 (vs not =): T-Value = 11.18 P-Value = 0.008 DF = 2

Boxplots of Initial and L-P
(means are indicated by solid circles)

105






095






0 85


Initial L-P



Figure 24: Significant Difference in Amount of Myrcene

Since the p value is 0.008, which is less than the alpha value of 0.01, we reject the


null hypothesis. The amount of myrcene in the initial juice is not equal to the amount in


the juice exposed to light in plastic.








60



Two-sample T for Initial vs D-P

N Mean StDev SE Mean
Initial 2 1.03376 0.00165 0.0012
D-P 3 0.9782 0.0331 0.019

Difference = mu Initial mu D-P
Estimate for difference: 0.0556
95% CI for difference: (-0.0268, 0.1380)
T-Test of difference = 0 (vs not =): T-Value = 2.90 P-Value = 0.101 DF = 2


Boxplots of Initial and D-P
(means are indicated by solid circles)

104-
103
102
1 01

1 00
0 99
098

097
096
0 95
0 94

Initial D-P





Figure 25: No Difference in Amount of Myrcene


Since the p value is 0.101, which is greater than the alpha value of 0.01, we fail to


reject the null hypothesis. The amount of myrcene in the initial juice is equal to the


amount in the juice protected from light in plastic.


















APPENDIX E
STATISTICAL TEST FOR CARVONE SIGNICANT DIFFERENCE

Two-sample T for Initial vs L-P

N Mean StDev SE Mean
Initial 2 0.04826 0.00196 0.0014
L-P 3 0.1750 0.0207 0.012

Difference = mu Initial mu L-P
Estimate for difference: -0.1267
95% CI for difference: (-0.1784, -0.0750)
T-Test of difference = 0 (vs not =): T-Value = -10.55 P-Value = 0.009 DF = 2

Boxplots of Initial and L-P
(means are indicated by solid circles)


0 20 -




015-




010-




0 05 -


w


Figure 26: Significant Difference in Amount of Carvone


Since the p value is 0.009, which is less than the alpha value of 0.01, we reject the


null hypothesis. The amount of carvone in the initial juice is not equal to the amount in


the juice exposed to light in plastic.











Two-sample T for Initial vs D-P

N Mean StDev SE Mean
Initial 2 0.04826 0.00196 0.0014
D-P 3 0.0857 0.0187 0.011

Difference = mu Initial mu D-P
Estimate for difference: -0.0374
95% CI for difference: (-0.0843, 0.0095)
T-Test of difference = 0 (vs not =): T-Value = -3.43 P-Value = 0.076 DF = 2

Boxplots of Initial and D-P
(means are indicated by solid circles)


011 -

010-

009 -

0 08 -

007 -

006 -

0 05 -


Figure 27: No Difference in Amount of Carvone


Since the p value is 0.076, which is greater than the alpha value of 0.01, we fail to


reject the null hypothesis. The amount of carvone in the initial juice is equal to the


amount in the juice protected from light in plastic.















APPENDIX F
GC-FID RESULTS FOR JUICES STORED IN PLASTIC














Light, In Plastic No Light, In Plastic
Week0 Week 4 Week 8 Week 12 Week0 Week 4 Week 8 Week 12 Identities
Ave Area Ave Area Ave Area Ave Area Ave Area Ave Area Ave Area Ave Area Ave Time DB-5 LRI
0.264 0.265 0.093 0.366 0.264 0.504 0.255 0.333 2.54 731
0.060 0.054 0.089 0.060 0.089 0.051 0.124 3.27 777
0.129 0.066 0.274 0.230 0.129 0.119 0.268 0.233 3.68 802 ethyl butanoate
1.000 1.000 0.400 1.000 1.000 1.000 0.400 1.000 5.55 901 ethyl valerate
0.306 0.260 0.285 0.272 0.306 0.294 0.305 0.277 6.29 936 a-pinene
0.995 0.814 0.892 0.863 0.995 1.003 1.052 0.978 7.54 993 b-myrcene
0.185 0.050 0.104 0.185 0.144 0.141 7.80 1005 octanal
0.038 0.039 0.017 0.038 0.036 0.019 0.034 8.00 1014 3-carene
54.076 44.197 47.998 47.173 54.076 51.387 53.610 50.653 8.64 1042 limonene
0.140 0.243 0.101 0.150 0.140 0.104 0.109 0.262 9.35 1073 p-cresol
0.553 0.461 0.513 0.615 0.553 0.549 0.591 0.674 10.03 1103 linalool
0.038 0.044 0.039 0.073 0.038 0.046 0.033 0.073 10.54 1126 trans-rose oxide
0.228 0.229 0.247 0.327 0.228 0.258 0.265 0.356 10.64 1130 ethyl 3-hydroxyhexanoat
0.111 0.111 0.204 0.106 0.135 0.123 0.216 11.82 1183
0.126 0.209 0.289 0.449 0.126 0.250 0.366 0.497 12.11 1197 a-terpineol
0.170 0.093 0.097 0.173 0.170 0.128 0.110 0.083 12.33 1207
0.033 0.040 0.049 0.089 0.033 0.036 0.039 0.054 12.72 1225 nerol
0.028 0.022 0.013 0.038 0.028 0.030 0.031 0.055 12.86 1231 neral
0.055 0.069 0.119 0.175 0.055 0.062 0.068 0.086 13.25 1250 carvone
0.027 0.018 0.096 0.027 0.040 0.058 14.73 1321 eugenol
0.045 0.055 0.069 0.137 0.045 0.053 0.057 0.107 15.27 1347 E-2-undecenal
0.032 0.026 0.038 0.064 0.032 0.033 0.030 0.071 16.51 1410 b-demascenone
0.062 0.053 0.065 0.084 0.062 0.065 0.073 0.098 16.93 1432
0.052 0.047 0.060 0.074 0.052 0.062 0.070 0.095 17.49 1461 wine lactone
0.075 0.067 0.077 0.098 0.075 0.081 0.089 0.129 17.99 1488
0.263 0.230 0.266 0.327 0.263 0.269 0.317 0.433 18.15 1497
3.192 2.788 3.311 4.104 3.192 3.339 3.761 5.040 18.37 1508 b-ionone
0.124 0.146 0.149 0.182 0.124 0.138 0.150 0.317 18.53 1517
0.217 0.214 0.244 0.314 0.217 0.237 0.283 0.386 18.81 1533
0.032 0.027 0.017 0.069 0.032 0.035 0.038 0.083 19.41 1565 dodecanoic acid
0.030 0.043 0.048 0.082 0.030 0.051 0.052 0.124 21.26 1672
0.031 0.023 0.021 0.034 0.031 0.023 0.041 21.73 1699 b-sinensal
0.071 0.063 0.094 0.153 0.071 0.128 0.113 0.215 22.76 1761 a-sinensal
0.233 0.196 0.256 0.393 0.233 0.232 0.300 0.529 23.78 1825 nootkatone
0.021 0.016 0.046 0.021 0.051 0.079 24.33 1859
1.397 0.740 1.034 1.903 1.397 1.134 0.652 2.439 24.65 1880
0.838 0.808 1.272 2.601 0.838 1.954 1.578 3.880 24.88 1895
0.138 0.124 0.175 0.254 0.138 0.139 0.178 0.346 29.97 2251
0.186 0.151 0.885 0.119 0.186 0.249 0.210 0.222 30.44 2286
0.046 0.041 1.136 0.193 0.046 0.137 0.045 0.273 30.74 2308
0.116 0.071 0.104 0.199 0.116 0.246 0.109 0.204 31.01 2329
0.056 0.037 0.045 0.056 0.060 0.053 0.058 31.24 2346
0.172 0.140 0.174 0.175 0.172 0.180 0.193 0.244 31.36 2356
0.093 0.057 0.067 0.087 0.093 0.134 0.085 0.182 31.69 2381





















APPENDIX G
GC-FID RESULTS FOR JUICES STORED IN GLASS


Light, In Glass No Light, In Glass
Week 0 Week 4 Week 8 Week 12 Week 0 Week 4 Week 8 Week 12 Identities
Ave Area Ave Area Ave AreaAve Area ve Area Ave Area Ave Area Ave Area Ave Time DB-5 LRI
0.223 0.505 0.310 0.650 0.223 0.271 0.273 0.872 2.54 731
0.048 0.098 0.032 0.163 0.048 0.052 0.055 0.216 3.27 777
0.117 0.114 0.223 0.299 0.117 0.068 0.207 0.183 3.68 802 ethyl butanoate
1.000 1.000 0.400 1.000 1.000 1.000 0.400 1.000 5.55 901 ethyl valerate
0.288 0.271 0.296 0.280 0.288 0.289 0.308 0.268 6.29 936 a-pinene
0.995 0.873 0.936 0.877 0.995 0.963 1.016 0.935 7.54 993 b-myrcene
0.167 0.118 0.061 0.167 7.80 1005 octanal
0.036 0.033 0.036 0.031 0.034 8.00 1014 3-carene
50.489 47.118 51.762 48.312 50.489 50.583 52.604 47.330 8.64 1042 limonene
0.206 0.094 0.189 0.102 0.206 0.231 0.184 0.128 9.35 1073 p-cresol
0.492 0.486 0.547 0.632 0.492 0.496 0.527 0.571 10.03 1103 linalool
0.036 0.060 0.058 0.155 0.036 0.037 0.034 0.152 10.54 1126 trans-rose oxide
0.224 0.244 0.287 0.365 0.224 0.253 0.269 0.284 10.64 1130 ethyl 3-hydroxyhexanoate
0.092 0.114 0.164 0.221 0.092 0.144 0.154 0.252 11.82 1183
0.108 0.222 0.289 0.463 0.108 0.238 0.347 0.477 12.11 1197 a-terpineol
0.141 0.121 0.090 0.153 0.141 0.038 0.057 0.090 12.33 1207
0.029 0.045 0.061 0.105 0.029 0.029 0.030 12.72 1225 nerol
0.023 0.027 0.023 0.031 0.031 12.86 1231 neral
0.048 0.094 0.167 0.241 0.048 0.040 0.056 0.066 13.25 1250 carvone
0.023 0.028 0.058 0.100 0.023 0.037 0.041 14.73 1321 eugenol
0.043 0.066 0.094 0.145 0.043 0.053 0.054 0.070 15.27 1347 E-2-undecenal
0.023 0.036 0.044 0.066 0.023 0.023 0.029 16.51 1410 b-demascenone
0.052 0.063 0.074 0.125 0.052 0.061 0.072 0.077 16.93 1432
0.045 0.060 0.070 0.090 0.045 0.058 0.067 0.069 17.49 1461 wine lactone
0.064 0.074 0.091 0.122 0.064 0.074 0.095 0.091 17.99 1488
0.229 0.267 0.321 0.402 0.229 0.251 0.297 0.294 18.15 1497
2.727 3.196 3.834 4.751 2.727 3.058 3.537 3.642 18.37 1508 b-ionone
0.112 0.144 0.135 0.190 0.112 0.149 0.144 0.160 18.53 1517
0.197 0.227 0.274 0.339 0.197 0.232 0.258 0.269 18.81 1533
0.025 0.039 0.052 0.073 0.025 0.040 0.033 19.41 1565 dodecanoic acid
0.027 0.054 0.057 0.103 0.027 0.047 0.049 0.070 21.26 1672
0.032 0.027 0.053 0.032 0.030 0.026 21.73 1699 b-sinensal
0.087 0.099 0.153 0.237 0.087 0.100 0.184 0.126 22.76 1761 a-sinensal
0.207 0.257 0.300 0.446 0.207 0.229 0.283 0.370 23.78 1825 nootkatone
0.015 0.023 0.082 0.078 0.015 0.055 0.067 24.33 1859
1.196 1.400 0.671 2.300 1.196 1.182 0.673 1.899 24.65 1880
0.691 1.358 1.213 3.544 0.691 2.768 3.851 2.216 24.88 1895
0.123 0.160 0.212 0.354 0.123 0.150 0.181 0.270 29.97 2251
0.151 0.383 0.272 0.176 0.151 0.236 0.321 0.139 30.44 2286
0.027 0.123 0.063 0.315 0.027 0.113 0.108 0.195 30.74 2308
0.081 0.193 0.141 0.246 0.081 0.188 0.270 0.192 31.01 2329
0.035 0.054 0.037 0.035 0.049 0.076 0.190 31.24 2346
0.126 0.185 0.110 0.273 0.126 0.177 0.261 0.246 31.36 2356
0.083 0.093 0.053 0.083 0.078 0.118 31.69 2381















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BIOGRAPHICAL SKETCH

Kristin Nelson graduated from Niceville High School in 1997. She received a

Bachelor of Science degree in Chemical Engineering from the Georgia Institute of

Technology in 2002, graduating with honors. Kristin completed her Master of Science in

food science and human nutrition at the University of Florida in 2005. Her research was

in the field of flavor chemistry and was conducted at the Citrus Research and Education

Center in Lake Alfred, Florida. She is now part of Kerry's graduate student management

training program working in Lakeland, Florida, in the flavor division. As part of the

program, Kristin trains in various areas of the company such as research and

development, beverage applications, pilot plant scale-up, production, quality control, and

analytical analysis.