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Examination of Aroma Volatiles Formed from Thermal Processing of Florida Reconstituted Grapefruit Juice


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EXAMINATION OF AROMA VOLATI LES FORMED FROM THERMAL PROCESSING OF FLORIDA RECONSTITUTED GRAPEFRUIT JUICE By WENDY ANN-MARIE BELL 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 2004

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Copyright 2004 by Wendy A. Bell

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To my Grandparents, with love.

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ACKNOWLEDGMENTS I would like to express my sincere gratitude to my committee chair, Dr. Rouseff, for his guidance, encouragement and support. It was a great learning experience. To my committee members, Dr. Charlie Sims, Bruce Welt and Kevin Goodner, I would like to extent my gratitude for their advice and assistance. I am also grateful to the Florida Department of Citrus for providing financial support of my graduate studies. I thank all my lab mates and Jack Smoot, for all their help. I would also like to thank April Elston and Kristin Nelson for sniffing my samples. Thanks go to my family for their support. To my grandmother, I would like to extend a special gratitude for her kindness and constant support. She has always encouraged me to work hard and to put God first in my endeavors. Finally I would like to acknowledge my best friend, James. He was always there, encouraging me with his kind words. I really appreciate all that he has done. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 Grapefruit......................................................................................................................3 Thermal Processing......................................................................................................3 Possible Interactions with Stainless Steel Pasteurization Tubes..................................4 Off-flavor......................................................................................................................5 Maillard Reaction Products...................................................................................5 Furans.............................................................................................................5 Furanones.......................................................................................................7 2,5 dimethyl 4-hydroxy3(2H) furanone (Furaneol).....................................7 4,5 Dimethyl 3-Hydroxy2(5H) Furanone (Sotolone)..................................8 Other Degradation Products..................................................................................9 Instrumental Analysis...................................................................................................9 GC Techniques......................................................................................................9 HPLC Methods....................................................................................................10 3 MATERIALS AND METHOD..................................................................................12 Reagents and Standards..............................................................................................12 Apparatus Setup..........................................................................................................12 Sample Preparation.....................................................................................................13 Sensory AnalysisDifference from Control Test.......................................................15 Instrumental Methods.................................................................................................17 Gas Chromatography-Flame Ionization Detector (GC-FID)..............................17 Gas Chromatography-Olfactometry (GC-O).......................................................17 Gas Chromatography-Mass Spectrometry (GC-MS)..........................................18 v

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HPLC Analysis of the Furans.....................................................................................18 Sample Preparation..............................................................................................18 HPLC Instrumentation.........................................................................................18 Identification and Quantification.........................................................................20 4 SENSORY ANALYSIS RESULTS AND DISCUSSION.........................................22 Statistical Tests for Significant Differences...............................................................23 Producing Heated/Cooked Flavor...............................................................................28 5 GAS CHROMATOGRAPHIC RESULTS AND DISCUSSION..............................31 GC-O...........................................................................................................................33 Aroma Active Compounds in Grapefruit Juice..........................................................35 Aroma Active Compounds Lost during Heating.................................................39 Aroma Active Compounds That Intensified after Heating..................................41 Caramel Aroma Compounds Formed from Sugar Degradation Reactions................42 Caramel Compounds Found in Grapefruit Juice.................................................45 3-Methyl 2(5H) Furanone....................................................................................46 Identification of Volatiles Using GC-MS...................................................................47 6 HPLC RESULTS AND DISCUSSION......................................................................53 7 CONCLUSION...........................................................................................................64 LIST OF REFERENCES...................................................................................................65 BIOGRAPHICAL SKETCH.............................................................................................70 vi

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LIST OF TABLES Table page 3-1 HPLC gradient conditions with flow rate at 1mL/min.............................................20 4-1 Difference from control sensory data from 22 panelists..........................................24 4-2 Difference from control sensory data for the second (rep 2) and third replication..25 4-3 Results of the analysis of variance for each of the three replications......................26 4-4 LSD comparisons of the means for the three samples.............................................28 4-5 Combination used in off-flavor duplication along with their descriptors................30 5-1 Preliminary identification of major grapefruit juice components based on.............33 5-2 Aroma active peaks with tentative identification ....................................................36 5-3 Aroma intensities based on normalized peak heights of samples............................38 5-4 LRI for ten sugar degradation products possessing caramel aroma.........................43 5-5 Components identified in grapefruit juice by GC-MS.............................................49 6-1 Retention time and maximum absorbance of furans standards................................55 6-2 Concentrations of 5-HMF, furfural and Furaneol....................................................60 vii

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LIST OF FIGURES Figure page 2-1 Formation of furans by either the acidic pathway (1,2enaminol) or by the basic pathway (2,3-enediol).................................................................................................7 3-1 Heating apparatus setup...........................................................................................14 3-2 Sample ballot for the grapefruit juice difference from control test using................16 3-3 Graphical representation of the HPLC gradient conditions.....................................20 5-1 Overlay of partial grapefruit juice FID chromatogram on a ZB-5 column..............32 5-2 Partial FID (top) and aromagram (bottom) overlay of grapefruit juice...................34 5-3 Aroma intensities for the unheated reconstituted juice and glass-heated samples...37 5-4 Aroma active volatiles whose intensity diminished due to heating.........................40 5-5 Aroma active volatiles (peak numbers 8, 11, 16, 19, 29, 26, 32, & 35)...................42 5-6 Structure of 3(2H) furanones ...................................................................................44 5-7 Structure of sotolone, a 5(2H) furanone...................................................................45 5-8 Structure of the 3-methyl-2(5H) furanone...............................................................46 5-9 Segmented MS total ion chromatogram using ZB-5 column...................................48 5-10 Mass spectra of myrcene (top) and -pinene (bottom)............................................50 5-11 Alpha-terpineol levels in the three samples.............................................................51 5-12 Formation pathway for -terpineol in the presence of limonene and linalool.........52 6-1 HPLC spectra of the seven standards.......................................................................55 6-2 Homofuraneol tautomers..........................................................................................56 6-3 Segment of HPLC chromatogram showing overlay................................................57 viii

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6-4 Spectra of 5-HMF in standard (top) and in the grapefruit juice sample...................58 6-5 Spectra of furfural standard (top) and sample (bottom)...........................................59 6-6 Linear calibration curve for 5-HMF analyzed in triplicate......................................60 6-7 Linear calibration curve for Furaneol at 290nm.......................................................61 6-8 Linear calibration curve for furfural standard at 290nm..........................................62 ix

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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 EXAMINATION OF AROMA VOLATILES FORMED FROM THERMAL PROCESSING OF FLORIDA RECONSTITUTED GRAPEFRUIT JUICE By Wendy Ann-Marie Bell December 2004 Chair: Russell L. Rouseff Major Department: Food Science and Human Nutrition Over fifty percent of Floridas grapefruit supply goes into juice production. Grapefruit juice is heated after reconstitution with water to minimize spoilage microorganisms. However, heating induces chemical changes that degrade juice flavor. Fruit juices are traditionally heated using stainless steel surface plates or tubes. Stainless steel contains appreciable amounts of transition metals such as nickel and chromium that are known to catalyze chemical reactions. Acidic (pH 2.9-3.5) juice may also promote leaching of these metals. Glass should be more inert than stainless steel and may offer a means to produce higher quality juice. This study was undertaken to examine changes in aroma volatiles in reconstituted grapefruit juice when heated on stainless steel and glass surfaces. Sensory analysis of grapefruit juices heated at 100C for 10 minutes indicated that both heated samples exhibited a heated, pineapple, metallic, and cooked off-flavor. Unheated reconstituted juice had a fresh grapefruit juice character. Using data from a ten x

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point rating system, the Difference from Control test indicated that significant differences (p<0.05) existed between the unheated juice and the two heat treatments. Analysis of heated juices with GC-O showed appreciable reductions in aroma intensity of compounds that are responsible for fresh grapefruit juice character. Intensities of -pinene, myrcene, and -sinensal were at least 45% lower in the heated juices compared to the unheated juice. There was also a corresponding increase in aroma intensity of compounds associated with flavor degradation such as 2,5-dimethyl-4-hydroxy-3(2H) furanone (Furaneol) and methional in heated samples. Two other Maillard reaction products, sotolone and 3-methyl-2(5H)-furanone, were detected by GC-O. Both high processing temperatures and extended times are required to produce cooked or heated off-flavors in grapefruit juice. Increases in 5-HMF concentration were observed with heating, suggesting that Maillard reactions were involved. Sensory experiments designed to induce cooked, heated off-flavor in unheated reconstituted juice indicated that a combination of Furaneol, homofuraneol and bis (2-methyl-3-furyl) disulfide, a thermal degradation product of thiamine (vitamin B1), could produce a cooked off-flavor similar to that observed in the excessively heated juices. xi

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CHAPTER 1 INTRODUCTION Grapefruit is the second largest citrus commodity in the State of Florida. Florida supplies over 30% of the worlds grapefruit production. Over 55% of Florida grapefruit goes into juice production while over 35% is used for fresh fruit. Grapefruit sales have been declining in recent years due to several factors ranging from uneven flavor quality to interactions with certain drugs. Grapefruit juice has a unique citrus flavor, but low sugar to acid ratios and high bitterness can overwhelm the pleasant flavor aspects. Red grapefruit is used mainly for fresh fruit or for not-from concentrate juice (NFC), while white grapefruit juice is generally converted to from concentrate (FCGJ) juice. Thermal processing alters the overall flavor of grapefruit juice but is necessary to reduce populations of viable spoilage microorganisms, inactivate pectinesterase and, ultimately to increase shelf life. Formation of off-flavors due to processing is highly undesirable to consumers; therefore research is necessary to identify critical factors that cause significant flavor alterations. Cooked and heated off-flavor was observed in both NFC and FCOJ and had profoundly negative impacts on perceived flavor quality. Traditional processing of grapefruit juice is achieved through the use of stainless steel tubes or plates. Transition metals can catalyze chemical reactions known to cause off-flavors. Glass is an inert material and should not catalyze such reactions. Previous studies have identified several compounds that contribute negatively to citrus juice flavor. One such class of compounds is the furans that are formed as a result of sugar degradation. These furans impart a caramel aroma depending on the 1

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2 concentration. Use of 5-HMF and furfural as markers of thermal abuse, have been suggested by several authors. Levels of these compounds in grapefruit juice typically do not approach their aroma threshold but as the level of these compounds increase, juice sensory quality declines. Furaneol, 2,5-dimethyl 4 hydroxy-3(2H) furanone, imparts an aged, pineapple like aroma to citrus juice at levels exceeding 50 g/L. In citrus based soft drinks, sotolone imparts a burnt spicy note at levels ranging from 1.38ng/mL to 209ng/mL. The objectives of this study were to determine if heating grapefruit juice in contact with glass or stainless steel resulted in distinguishable sensory flavor differences as well determining there were differences in individual aroma volatiles. A secondary objective was to determine what compounds were responsible for heated or cooked off flavor in grapefruit juice.

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CHAPTER 2 LITERATURE REVIEW Grapefruit Grapefruit is the second largest citrus commodity in the State of Florida, accounting for over 30% of the worlds grapefruit production. The entire United States of America produces 40% of the world grapefruit production. For the 2002-2003 season, 36% of the grapefruit was used for fresh fruit, 16% for the chilled juice sector and over 40% to frozen concentrate products. With the exception of 1999-2000 season, there has been a continuous decline in grapefruit production. Sales have been declining due to factors ranging from poor flavor quality to medical precautions. Significant changes in the overall flavor of processed grapefruit are of concern to the grapefruit processors as well as the consumers. Thermal Processing Citrus juice generally undergoes heat treatment in order to achieve reductions in viable spoilage organisms, to inactivate pectic enzymes, and ultimately to extend the shelf life. Inactivation of pectinesterase is important since it prevents the cloud separation and associated reactions that may alter the flavor profile. Temperatures of at least 70C to 90C with hold times varying from a few seconds to 1 minute are necessary for inactivation of most pectic enzymes. Pectin methyl esterase is a heat resistant enzyme found in grapefruit juice and, is inactivated at temperatures exceeding 90C (1). Most processors pasteurize not from concentrate (NFC) or chilled juice at temperatures 3

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4 exceeding 90C and majority of the time juice is being heated twice. Grapefruit juice from concentrate (FCGJ) is subjected to at least two heat treatments. The first occurs during the evaporator concentration process where water is removed with a multi effect evaporator. The second heat treatment occurs after the reconstitution of the juice and the add-back of important flavor volatiles. Possible Interactions with Stainless Steel Pasteurization Tubes Pasteurization and concentration processes involve heating the juice in stainless steel tubes or plates. Transition metals in food grade stainless steel tubes may catalyze reactions throughout the heating processes. Food grade stainless steel, 316, typically has 16-18% Cr, 10-14% Ni and 2-3% Mo. Since juice contacts steel directly, leaching of metals may be enhanced by factors such as low pH, high temperatures, and contact time. Studies on effect of pH and temperature on chromium release from stainless steel showed that after heating fruit juices with pH ranging from 2.5-3.0 at 95C for 1hr, 31-50g/L chromium was released (2). Residence times in tubes for NFC pasteurized juices are typically in the range of 6-30s. The thermally accelerated short time evaporator (TASTE) developed by Cook Machinery (Dunedin FL) is generally used to concentrate citrus juices. This evaporator utilizes high temperature with residence times of less than one minute (3). Although residence time is not long, high processing temperatures, combined with the low pH may be more damaging than either factor alone. High temperature pasteurization induces chemical changes that may affect the quality of the juice immediately or will initiate a chain of chemical reactions that will ultimately produce flavor degrading compounds.

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5 Off-flavor High heating temperatures along with prolonged storage have been shown to be contributing factors to off-flavor development (4;5;6). Studies into heated/cooked off flavor found in orange juice was carried out by heating orange juice from concentrate at various time-temperature combinations and then subjecting the samples to varying storage times. Sensory evaluation revealed that orange juice heated at 100C for 10 min produced a heated, cooked and over processed flavor but was not predominant. Samples were rated either dislike slightly or lower by majority of the panelists (7). Consumer testing of commercial orange juices revealed that that several had a cooked flavor. Juices with this cooked flavor were given the lowest quality ratings. Both chilled (NFC) and from concentrate products were evaluated (8). Maillard Reaction Products Off flavor formation in citrus due to thermal degradation of sugars is of high importance. Non-enzymatic browning products may be directly or indirectly contributory to the flavor. Furans In citrus juices, furans are important indicators of thermal abuse. Furfural and 5-hydroxy methyl furfural (5-HMF) have been reported in citrus juices that have undergone heat treatment and high storage temperatures (4;6). Commercial canned and glass packed orange juice samples were stored at temperatures ranging from 5C to 30C over a period of 16 weeks. Results showed that storage temperatures above 16C increased the rate of furfural formation more rapidly than at lower temperatures. Concentrations of furfural observed in canned orange juices stored at 30C increased 28 times compared to juices stored at 5C. For every 5C

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6 increase in storage temperature, the level of furfural doubled, thus demonstrating a hight correlation between storage temperatures and furfural accumulation. Glass packed orange juice samples showed a more rapid increase in furfural formation. In fact, a 72-fold increase in furfural levels were noted in samples ranging from 5C 30C storage temperatures (6). A similar study utilizing commercially canned grapefruit juice revealed that storage temperatures from 10C to 40C were enough to induce a 390-fold increase in furfural levels over a 15 week storage time. Panelists were able to detect significant flavor changes in juices in which furfural levels exceeded 0.175 ug/ml. Since its taste threshold in orange juice was 80 g/mL and thus not directly flavor active, furfural can only be used as an indicator for off flavor (9). Amino acids can increase the rate of ascorbic acid degradation. Furfural is formed through acid catalysis of ascorbic acid (6). Ascorbic acid is degraded more rapidly in single strength juice than in concentrated juices (10). Levels of 5-hydroxy methyl furfural (5-HMF) in grapefruit juice samples increased up to 3000 times with a 40C rise in temperature over 15 week storage study (9). Formation of 5-HMF increased slightly between 10C and 20C but rose exponentially above 30C. Under acidic conditions, 5-HMF is formed by the 1,2 enolization and dehydration of hexose sugars (fig.2-1). The R group in figure 2-1 is replaced by a methyl group. It has a high threshold of 100 g/mL (11) and therefore is not flavor active (12). In fact, sensory results did not correlate with 5-HMF concentration (9). Thus other compounds must be responsible for the observed off-flavor.

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7 CCHOH CHOH CH2NHRO C-OHCHOH CHOH O R CHO CH-NHRC-OHC-OH CHOH CH2NHR O O OHO OH O R Amadori Compound 1,2-enaminol undergoes series of Dehydration & deamination with pentose R=Hwith hexose R= CH2OH 2,3-enediol undergoes series of dehydration & deamination from hexose with pentose R=Hwith hexose R= CH3CCHOH CHOH CH2NHRO C-OHCHOH CHOH O R CHO CH-NHRC-OHC-OH CHOH CH2NHR O O OHO OH O R Amadori CompoundAmadori Compound 1,2-enaminol undergoes series of Dehydration & deamination with pentose R=Hwith hexose R= CH2OHwith hexose R= CH2OH 2,3-enediol undergoes series of dehydration & deamination from hexose with pentose R=Hwith hexose R= CH3with hexose R= CH3 Figure 2-1 Formation of furans by either the acidic pathway (1,2enaminol) or by the basic pathway (2,3-enediol) Furanones Furanones are important flavor compounds that are found both naturally and as a result of thermal processing in foods and beverages. Two important furanones are Furaneol and sotolone. 2,5 dimethyl 4-hydroxy3(2H) furanone (Furaneol) Furaneol gives a caramel-like, burnt pineapple aroma in high concentrations and a sweet, strawberry like aroma at low concentrations. It is one of the major character impact compounds in pineapple (13) and muscadine grapes (14). Furaneol can exist either free or glycosidically bound in other fruits such as mango, grapes, tomato (15), and strawberry (16;17). It is found in thermally processed foods such as beef broth (18), and fruits such as pineapple. This furanone has been shown to mask the flavor of orange

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8 juice, giving an aged, cooked aroma when present at 0.05 g/mL or greater (4). In grapefruit juice, a pineapple-like aged flavor develops with high levels of Furaneol (19). It has a threshold of 0.03 g/L in water (20). Orange juice samples stored at 40C had 5 times more Furaneol compared to samples stored at 4C (21). Grapefruit juice samples stored at 50C for 15 weeks showed an increase up to 28 times that of samples stored at 20C (19). Furaneol formation in citrus juices occurs as a result of a Maillard sugar degradation reaction. Studies using model solutions of citrus juice showed that Furaneol is formed over a range of pH values in the presence of rhamnose. At higher pH values Furaneol is formed through the 2,3-enolization pathway of the Amadori compound (fig. 2-1) followed by dehydration and molecular rearrangement (22). The amino acid arginine is also a necessary substrate for its formation under acidic conditions. Rhamnose is a 6-deoxy hexose sugar formed from enzymatic degradation of pectin during processing and storage. Flavanone glycosides such as hesperidin and naringin can also degrade to form free rhamnose (23). Acid-catalyzed fructose degradation is also a possible formation pathway for Furaneol. In heated solutions it can decompose to smaller molecules (24) or it can react with other groups such as thiols to produce flavorful compounds. Like most Maillard reactions, this reaction requires only a small amount of substrate produce potent flavors; thus; it is hard to measure the depletion of these compounds. 4,5 Dimethyl 3-Hydroxy2(5H) Furanone (Sotolone) Sotolone is a powerful aromatic compound that produces a curry-like aroma in high concentrations, however, at low levels imparts a burnt, spicy aroma (25). Sotolone has an aroma threshold of 0.02 g/L in air (26) and is found in wines (25), sherry and roasted

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9 coffee (26). Recently, it has been reported in orange essence oil (27) and was found to be a source of off-flavor in citrus soft drinks (28) producing a spicy, burnt aroma. Its formation was monitored in model soft drinks both with and without ascorbic acid. It was postulated that sotolone was formed from ascorbic acid or dehydroascorbic acid in the presence of ethanol. Oxygen and metal ions are also necessary for its formation. Small amounts of ethanol are naturally found in citrus juices. Other Degradation Products Methional and 2-methyl-3-furanthiol are possible off-flavor contributors formed from strecker and thiamine thermal degradation, respectively. Methional has a cooked potato aroma while 2-methyl-3-furanthiol has a cooked, meaty aroma. Another thiamine degradation product, bis (2-methyl3-furyl) disulfide, a dimer of 2-methyl-3-furanthiol (MFT), which also produces a meaty aroma. Both MFT and its dimer have been reported in grapefruit juice from concentrate (29). Thermal processing can also degrade -carotene; producing norisoprenoids such as -damascenone, -ionone and -ionone. Instrumental Analysis GC Techniques Gas chromatography can separate complex mixtures of volatiles using high resolution capillary columns. GC works on the principle that volatiles are sequentially eluted in the general order of boiling point. Thus they are thermally desorbed from the capillary column as the column (oven) temperature increases. Separation efficiency also depends on polarity of both the compound and the stationary phase, which lines the column. On a polar column such as DB-wax, polar compounds will be retained by the stationary phase more than they would on a non-polar DB-5 column.

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10 Gas chromatography-olfactometry (GC-O) is a powerful analytical technique that combines an instrumental detector as well as human response. It offers the ability to define aroma active volatiles in terms of odor and intensity (30). The human nose has an odor detection threshold of 10 -19 moles (31) compared to 10 -12 grams for a mass spectrometer (32). Isolation of the compounds of interest from the food matrix is very important (31). GCO analysis can involve flavor dilution techniques such as Charm or aroma extraction dilution analysis. These techniques are useful in determining flavor threshold of important aroma active compounds. Another technique, OSME or time-intensity, is based on evaluating intensities of aroma over the course of the GC analysis and in most cases is not as time consuming as the flavor dilution techniques. Drawbacks to sniffing include fatigue, saturation and adaptation (31) therefore assessors should limit repetitions that are done in a single day and also lengthen the time between sniffs. HPLC Methods HPLC is an excellent alternative method of analysis. It is a non-thermal chromatographic technique, which allows the separation of both volatiles and non-volatiles. Reversed phase chromatography has been one of the choice methods for furan analysis in citrus products (19;21;28;33). Grapefruit juice is a complex mixture and efficient separation of its components lies in sample preparation and chromatographic conditions. There have been several sample preparation procedures for furan analysis in citrus juices. Use of the Carrez clarifying reagent was found to be effective in removing pulp, fat, protein and carotenoids that may co-elute with the furans (34). The same author (9) employed a simplified method of centrifuging the juice to remove the pulp layer. In both cases passing the remaining solution through a C-18 cartridge to selectively remove the desired polar compounds (34). Previous studies used mobile phases of consisting of

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11 acetonitrile-water (85:15), (34), acetonitrile-glacial acetic acid-water (9) to effectively isolate 5-HMF and furfural. Methanol and sodium acetate buffer were found to satisfactorily isolate Furaneol. Initial efforts to quantify Furaneol in grapefruit juice using aqueous methanol (30%) or phosphate buffer with 30% methanol were unsuccessful due to co-elution with another compound (19). A linear gradient and mobile phase consisting of methanol, acetonitrile and pH4 acetate buffer provided good separation of Furaneol (21). However, in citrus juices another compound will co-elute with Furaneol. The technique of creating difference chromatograms by subtraction of two detection wavelengths (35) has been employed to isolate Furaneol. Furaneol has minimal absorbance at 335nm and appreciable absorbance at 292nm whereas the interfering compound had an absorbance maximum at 335 nm. Thus, the difference chromatogram should provide a peak which is due almost entirely to Furaneol. Sotolone has previously been isolated using both reversed and normal phase chromatography. Reversed phase chromatography using a mobile phase consisting of acetonitrile and water adjusted to pH 2.5 with sulfuric acid was used to quantify sotolone in citrus soft drinks (28). This compound was monitored at 235nm, a wavelength close to its maximum absorbance. Normal phase chromatography has been the predominant method of isolating or analyzing sotolone. The sotolone content in wines was determined with a detection limit of 10g/L using a diol column and hexane/ dichloromethane as the mobile phase (25). HPLC can also be used as a preparative step by collecting fractions of sotolone and then using GC-MS to identify and quantify sotolone (36).

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CHAPTER 3 MATERIALS AND METHOD Reagents and Standards Pentane, an organic solvent used in the gas chromatographic analysis was obtained from Fisher Scientific (Pittsburgh, PA). Acros Organics (New Jersey) supplied diethyl ether used for solvent extraction, and the methanol used in HPLC solvents and for preparation of standards. Standards of 3-methyl-2(5H) furanone, 4-hydroxy-5-methyl-3(2H) furanone, 2,5-dimethyl-4-hydroxy-3(2H) furanone, 2,5-dimethyl-4-methoxy-3(2H) furanone, 2-ethyl-4-hydroxy-5-methyl-3(2H) furanone, 5-hydroxy methyl furfural, 4,5-dimethyl-3-hydroxy-5(2H) furanone, E-2-decenal, E,E-2,4-decadienal, methional, E,Z-2,4-decadienal, E,E-2,4-nonadienal, E,Z-2,6-nonadienal, 1-octen-3-one, maltol and 2-methyl-3-furanthiol were obtained from Aldrich Chemical Co. (Milwaukee, WI). Vanillin was obtained from Sigma Chemical Co (St. Louis, MO). Linalool, octanal, decanal, nootkatone, -sinensal, -terpineol, myrcene, and -pinene Sodium phosphate was obtained from Sigma while the phosphoric acid from Fisher Scientific. Apparatus Setup Figure 3-1 illustrates the heating apparatus setup. An oil bath consisting of light mineral oil (Fisher Scientific) was heated using a hot plate. A condenser with chilled (-5C) 50:50, ethylene glycol: water, was attached to the round bottom flask to allow for refluxing of volatiles through condensation. The mouth of the condenser was also covered with aluminum foil. For each heating in the stainless steel and the glass containers, temperatures of both the oil bath and the juice samples were monitored. 12

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13 Sample Preparation Mid Season White Grapefruit Concentrate was obtained from a local processor. The concentrate was reconstituted to 10Brix using deionized water. 300ml of the reconstituted grapefruit juice was placed in a 1000ml stainless steel round bottom flask. This was then submerged into a pre-heated oil bath. A hold time of 10 min was maintained once the sample reached a temperature of 100C. After heating, samples were placed in a pre-sterilized 10oz glass flint bottle (All American Container, Tampa, FL), and immediately cooled on ice. The heating was repeated using a glass round bottom flask. Sample preparation for GC analysis was carried out using liquid-liquid extraction. 20 ml of grapefruit juice were added to 10 ml of 1:1 pentane and diethyl ether for extraction. A Mixxor-like apparatus consisting of two 50 ml syringes connected via a stainless steel luer-lock connector was used to extract the volatiles. Extraction was facilitated by moving syringes in a back and forth motion twenty times. The mixture was then centrifuged at 4000 rpm for 10 minutes to break the emulsion. The solvent layer was removed and retained, while the aqueous portion was extracted a second time. After the second extraction, both solvent fractions were combined. The extract was dried with sodium sulfate.

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14 Condensor with ethylene gylcol Oil bath Figure 3-1 Heating apparatus setup A 50 l volume of 2000 g/mL ethyl valerate and 2-heptadecanone were added as internal standards. The solvent layer was concentrated to approximately 100l using a stream of high purity nitrogen and placed in a 100l limited volume insert, placed in a 1.8 mL sampling vial and sealed with a screw top lid. Each sample was refrigerated until GC-O/GC-FID (GC) and GC-MS analysis. Each sample was analyzed four times for GC-O analysis and twice for GC-MS.

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15 Sensory AnalysisDifference from Control Test Six batches of grapefruit juice were heated in stainless steel and glass containers. The batches were then combined so as to present a uniform mixture to panelists. Twenty-one to twenty-five untrained panelists from the Citrus Research and Education Center were recruited for the study. A control and three samples, each coded with a randomly selected 3-digit number were presented to the panelists. Order of presentation randomized for each panelists based on six orders of presentation (ABC, ACB, BCA, BAC, CBA, BAC). Panelists were then asked to taste the control then to taste the sample and to rate the degree of difference between them. They were also told that a hidden control was present among the samples. A sample ballot is shown in figure 3-2. The control was unheated reconstituted grapefruit juice. Panelists were also asked to cleanse their palettes by eating a piece of unsalted cracker and drinking a sip of water between samples to prevent any carryover from the previous sample. A numerical ten-point scale similar to the hedonic scale was used, with scale ranging from no difference to extreme difference. Color changes during the heating process prompted the use of red lighting so as to mask the color difference. The above procedure was repeated two additional times yielding a total of the three sensory evaluation sessions.

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16 DIFFERENCE FROM CONTROL TEST Name: ________________ Date: ____________ Today you will be tasting grapefruit juice. Please eat a piece of cracker and drink a sip of water in between each sample to cleanse your palette. Three samples and one control are presented to you. Please circle the degree of difference against the control for each sample. It is possible that there may be no difference between the control and one of the samples. Control vs. # Control vs. # Control vs. # 0 =No difference 0 =No difference 0 =No difference 1= slight difference 1= slight difference 1= slight difference 2 2 2 3 = mod difference 3 = mod difference 3 = mod difference 4 4 4 5 = large difference 5 = large difference 5 = large difference 6 6 6 7 = very large difference 7 = very large difference 7 = very large difference 8 8 8 9 = extreme difference 9 = extreme difference 9 = extreme difference Comments Figure 3-2 Sample ballot for the grapefruit juice difference from control test using a ten-point rating scale

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17 Instrumental Methods Gas Chromatography-Flame Ionization Detector (GC-FID) Volatiles from grapefruit juice extracts were separated using an HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, Ca, USA) equipped with a flame ionization detector. One-half micro liter sample was injected onto a ZB-5 or DB-Wax capillary column (30m x 0.32mm I.D., 0.5m film thickness) (J&W Scientific, Folsom, CA). Oven temperature was programmed with an initial temperature of 40C and ramped up to 240C for wax column and 265C for ZB-5 column, at 7/min with a final hold time of 5 minutes. Samples were injected using splitless mode with an injector port temperature of 220C and the detector at 250C. The effluent was split 1:2 between a flame ionization detector and a sniff port (Datu, Geneva, NY). Data were collected and integrated using the ChromPerfect v. 5.0software (Justice Innovations, Denville, NJ). Gas Chromatography-Olfactometry (GC-O) A sniff port was attached to the GC in order to facilitate the identification of aroma active volatiles. The GC temperature program was the same as that for the GC-FID. Two assessors were used to evaluate the aroma quality of the samples. Each assessor analyzed the samples in duplicate accounting for four sniffs per sample. Only those odors that were detected in 50% of the sniffs were considered to be aroma active. Sniffing began after the elution of the solvent peak, approximately three minutes into each run. A sliding scale ranging from none to extreme was used to indicate the aroma intensity. Assessors responses were captured using a variable potentiometer, which was collected on the ChromPerfect software. Sniffing time for each run was 25 and 29 minutes for the ZB-5 and DB-wax column respectively. Aroma quality of each sample was recorded.

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18 Gas Chromatography-Mass Spectrometry (GC-MS) Data were collected using a Finnigan GCQ Plus GC-MS system (Thermo Electron, San Jose, CA) using a 99.999% pure helium as the carrier gas. A DB-5 capillary column (60m x 0.25 mm id, 0.25m film thickness column (J&W Scientific) was used. Oven temperature was programmed from 40C to 250C at a rate of 7C/min. Samples were injected using split less mode with an injector port temperature of 220C. Electron impact (EI) mode was used with ionization energy of 70eV. Scans were obtained after 7 minutes to avoid the solvent peak and were measured from 40-300 m/z. Data were analyzed with XCalibur v.1.3 software (Thermo Electron, San Jose, CA). HPLC Analysis of the Furans Sample Preparation Solid Phase Extraction using a method modified by Walsh was employed (21). 10ml of grapefruit juice were centrifuged for 5 min at 3000 rpm. Three milliliters supernatant were passed thru a C-18 cartridge (J & W Scientific, SPE Accubond 500mg/6ml, Folsom, CA) that had been pre-conditioned with 2.5 ml methanol and washed with 6 ml water. The cartridge was then washed with 2 ml water to remove the sugars and eluted with 2 ml methanol at a rate of 1 drop per 10s. Samples were filtered thru a 0.45 nylon filter (Fisher Scientific) and stored in a 1.8 ml amber vial until injected into the HPLC. HPLC Instrumentation Separation was achieved by injecting 20 l of the juice extract onto a Phenomenex 5 C-18 column (4.6mm id x 250mm) (Phenomenex, Torrance, CA). A 10ul injection volume was used for standards. Column temperature was kept at 25C. Chromatographic analysis was carried out using Surveyor HPLC equipped with a PDA

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19 detector. Versatility of the PDA detector enabled the monitoring of three individual wavelengths: 235nm, 290nm and 335nm, while obtaining a spectral scan 220-380nm. Data was collected and analyzed using Atlas software v.1.0 at an acquisition rate of 10Hz (Thermo Electron) over a 40 minute run time. The mobile phase consisted of methanol and phosphate buffer. Phosphate buffer (pH4.1 1) was made using a 0.05M sodium phosphate solution and phosphoric acid. Linear gradient conditions are summarized in table 3-1 while figure 3-3 shows the graphical gradient conditions. A flow rate of 1ml/min was maintained and a re-equilibration time of 10 min was applied.

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20 Table 3-1 HPLC gradient conditions with flow rate at 1mL/min Time % Methanol % phosphate 0 5 95 10 15 85 27 30 70 33 40 60 40 5 95 50 5 95 0204060801000102030405Time (min)solvent strength (%) 0 % methanol buffer Figure 3-3 Graphical representation of the HPLC gradient conditions Identification and Quantification Identification of aroma active components was achieved by comparing linear retention indices and aroma descriptors with those from a database or published values. Confirmation was achieved by matching fragmentation pattern of the sample with that of standards or library. A series of alkanes (C 5 -C 25 ) analyzed under the same GC conditions

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21 were used to calculate linear retention indices (37). A scatter plot of retention time along with retention indices of alkane standards were graphed on Excel. Retention indices for the standards were obtained by multiplying the carbon number by a factor 100. A polynomial was obtained from a regression. This equation was then used to calculate linear retention indices for individual components in grapefruit juice. Alkane standards were analyzed for both DB-wax and ZB-5 columns, as well as for GC-MS. Components from HPLC analysis were identified using a combination of comparing retention times and spectra of standards with that of sample. Quantification was done using external standard method. Sets of standards with known concentrations were analyzed in triplicate. Areas of those standards were plotted against the amounts and a linear regression was obtained along with a linear equation. Responses from unknown components were then substituted in the linear equation in order to find the amounts and ultimately the concentration of components in juice samples.

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CHAPTER 4 SENSORY ANALYSIS RESULTS AND DISCUSSION There are several sensory analysis tools that are used in difference testing of foods. Difference from Control test was used in this study as it enabled the evaluation of three treatments in one sitting. There are two objectives of the difference test. First, it determines if a difference exists between samples and the control. Secondly, it estimates the magnitude of the difference (38). The three samples that were evaluated were Unheated unheated reconstituted grapefruit juice (as well as hidden control) Glass FCGJ heated 10 min. on glass surface Metal FCGJ heated 10 min. on stainless steel surface The sensory evaluation data for each replicate are shown in tables 4-1 and 4-2. Except for one (panelist #12, table 4-2 rep 3), all panelists were able to identify the hidden control, as having little or no difference from the control. Although overall scores (table 4-2 rep3) ranged from no difference (0) to very large or extreme (8) difference, only 68% of the panelists were able to distinguish differences between the control and the juice heated in metal. A difference score of three and greater was considered to show difference, difference scores of less than three was not considered to be different from the control. Scores for juice heated in glass ranged from moderate difference to very large difference (7) for over 86% of the panelists. Seven of the twenty-two panelists rated the juice heated in metal with a score of two or less compared to only three for the juice heated in glass. Perhaps, these panelists could not distinguish the differences due to their 22

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23 low bitterness threshold and hence high sensitivity. This sensitivity incapacitates panelists from being able to distinguish flavor differences among samples. An example of this is panelist number 15 (table 4-1 rep 1) and panelist # 4 9table 4-2 rep 2) who was unable to detect much difference and commented that all three samples had high bitterness levels. Also they could be anosmic or unable to detect heated off-flavors. Although panelists scores improved for the second and third evaluations, some panelists were still unable to detect flavor differences in the juices. Average difference score for the unheated reconstituted juice ranged from 1.96 in rep 1 to 0.45 in rep 3 suggesting an improvement in identifying the hidden control. Most panelists had difficulty detecting differences between the two heated samples e.g. panelists #1, 2, 5, 6, 8 & 10 from table 4-1 rep1 indicated at most, a one-point difference between the glass and metal samples. Although there may have been a difference in overall flavor between the two heated sample types, the severe heating produced such a strong off flavor that it was difficult for panelists judge the degree of difference. Both samples had very strong heated/ cooked off-flavors. Statistical Tests for Significant Differences Statistical differences between samples were determined by using a two-way analysis of variance (ANOVA) test, at the 95% confidence level, as it allows for the comparisons of more than two samples (table 4-3). The advantage of using a two-way ANOVA is that any error due to the panelists can be distinguished.

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24 Table 4-1 Difference from control sensory data from 22 panelists between a control and three samples, a hidden control (unheated reconstituted), grapefruit juice heated in glass, and juice heated in metal using a 10-point scale. Rep 1 Panelists Unheated reconstituted Glass Metal 1 1 5 5 2 0 6 5 3 4 5 1 4 0 5 3 5 5 8 7 6 2 6 5 7 2 3 5 8 1 6 7 9 0 3 3 10 0 5 4 11 0 3 5 12 1 0 3 13 4 6 4 14 1 2 5 15 0 0 0 16 4 3 2 17 3 5 3 18 4 7 2 19 3 5 1 20 2 2 4 21 2 5 7 22 1 7 7 23 5 7 7 24 3 9 1 25 1 1 3 Average 1.96 4.56 3.96 Median 2 5 4 St Dev 1.62 2.46 2.09 Min 0 0 0 Max 5 9 7

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25 Table 4-2 Difference from control sensory data for the second (rep 2) and third replication (rep 3). Rep 2 Rep 3 Panelist No. Unheated reconstituted Glass Metal Unheated reconstituted Glass Metal 1 0 5 3 0 5 5 2 4 9 6 0 7 3 3 1 3 1 0 3 1 4 1 0 0 1 1 3 5 1 4 6 0 3 3 6 0 3 5 1 6 0 7 1 3 3 1 3 1 8 0 5 8 0 4 5 9 0 7 5 1 4 7 10 1 3 5 0 1 2 11 3 5 7 1 7 7 12 2 1 3 2 7 8 13 1 3 3 1 5 8 14 1 6 5 0 3 4 15 0 5 5 1 5 5 16 0 5 3 0 5 1 17 1 3 0 0 5 6 18 1 3 1 0 5 0 19 1 0 5 0 7 3 20 3 5 1 0 5 5 21 3 3 3 0 3 1 22 1 1 3 Average 1.19 3.86 3.71 0.45 4.32 3.68 Median 1 3 3 0 5 3 St Dev 1.14 2.10 2.21 0.58 1.87 2.46 Min 0 0 0 0 1 0 Max 4 9 8 2 7 8

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26 Table 4-3 Results of the analysis of variance for each of the three replications. Rep1-table 4-1, reps 2 and 3 data are shown in table 4-2. Rep1 Source of Variation SS df MS F P-value F crit Panelists 158.08 24 6.59 2.08 0.015 1.75 Treatments 92.67 2 46.33 14.63 1.09E-05 3.19 Error 152 48 3.17 Total 402.747 74 Rep2 Source of Variation SS df MS F P-value F crit Panelists 107.94 20 5.40 1.89 0.043 1.84 Treatments 94.51 2 47.25 16.56 5.77E-06 3.23 Error 114.16 40 2.85 Total 316.603 62 Rep3 Source of Variation SS df MS F P-value F crit Panelists 105.15 21 5.01 1.88 0.040 1.81 Treatments 188.82 2 94.41 35.45 9.58E-10 3.22 Error 111.85 42 2.66 Total 405.82 65 The F test is a statistical technique that was utilized to compare multiple means and to test for variability among the means for the three samples (treatments) against the variability of the observations (error). In this study the null hypothesis assumed that the treatment means were the same, however, if the mean for the treatments were different then the null hypothesis would be rejected. The maximum ratio between variations among the treatments was measured using the F crit value. If the calculated F value were greater than the F crit then there would be significant variations, which would lead to differences among the sample means.

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27 From table 4-3 it can be seen that the F calc value is greater than the F crit, in all three replicates, therefore we reject the null hypothesis and conclude that at least one of the treatments means is different. Critical F values were approximately 3 compared to calculated F values of 15, 17 and 35 respectively for reps 1, 2, & 3. Since the analysis of variance test only suggests that a difference exist among the populations, a multiple comparison test was used to rank the means and identify the means that were different. Fisher LSD multiple comparison test was applied to the means because it is the least conservative test in comparison to the Tukey and Duncans test and should produce the most difference. The LSD test is defined as any observed difference necessary to declare the corresponding population means different(39). The following equation was used to calculate the LSD: LSD = t /2 (2*EMS)/n where t /2 was found at the degree of freedom of the error and an alpha level of 0.025 from a t-table, EMS, error mean square was taken from the ANOVA results and n was the number of panelists. Means for each population were subtracted from each other. Those with a difference greater than the LSD value, were considered to be significantly different, while those that were similar or less than the LSD value were not significantly different. From the data in table 4-4 rep3, the difference between the glass and the metal was only 0.64 while the difference between the unheated reconstituted juice and glass was 3.87. The calculated LSD value was 0.99.

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28 Table 4-4 LSD comparisons of the means for the three samples Unheated reconstituted Glass Metal LSD Means Rep 1 1.96a 4.56b 3.96b 1.02 Means Rep 2 Means Rep 3 1.19a 0.45a 3.86b 4.32b 3.71b 3.68b 0.50 0.99 Since the difference between the unheated reconstituted juice and two heat treatments in all three replications were greater than LSD values, it can be concluded that a significant difference existed between unheated reconstituted and heated samples. However the difference of means for two heated samples were less than the LSD value, therefore they were not statistically different. Cooked, heated, processed, pineapple flavor were some comments recorded by panelists for heat-treated samples while the unheated reconstituted sample was identified as having good grapefruit flavor. Panelists who had difficulty recognizing differences between the control and two heat-treated samples recorded comments such a high bitterness, acidity. Producing Heated/Cooked Flavor Attempts to reproduce the heated cooked off-flavor were carried out using three furans known to produce a caramel aroma, Furaneol, sotolone, homofuraneol. In addition, bis (2-methyl-3-furyl) disulfide, a thiamine degradation product sometimes found in thermally abused citrus juices, was also added. Table 4-5 shows the combination of the compounds used as well as their sensorial characteristics when added to grapefruit juice based on a 6-member panel. It has been previously reported that

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29 concentration greater than 0.05 g/mL Furaneol masked the flavor of grapefruit juice. This was seen in the combinations that had 1g/mL Furaneol. Little or no grapefruit juice character detected by the panelists. However, in the combination containing 0.5 g/mL, a slight grapefruit juice character was noticed, but caramel and sweet flavor impression were more prominent. The combination of homofuraneol (1g/mL ), Furaneol (1g/mL ) and bis (2-methyl3-furyl) disulfide (2g/mL ) produced a cooked off-flavor. Extremely low concentrations of sotolone (0.01g/mL ) also contributed to over-processed flavor. Sotolone is a very potent aroma active compound and would not require a high concentration to produce a pronounced off-flavor.

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30 Table 4-5 Combination used in off-flavor duplication along with their descriptors Combination No. Concentration Flavor Descriptor 1 1g/mL homofuraneol + 1g/mL Furaneol + 1g/L MFT-MFT Sl cooked fl, caramel aroma, no gfj character, sweet, less acid 2 1g/mL homofuraneol +1g/mL Furaneol + 2g/mL MFT-MFT More cooked/caramel aroma, less caramel aroma, less tart, sl gfj character 3 0.5g/mL Furaneol + 1g/mL homofuraneol +2g/L MFT-MFT Less cooked aroma, more gfj character, more sweet 4 0.5g/mL sotolone + combination #1 Spicy, strong caramel, spoiled juice, no gfj character 5 0.01g/mL sotolone Processed, spicy aroma

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CHAPTER 5 GAS CHROMATOGRAPHIC RESULTS AND DISCUSSION Gas Chromatography is a useful method of separating volatiles based on several factors including polarity and boiling point. In this study, juice volatiles were separated using high-resolution capillary gas chromatography. The use of at least two dissimilar chromatographic phases is advantageous in that compounds that are not well separated on a polar phase column can often be better separated on a non-polar column and the reverse. Polar compounds tend to remain on a polar column longer and thus would be better separated. Since GC is a separation technique, these components cannot be identified without additional information. From the overlay of the FID chromatograms (fig. 5-1) obtained from the three sample types, it was seen that some peaks were diminished due to heating while others increased. Peaks I, II and III showed a higher FID response for the unheated reconstituted juice compared to that of the two heated treatments. In contrast, peak VII had a higher response in the two unheated reconstituted samples. Table 5-1 lists some of the volatiles that were tentatively identified using the linear retention indices on two columns. 31

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FID ResponseTime (min) FID Chromatogram of Grapefruit JuiceZB-5 columnIIIIVVIVIIIIIXIVVIIIXXIInternal StandardInternal StandardABC FID ResponseTime (min) FID Chromatogram of Grapefruit JuiceZB-5 columnIIIIVVIVIIIIIXIVVIIIXXIInternal StandardInternal StandardABC 32 Figure 5-1 Overlay of partial grapefruit juice FID chromatogram on a ZB-5 column. A-unheated reconstituted juice, B-juice heated in glass, C-juice heated in metal.

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33 Table 5-1 Preliminary identification of major grapefruit juice components based on standardized retention times using FID responses from both polar (wax) and non-polar (ZB-5) columns. Peak RT (min) LRI (ZB-5) Lit LRI (ZB-5) Preliminary Identification LRI (wax) Lit LRI (wax) i 4.61 847 846 Ethyl 2-methyl 1022 1041 ii 6.39 939 939 -pinene 978 1011 iii 7.49 989 989 myrcene 1172 1168 iv 8.5 1032 1025 limonene 1214 1205 v 9.82 1087 1074 cis-linalool oxide 1480 1448 vi 10.16 1100 1100 linalool 1552 1552 vii 12.37 1192 1207 -terpineol 1713 1713 viii 14.75 1315 1376 -copaene 1480 1472 ix 16.97 1432 1418 -caryophyllene 1644 1624 x 21.38 1677 1706 -sinensal 2254 2251 xi 23.81 1834 1814 nootkatone 2587 2595 GC-O Since the invention of GC-O as a tool to evaluate flavor volatiles, it has been adapted by the food, environmental, and flavor industry as a major tool used to identify those compounds that are aroma active. The selectivity and sensitivity of the human nose combined with the resolving power of the GC provides useful information about those volatiles that produce any aroma sensation. This is a particularly useful tool for those potent aroma compounds that have extremely low thresholds. It can be seen that little or no FID peaks were detected for the two peaks, 1-octen-3-one and 1-octen-3-ol, in the region of 7-8 minutes (fig. 5-2). Their analytical concentrations were below detection levels of the FID detector, yet their concentrations exceeded their aroma thresholds so they were aroma active.

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limonene caryophyllenelinalool oxide FID ResponseAroma Intensity 6 8 10 12 14 16 18 20 22 24 26 28octen-3-one limonene caryophyllenelinalool oxide FID ResponseAroma Intensity 6 8 10 12 14 16 18 20 22 24 26 28 limonene caryophyllenelinalool oxide limonene caryophyllenelinalool oxide limonene caryophyllenelinalool oxide FID ResponseAroma Intensity 6 8 10 12 14 16 18 20 22 24 26 28octen-3-one 34 Figure 5-2 Partial FID (top) and aromagram (bottom) overlay of grapefruit juice analyzed on a ZB-5 column.

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35 Similarly, not all volatiles found in relatively high concentrations or large FID peaks are aroma active. Large FID peaks such as limonene and -caryophyllene shown in fig. 5-2 had little to no aroma activity. In the region between 24-28 min there were several prominent FID peaks that did not produce detectable aroma activity. Aroma Active Compounds in Grapefruit Juice GC-O is particularly useful in explaining sensory data. However, not everyone has the same sensitivity to all aroma compounds and there will be variability between individuals in assessing these aromas. To compensate for individual sensory variability, each sample was sniffed at least four times, twice per assessor. A compound was considered aroma active only if it was detected in 50% of the sniff runs. By using two panelists, effects of individual hypersensitivities and specific anosmias are minimized. If one assessor is anosmic to a certain class of compounds and the other isnt, then there is a better chance of detecting these compounds in the averaged assessor results. GC-FID and GC-O were carried out on two dissimilar columns. The advantage to this method is that volatiles are eluted in a different order with each column type. Tentative identification is based upon cross-referencing calculated retention indices with those from a library, published results or with standards. Over forty aroma active grapefruit juice volatiles were detected using both wax and ZB-5 columns. Shown in table 5-2 are twenty-six aroma active compounds that were identified by matching retention indices along with aroma descriptors of components on both columns. Standards were run under similar conditions to confirm retention values and odorant descriptors.

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36 Aroma intensities were normalized for each assessor to minimize differences between assessor uses of the intensity scale. Aroma intensities detected on wax column were normalized based on vanillin, which had the highest peak height among all the volatiles (see table 5-3 and fig. 5-3). Table 5-2 Aroma active peaks with tentative identification based on aroma attributes and retention characteristics for grapefruit juice samples as detected by GC-O on polar and non-polar columns. DB-Wax ZB-5 Component Name Descriptor 978 939 -pinene piney 1001 800 ethyl butyrate Sweet, fruity 1022 847 ethyl-2-methyl butyrate fruity 1172 989 myrcene green, earthy, metallic 1214 1032 limonene impurity minty, licorice 1306 1003 octanal green, fresh, lemony 1315 978 1-octen-3-one mushroom 1390 943 4-mercapto-4-methyl-pentanone cat litter 1470 906 methional Cooked potato 1509 1206 decanal green, fatty, lemony 1514 1095 Z-2-nonenal metallic, green 1544 1544 Z-4-decenal fresh, floral, fatty 1552 1103 linalool floral, orange, greenish 1596 1194 E,Z-2,6-nonadienal faint cucumber, green 1633 820 butanoic acid skunky, rotten, fresh 1773 1232 citronellol sweet, roasted 1828 1321 E,E-2,4-decadienal fatty, sl orange 1840 1393 -damascenone tobacco(faint), piney 1861 1264 geraniol rose, fresh, lemon pledge 2017 1383 4,5-epoxy-(E)-2-decenal metallic, cooked, burnt 2043 1062 Furaneol caramel 2201 1325 4-vinyl guaiacol spice 2254 1677 -sinensal grapefruit, citrusy 2259 1470 wine lactone nutty, musty 2587 1834 nootkatone tropical, sour 2608 1418 vanillin vanillin

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Retention Time (min)GC-O Aromagram -10-505103691214171922242528Normalized Aroma IntensitiesUnheatedGlass123456781110129211918171615141320242223252627282930323133343536373839414042vanillinRetention Time (min)GC-O Aromagram -10-505103691214171922242528 -10-505103691214171922242528Normalized Aroma IntensitiesUnheatedGlass123456781110129211918171615141320242223252627282930323133343536373839414042vanillin 37 Figure 5-3 Aroma intensities for the unheated reconstituted juice and glass-heated samples analyzed on wax column. Intensities were normalized peak height based on vanillin, which had the highest peak height. Peak numbers correspond to those listed in table 3.

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38 Table 5-3 Aroma intensities based on normalized peak heights of samples analyzed using wax column. Descriptors are listed in ascending order of LRI. Normalized Aroma Intensities Component No Calculated LRI Descriptors Unheated reconstituted Glass Metal 1 978 piney 6.06 2.91 2.36 2 1001 sweet, fruity 2.24 3 1022 fruity 4.94 5.22 6.88 4 1098 sulfury, stale 6.26 5.81 5 1172 green, earthy, metallic 6.09 1.88 0.83 6 1214 minty, licorice 5.45 6.52 3.83 7 1306 green, fresh, lemony 3.91 5.84 5.38 8 1315 mushroom 2.70 5.74 5.65 9 1381 green, grassy, piney 6.85 8.66 8.40 10 1390 cat litter 6.11 11 1470 cooked potato 0.65 9.01 8.12 12 1509 green, fatty, lemony 6.98 5.58 7.17 13 1514 metallic, green 2.53 6.91 2.81 14 1544 fresh, floral, fatty 4.24 3.41 4.06 15 1552 floral, orange, greenish 8.37 9.32 4.09 16 1596 faint cucumber, green, sweet 2.06 4.30 3.59 17 1633 skunky, rotten, fresh 3.72 3.31 3.06 18 1773 sweet, roasted 3.43 19 1828 fatty, sl orange 3.27 7.95 6.55 20 1840 tobacco(faint), piney 4.37 2.87 1.53 21 1861 rose, fresh, lemon pledge 2.88 7.52 6.30 22 1868 bug spray,spice 3.64 4.36 5.92 23 1875 caramel, sweet, fresh 3.34 3.57 3.90 24 1891 fresh cucumber,floral, fresh 3.18 2.36 25 1940 metallic 3.16 26 2000 burnt, spicy 5.85 2.72 27 2017 metallic,cooked, burnt 3.95 3.16 3.30 28 2038 spice 3.92 29 2043 caramel 4.35 8.00 8.61 30 2059 spicy(strong), curry 3.93 5.48 31 2154 sweet caramel 3.90 1.48 32 2163 sweet, caramel 7.35 7.84 33 2179 charred, sweet 5.07 34 2188 grapefruit, spice(lingering) 3.14 7.94 5.82 35 2201 spice 4.68 5.11 8.78 36 2207 sweet, burnt wood 2.85 37 2243 rancid fruit 3.27 38 2254 grapefruit, citrusy 7.55 4.37 4.87 39 2259 nutty, musty, crayons 1.05 40 2298 grapefruit 5.86 2.37 41 2587 tropical, sour 5.20 2.89 42 2608 vanillin 9.73 4.26 10.00

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39 Aroma Active Compounds Lost during Heating Many of the components listed in table 2 have been previously reported in fresh grapefruit juice (40). However, their concentrations have probably been altered due to the heating process. During the evaporation process when making concentrate, most volatiles are lost along with water. Although it has been reported that over 95% of the volatiles in the fresh juice are lost in the process of concentrating grapefruit juice, over 57% of the total aroma activity remains (29). Typically, fresh juice flavor characteristics are restored to the concentrated juice by the addition of juice essence oil, aqueous essence and peel oil. The juice concentrate used in this study had 0.012% cold pressed grapefruit oil added to it. This oil was the source for most of the volatiles and many aroma active volatiles. Since essence oil and aqueous essence are obtained from the condensate of the evaporation process, they will contain volatile heat reaction products and thus were not used for this study. Therefore, any heated, cooked, caramel aromas were produced during the specific heating conditions in this study. Normalized intensities of aroma active volatiles (table 5-3) present in both the unheated reconstituted juice as well as the two heat-treated juice samples were compared in order to identify aroma differences due to heat treatment and contact surface. Three aroma active compounds, -pinene (1), myrcene (5) and -sinensal (38), exhibited greater aroma intensities in the unheated reconstituted juice compared to that of the two heated juice samples (fig. 5-4). Another aroma active compound, ethyl butyrate (2), was detected only in the unheated reconstituted juice with a normalized intensity of 2.24 on a 10-pt scale. Ethyl butyrate imparts a sweet, fruity aroma and is considered to be a positive contributor to grapefruit juice aroma (40). It is a low

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40 molecular weight compound and is highly volatile, thus it likely to be degraded during extended heating times. 0510Retention Time (min)Aroma Intensity unheated Glass Metal--pinene-sinensalethyl butyratemyrcene 0510Retention Time (min)Aroma Intensity unheated Glass Metal--pinene-sinensalethyl butyratemyrcene Figure 5-4 Aroma active volatiles whose intensity diminished due to heating As seen in table 5-3, alpha-pinene (1) and myrcene (5) are two early eluting, low boiling point compounds, which had higher aroma intensities in the unheated reconstituted juice compared to the two heated juice treatments. In both heated samples, the response for -pinene was less than 45% that of the unheated reconstituted juice while that for myrcene was less than one-third of the intensity in the unheated reconstituted juice. A piney aroma was elicited by -pinene while myrcene imparted a green, metallic, earthy aroma. Both these compounds are positively correlated with citrus juice flavor (41). They are responsible for the green notes in citrus juices. Beta sinensal (38) is a rather high boiling point volatile, which was reduced through heating. The aroma intensity of this aliphatic aldehyde was 42% higher in the unheated reconstituted juice compared to that of the juice heated in glass and 36% higher than the juice heated in metal. Beta sinensal possesses a grapefruity, citrusy

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41 aroma and is one of the important compounds responsible for the fresh grapefruit juice flavor. Aroma Active Compounds That Intensified after Heating Eight aroma active compounds (1-octen-3-one (8), methional (11), (E,Z)-2,4-nonadienal (16), (E,E)-2,4-decadienal (19), Furaneol (29), and 4-vinyl guiaicol (35) and two unknowns (26 & 32) were present in intensities greater than 50% in comparison to the unheated reconstituted juice (fig 5-5). Both Furaneol (29)(discussed later) and methional (11) are thermally generated. Furaneol is formed from the reaction of rhamnose and an amino acid in the presence of heat. Methional is believed to be a Strecker degradation product of methionine in the presence of ascorbic acid. Its normalized intensity in the two heated samples was 8 and 10 compared to 0.65 in the unheated reconstituted juice. (E,Z)-2, 6-nonadienal (16) and (E,E)-2,4-decadienal (19) are unsaturated aldehydes and are likely to be formed from fatty acid degradation (42). Both of these aldehydes are essential flavor components of citrus oils. A faint cucumber, green aroma was detected from (E,Z) 2,6 nonadienal while the aroma of (E,E)-2,4-decadienal was described as fatty with a slight orange character. The two unknown compounds (26 & 32) have a spicy and sweet, caramel aroma respectively. Neither of the two unknowns was detected in the unheated reconstituted juice. However the intensity of unknown # 26 in the juice heated in glass was 54% higher than the juice heated in the metal. Unknown # 32 had similar aroma intensity in both the heated samples. Their linear retention index values on wax were 2000 and 2163 respectively. Maltol, a Maillard reaction product elutes at an LRI close to 2000 possesses a caramel aroma and could be a possible identity. Identification of this compound would be satisfied through the use of GC-MS and by matching the spectra of

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42 the compound alongside that of the pure standard. However, maltol does not have a unique mass spectrum and may be present in concentrations lower than the detection threshold of the GC-FID or GC-MS, but not of the human nose. Retention Time (min)Aroma Intensity Unheated Glass Metal 05101-octen-3-oneE,Z-2,6-nonadienalmethionalE,E-2,4-decadienalUnknown #26FuraneolUnknown # 324-vinyl guiaicolRetention Time (min)Aroma Intensity Unheated Glass Metal 05101-octen-3-oneE,Z-2,6-nonadienalmethionalE,E-2,4-decadienalUnknown #26FuraneolUnknown # 324-vinyl guiaicol Figure 5-5 Aroma active volatiles (peak numbers 8, 11, 16, 19, 29, 26, 32, & 35) whose intensity increased after heat treatment was applied. Caramel Aroma Compounds Formed from Sugar Degradation Reactions From table 5-3, it can be noted that caramel burnt aroma was detected several times throughout the sniff run. In some cases the volatiles lingered for up to 20 sec. In order to identify these compounds, several standards that are known to impart caramel aromas were analyzed under similar conditions on both the wax and ZB-5 columns. Table 5-4 lists the furans and a pyranone standard that were examined to identify the unknown compounds responsible for the caramel aroma.

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43 Table 5-4 LRI for ten sugar degradation products possessing caramel aroma analyzed by GC on both polar (wax) and non-polar (ZB-5) columns. 1 Identified in all three samples Compounds LRI (ZB-5) Present (ZB-5) Present (Wax) LRI (DB-Wax) Furfural 850 + 1215 5-methyl furfural 967 1560 3-methyl2(5H) furanone 1 989 + + 2153 Furaneol 1 1064 + + 2096 Norfuraneol 1054 2124 Mesifurane 1064 1602 Maltol 1114 2002 Sotolone 1 1118 + 2203 Homofuraneol 1145 2045 5-HMF 1 1236 + 2526 1 2 found in unheated reconstituted only All the furans listed are oxygenated. Furaneol, mesifurane and norfuraneol elute in close proximity on the ZB-5 column. Both Furaneol and mesifurane had identical retention indexes values (1064) while norfuraneol had a linear retention index value of 1054. The ease of volatilization of a compound is one of several factors influencing GC separation. Most of the furans are structurally similar and, would be expected to volatilize easily. Also, column chemistry plays a key role in separation. Polar compounds elute more quickly on a non-polar column but will be better retained on a polar column. The rule like attracts like applies. On the ZB-5 column it was seen that the 3(2H) furanones elute in close proximity to each other e.g. Furaneol and mesifurane eluted at 1064, and norfuraneol eluted at 1054. The structures of these compounds are shown below (fig. 5-6).

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44 Figure 5-6 Structure of 3(2H) furanones O CH3 OH CH3CH2 O O R3 R2 R1 O O CH3 OH CH3 O O CH3 CH3 O MeO O CH3 OH O 3(2H) furanone Furaneol2,5 dimethyl-4-hydroxy-3(2H) furanone Homofuraneol 2 ethyl 4-hydroxy 5 methyl-3(2H) furanone Mesifurane Norfuraneol 4 hydroxy-5-methyl-3(2H) furanone 4-methoxy-2,5dimethyl-3(2H) furanone Homofuraneol has an ethyl group as one of the side groups and this additional increase in molecular weight and boiling point is probably the reason for its later elution compared to the other furanones. Sotolone (fig.5-7), a 2(5H) furanone, also has a molecular weight of 128 and combined with the different position of the R groups could be responsible for the decrease in its volatility.

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45 O O OH CH3 H3C Sotolone Figure 5-7 Structure of sotolone, a 5(2H) furanone The use of a dissimilar column such as the wax column is an excellent choice in situations like these, as the order of elution will be different. By applying the like attracts like principle, polar compounds would be retained for a longer time on the polar wax column. Caramel Compounds Found in Grapefruit Juice Furaneol and sotolone were detected by GC-O while 5-HMF was detected by GC-FID. Furaneol has previously been reported in grapefruit juice and has been known to mask the flavor of orange juice (4;11). Its intensity was over 50% higher in the two heat treated samples as opposed to the unheated reconstituted juice (table 5-3). Furaneol is formed from rhamnose through the 2,3-enolization pathway of the Amadori compound followed by dehydration and molecular rearrangement (22). The amino acid arginine has been shown to be a necessary substrate for its formation under acidic conditions. Sotolone has recently been reported as an off-flavor to citrus sodas containing ethanol and ascorbic acid (28). Sotolone imparted a caramel, cooked flavor to grapefruit juice. The 5-HMF has been reported in citrus juices and has been suggested as a marker for thermal abuse. This compound is formed from the 1,2-enolization pathway and can be polymerized to form colored pigments or other flavor compounds. Unlike the other furanones, 5-HMF has a high threshold and consequently does not have a direct aroma impact. However, it produced substantial FID response.

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46 Furfural was detected by GC-MS. It has a unique fragmentation pattern with the major ion found at mass 95, indicating the cleavage of a hydrogen atom. By using this knowledge as well as selecting mass peaks 96 and 95, the spectra was deduced and then matched with that of the standard at the expected retention time. 3-Methyl 2(5H) Furanone A maltol degradation product, 3-methyl-2(5H) furanone (fig. 5-8), (43) not previously reported in grapefruit juice was tentatively identified on both polar and non-polar columns. Its LRI on a the non-polar column was 982 on and 2153 on the polar column. It had a weak-moderate caramel aroma. It has been reported in mammee apples (44). O O CH3 3-methy 2(5H) furanone Figure 5-8 Structure of the 3-methyl-2(5H) furanone When the standard was analyzed on GC-MS, the retention time and linear retention index suggested that it eluted with peak # II (fig. 5-9). Commercial FCGJ undergoes at least two heat treatments, once during the evaporation step and second after reconstitution. However, the untreated juice, which was reconstituted grapefruit juice, underwent only the first heat treatment, which involves heating temperatures as high as 93C. A high temperature such as this is sufficient to induce the formation of these furanones. The heating of the juice in both the glass and stainless steel containers may have increased the formation or

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47 concentration of these compounds, but due to their extremely low threshold the aroma remains quite intense. The Maillard reaction is initiated by high temperature treatment of foods, and its products are increased with extreme heat treatment and long-term storage. The first step involves the reaction of a sugar with an amine and subsequent loss of water. There is only a slight loss of reactants since sugars are relatively plentiful in grapefruit juice. Identification of Volatiles Using GC-MS Since MS is an identification tool, it was used to identify the volatiles based on their specific fragmentation spectra as well as their linear retention indices. Most compounds fragment in a unique pattern and by comparing this pattern with standards analyzed under the same conditions or by comparing to library databases with known spectra, compounds can usually be identified. Fig. 5-9 shows the total ion chromatograph of grapefruit juice from 10-33 minutes while table 5-5 lists the identities of a few of the major components in grapefruit juice. The areas were normalized to internal standard #2. Matching linear retention indices was used for a secondary confirmation. This is particularly important in the detection of terpenes. Terpenes are C 10 H 16 hydrocarbons and have a molecular weight of 136. It is very difficult to identify these compounds by solely matching mass spectra as they tend to exhibit similar fragmentation patterns particularly for the major mass peaks.

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Retention Time (min)TIC Response 10 16 20 24 28 32Total Ion Chromatogram for Grapefruit Juice VIIIIIVIVIVIXXXIXIIXIIIIIIIInternal StandardInternal Standard IIVIIACBRetention Time (min)TIC Response 10 16 20 24 28 32Total Ion Chromatogram for Grapefruit Juice VIIIIIVIVIVIXXXIXIIXIIIIIIIInternal StandardInternal Standard IIVIIACB 48 Figure 5-9 Segmented MS total ion chromatogram using ZB-5 column for unheated reconstituted grapefruit juice (A), juice heated in glass (B) and juice heated in stainless steel (C).

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49 Table 5-5 Components identified in grapefruit juice by GC-MS and analyzed on a DB-5 column. Peak Number Retention Time (min) LRI Component Name Unheated reconstituted Glass Stainless Steel %Area %Area %Area I 12.47 941 -pinene 15.68 2.63 0.74 II 13.68 987 myrcene 45.93 7.52 4.66 III 14.93 1035 limonene 2512.60 611.60 364.36 IV 15.14 1043 E-ocimene 2.25 0.74 0.73 V 15.92 1073 linalool oxide 6.72 27.23 18.83 VI 16.31 1089 linalool 3.79 16.42 11.51 VII 19.05 1201 -terpineol 10.57 20.67 11.74 VIII 23.19 1385 -copaene 6.80 0.32 3.39 IX 24.24 1435 -caryophyllene 30.21 1.73 12.61 X 25.55 1499 -humulene 12.40 7.43 7.75 XI 26.00 1522 bisabolene 11.22 1.02 6.49 XII 26.14 1529 -cadinene 1.43 1.77 0.96 XIII 31.44 1823 nootkatone 80.76 43.69 41.95 An example of this can be seen in peaks number I and III, which were identified as -pinene and myrcene respectively. Both these compounds are terpenes having piney and musty aroma respectively. From the spectra of these two compounds, fragments 121, 91 and 77 (fig. 5-10) were common to both and appeared to have the same intensity. Mass peaks at 92, 93 and 79 had slightly different intensities and may be used as distinguishing fragments. However, by using the linear retention indices of 939 and 989 respectively, these two compounds could be easily distinguished.

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50 TIC Relative Abundance406080100120406080100120140m/z 91.177.279.392.181.251.265.3105.1 109.240.9121.2137.2 -pinene m/z 91.277.493.179.341.251.480.367.365.3107.1121.2135.2 myrceneMass/Charge (m/z) ratioTIC Relative Abundance406080100120406080100120140m/z 91.177.279.392.181.251.265.3105.1 109.240.9121.2137.2 -pinene406080100120140m/z 91.177.279.392.181.251.265.3105.1 109.240.9121.2137.2 -pinenem/z 91.177.279.392.181.251.265.3105.1 109.240.9121.2137.2 -pinene m/z 91.277.493.179.341.251.480.367.365.3107.1121.2135.2 myrcene m/z 91.277.493.179.341.251.480.367.365.3107.1121.2135.2 myrceneMass/Charge (m/z) ratio Figure 5-10 Mass spectra of myrcene (top) and -pinene (bottom) taken from a sample analyzed on a DB-5 column. Limonene (peak III) was present in the highest amount as expected. Limonene is the most abundant terpene present in citrus juices. Peak VII was identified as -terpineol. Figure 5-11 is an expanded view of the chromatogram indicating the differences among the three samples. Alpha terpineol content of juice heated in glass was higher than both the stainless steel (metal) and unheated reconstituted from concentrate juice. It 63%

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51 higher than the unheated reconstituted juice and 43% higher than the juice heated in metal. Retention Time (min)TIC Relative Abundance 18.95 19.05 19.15 19.25 -terpineol levels in Grapefruit JuiceGlassMetalUnheated Retention Time (min)TIC Relative Abundance 18.95 19.05 19.15 19.25 -terpineol levels in Grapefruit JuiceGlassMetalUnheated Figure 5-11 Alpha-terpineol levels in the three samples, unheated reconstituted juice, juice heated in glass and juice heated in metal Alpha-terpineol can be formed from either (or both) the acid-catalyzed reaction of limonene or linalool (fig. 5-12) in the presence of heat (45). Alpha terpineol has also been shown to negatively correlate with the flavor of citrus juices (4) and has been suggested as an indicator for over-processed or stored orange juices.

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52 CH3 CH2 CH3 CH3 CH3 CH3 OH CH3 CH3 CH3 OH CH3 CH3 CH3+ CH3 CH3 CH3 + d-limonene a-terpineol Linalool H+, -HOH H+, -HOH Figure 5-12 Formation pathway for -terpineol in the presence of limonene and linalool in grapefruit juice

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CHAPTER 6 HPLC RESULTS AND DISCUSSION Unlike GC, HPLC is a non-thermal method of analysis used in the identification of both volatiles and non-volatiles. HPLC is useful in situations where polar volatiles have strong UV chromaphores and are difficult to extract using typical GC extraction procedures. For example, Furaneol in grapefruit juice is usually found at levels below the detection threshold of the flame ionization detector, making detection and quantification difficult. Solid phase extraction employing C-18 chemically bonded silica, can trap polar volatiles onto the modified silica for later analysis on reversed phase HPLC. Since furans are thought to be responsible for heated off-flavor, HPLC was used to identify and measure furans in grapefruit juice. Over the years reversed phase HPLC has been favored in the isolation of furans in several fruit juices (19;21;46). Reversed phase LC is the most popular liquid chromatographic method used today (47). Compounds with increasing hydrophobicity or non-polar behavior will be retained on the column more strongly. Water is the weak solvent and a methanol or acetonitrile gradient is employed to selectively elute the materials off a reverse phase column. In citrus juices several mobile phase compositions have been used ranging from methanol or acetonitrile/water, acetonitrile/methanol/buffer, methanol/acetate buffer. These solvent systems either did not produce a good separation or did not exhibit good reproducibility. Acetate buffer has an absorbance maximum at 235nm, which interferes with the spectra of sotolone whose maximum absorbance is 237nm. Although previous studies (19) reported that methanol/phosphate buffer condition could not resolve furfural 53

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54 and Furaneol, this mobile phase composition was re-examined since the phosphate buffer had a lower UV absorbance background. It had been reported that 30% methanol/phosphate buffer could not resolve furfural and Furaneol. Thus several gradients were evaluated until a suitable separation was between 5-HMF, furfural, and Furaneol was achieved. These three compounds had been previously been identified in grapefruit juice using RP-HPLC (19). In order to increase retention time and promote resolution, the amount of modifier, methanol was decreased to 5% with a slow increase up to 30% in 27 minutes. It was found that at approximately 20% methanol Furfural was eluted and Furaneol close to 24% methanol. Additionally, the temperature of the column was kept at 25C and the sample 24C. Since the three compounds were completely resolved, other Maillard reaction products, (mesifurane, homofuraneol, sotolone, and maltol) were evaluated under similar conditions and the result shown in fig. 6-1. The small humps at the end of the late eluting peaks were apparently due to a small void at the head of the HPLC column. These standards are all products of the Maillard reaction and possess caramel or burnt sugar aroma that could possibly be formed in grapefruit juice during heating. These seven compounds along with their retention times and maximum absorbance are listed in table 6-1.

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55 Table 6-1 Retention time and maximum absorbance of furans standards on a C-18 column and using a methanol/phosphate buffer at flow rate of 1mL/min Furan Standards Retention Time (min) Max Wavelength (nm) 5-HMF 13.04 283 Furfural 14.95 276 Maltol 17.41 274 Furaneol 18.86 287 Sotolone 20.24 235 Mesifurane 27.63 278 Homofuraneol A Homofuraneol B 30.40 31.80 287 287 ResponseRetention Time (min) 15 20 25 30 5-HMFFurfuralMaltolFuraneolSotoloneMesifuraneHomofuraneolAHomofuraneolB283nm234nm284nm274nm276nm278nm287nmResponseRetention Time (min) 15 20 25 30 5-HMFFurfuralMaltolFuraneolSotoloneMesifuraneHomofuraneolAHomofuraneolB283nm234nm284nm274nm276nm278nm287nm Figure 6-1 HPLC spectra of the seven standards (5-HMF, furfural, Furaneol, sotolone, mesifurane and homofuraneol) monitored from 230-380nm. Homofuraneol has been known to exist in two tautomeric forms accounting for the presence of an A and B form (48) and produces two distinct HPLC peaks. As can be

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56 seen from fig. 6-2, homofuraneol has an ethyl group that can be interchanged with the methyl group so that it can be either in the 2 or 5 position. O O CH3CH2 CH3 OH O O CH3 CH3CH2 OH B A Figure 6-2 Homofuraneol tautomers, A2-ethyl-4-hydroxy-5-methyl-3(2H) furanone; B5-ethyl-4-hydroxy-2-methyl-3(2H) furanone The value of the PDA detector lies in its ability to distinguish compounds based on their spectra as well as retention time. This feature can be useful in identifying unknown peaks and determining if there are co-eluted compounds in a single HPLC peak. With the exception of sotolone, which has a maximum absorbance at 235nm, most furans exhibit maximum wavelength absorbance between 270-290nm. For this reason dual wavelength monitoring at 235nm and 280nm was carried out. Additionally, monitoring was done at 335nm as these furans had minimum absorbance at that wavelength. This would be useful in distinguishing furans from other co-eluting compounds. A comparison of the spectra and the retention times of components in the sample with those of standards, suggest the presence of 5-HMF, furfural, and Furaneol in the grapefruit juice (fig. 6-3). As discussed earlier, these three compounds are heat induced hexose sugar degradation products that have been previously identified in citrus juices.

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57 Retention Time (min)Response 13 14 15 16 17 18 19 5-HMFFurfuralFuraneolunheated Glass Metal Retention Time (min)Response 13 14 15 16 17 18 19 5-HMFFurfuralFuraneolunheated Glass Metal Figure 6-3 Segment of HPLC chromatogram showing overlay of unheated, juice heated in glass and juice heated in metal and monitored at 290nm By looking at the spectra of each of these compounds in the sample and comparing it with that of the standard, it was found that 5-HMF was the only compound of interest that was well resolved from other grapefruit juice components (fig. 6-4).

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58 Figure 6-4 Spectra of 5-HMF in standard (top) and in the grapefruit juice sample (bottom) analyzed on C-18 column and monitored at 290nm. Spectral data for both Furaneol and furfural peaks suggested the presence of coeluted interfering compounds. Figure 6-5 shows the spectra of furfural and it can be seen that the peak characteristics are different from the standard. An absorbance maximum of 276nm was seen for the standard while a maximum of 326nm was seen for the sample. Between 260 nm and 300nm, there appears to be another peak indicating that co-elution might have occurred. It was not possible to obtain an absorbance difference chromatogram using output from the two wavelengths (326nm and 278nm) at this time. Previous studies had used this technique to identify and characterize Furaneol in a grapefruit juice matrix (21).

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59 Figure 6-5 Spectra of furfural standard (top) and sample (bottom) showing a difference in the wavelength maxima at 15.15 min Grapefruit juice furan and furanones were quantified using the external standard method, wherein different concentrations of standards were injected onto the LC in triplicate. Area responses were then plotted against the amounts injected and linear responses were obtained. The 5-HMF amounts ranged from 1.07ng to 21.4ng to ensure that the range found in samples was covered. From calibration plot (fig. 6-6), the r 2 value which shows how well the data fit a linear regression was 0.9993, indicating good linearity.

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60 Calibration Curve for 5-HMF R2 = 0.999301000002000003000004000005000006000000510152025Amount (ng)Peak Area Figure 6-6 Linear calibration curve for 5-HMF analyzed in triplicate. Based on the linear equation, the amount of the each furan in grapefruit juice samples could be calculated. Juice heated in the presence of stainless steel had a 5-HMF concentration of 0.47ng/ul, while the same juice heated in glass had 0.34ng/ul (table 6-2). Unheated juice contained the least amount of 5-HMF (0.03ng/ul). Table 6-2 Concentrations of 5-HMF, furfural and Furaneol in the three grapefruit juice samples 5-HMF (ng/ul) Furfural (ng/ul) Furaneol (ng/ul) Unheated 0.03 0.40 0.22 Glass 0.34 0.25 0.08 Metal 0.47 0.25 0.08

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61 A similar plot (fig.67) was conducted for Furaneol with amounts ranging between 1.8ng to 18ng and an r 2 value of 0.92 was found. Unheated reconstituted juice had a concentration of 0.22ng/ul while the two heated juice samples had levels of 0.08ng/ul. Furaneol Calibration curve R2 = 0.92610250000500000750000100000005101520Amount (ng)Peak Area Figure 6-7 Linear calibration curve for Furaneol at 290nm. The standard curve (fig. 6-8) for furfural also had a good fit with an r 2 value of 0.99 using a range of 2ng to 200ng. Concentration of furfural in the unheated reconstituted juice was 0.40ng/ul while juices heated in glass and metal had 0.25ng/ul.

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62 Furfural Calibration Curve R2 = 0.998901000000200000030000004000000050100150200250Amount (ng)Peak Area Figure 6-8 Linear calibration curve for furfural standard at 290nm. All three compounds, 5-HMF, furfural and Furaneol are sugar degradation products. Formation of 5-HMF occurs through the 1,2 enolization at low pH values in the presences of a hexose sugar. The pH of grapefruit juice is usually between 2.93.2, which is highly acidic, and provides an explanation for the increase in the 5-HMF formation in the two heated samples. Studies have shown when grapefruit juice that under similar conditions the rate of formation of 5-HMF was far greater than that of furfural (9). Furfural can be formed from acid catalysis of ascorbic acid in the presence of an amine. Since it can be formed from ascorbic acid, which is known to degrade with heating, it was expected that this compound would have higher concentrations in the two heated samples. However, it is a very reactive compound and, in the presence of acids can react with aldehydes, ketones and amino acids (49). This could possibly be a reason for the lower levels in the heated samples. Furaneol is also a sugar degradation product and its concentration was expected to increase as a result of the heating. Its formation is favored at high pH and goes through

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63 the 2,3-enolization pathway, however at low pH values it is formed in the presence of alanine. Studies have shown that Furaneol is unstable and can be degraded at high temperatures (24) to form highly reactive dicarbonyls, ketones and alcohols. In equilibrium, Furaneol can exist in its open chain form, thereby facilitating attack of sulfur groups such as thiols on the carbonyl (50). It can also react with sulfur compounds, such as cysteine at low pH, and hydrogen sulfide during thermal processing (51;52). Consequently, at high temperatures, the concentration of Furaneol would be lower than expected.

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CHAPTER 7 CONCLUSION No significant flavor difference was detected between juices heated 10 min in contact with stainless steel and the same juice heated in glass. However, this excessive heating induced a flavor change that was significantly different from the unheated reconstituted grapefruit juice at the 95% confidence interval. Thermal processing produced heated, cooked, pineapple and metallic off-flavors. Similar volatiles were detected in both heat-treated samples, suggesting that high processing temperatures and long times appeared to be major contributing factors for cooked off flavor formation. The quality of the juice heated on glass surface was not of superior quality to that of metal surface, therefore, the current practice of using stainless steel is agreeable. Intensities of compounds such as -pinene and myrcene that are known to positively contribute to fresh citrus juice character were diminished during the heating process, while aroma intensity of heat induced compounds such a methional and Furaneol increased during heating. Sugar degradation products such as sotolone, and 3-methyl-2 (5H) furanone were detected by GCO. The 3-methyl-2 (5H) furanone was tentatively identified in grapefruit juice for the first time. Concentrations of 5-hydroxy methyl furfural, another sugar degradation product, increased with heating. 64

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LIST OF REFERENCES 1. Dugo, G.; Giacomo, A. D. Citrus, The Genus Citrus; Taylor & Francis: New York, NY, 2002. 2. Offenbacher, E. G.; Pi-Sunyer, F. X. Temperature and pH effects on the release of chromium from stainless steel into water and fruit juices. Journal of Agricultural and Food Chemistry 1983, 31, 89-92. 3. Woodroof, L. Commercial Fruit Processing; The AVI Publishing Company, Inc: Westport, Connecticut, 1975. 4. Tatum, J. H.; Nagy, S.; Berry, R. E. Degradation products formed in canned single-strength orange juice during storage. Journal of Food Science 1975, 40, 707-9. 5. Naim, M.; Rouseff, R. L.; Zehavi, U.; Schutz, O.; Halvera-Toledo, E. Chemical and sensory analysis of off-flavors in citrus products. ACS Symposium Series 1998, 705, 303-319. 6. Nagy, S.; Randall, V. Use of furfural content as an index of storage temperature abuse in commercially processed orange juice. Journal of Agricultural and Food Chemistry 1973, 21, 272-5. 7. Fellers, P. J.; Carter, R. D. Effects of thermal processing of chilled orange juice on flavor quality. Food Processing 1993, 436-441. 8. Orange juice: How far from fresh? Consumer Reports, 1995. 9. Lee, H. S.; Nagy, S. Quality changes and nonenzymic browning intermediates in grapefruit juice during storage. Journal of Food Science 1988, 53, 168-172. 10. Kanner, J.; Harel, S.; Fishbein, Y.; Shalom, P. Furfural accumulation in stored orange juice concentrates. Journal of Agricultural and Food Chemistry 1981, 29, 948-9. 11. Lee, H. S.; Nagy, S. Chemical degradative indicators to monitor the quality of processes and stored citrus products. ACS Symposium Series 1996, 631, 86-106. 12. Scarpellino, R.; Soukup, R. J. Key flavors from heat reactions of food ingredients. Flavor Sci. [Discuss. Flavor Res. Workshop] 1993, 309-35. 65

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66 13. Rodin, J. O.; Himel, C. M.; Silverstein, R. M.; Leeper, R. W.; Gortner, W. A. Volatile flavor and aroma components of pineapple. I. Isolation and tentative identification of 2,5-dimethyl-4-hydroxy-2,3-dihydro-3-furanone. Journal of Food Science 1965, 30, 280-5. 14. Baek, H. H.; Cadwallader, K. R.; Marroquin, E.; Silva, J. L. Identification of predominant aroma compounds in muscadine grape juice. Journal of Food Science 1997, 62, 249-252. 15. Krammer, G. E.; Takeoka, G. R.; Buttery, R. G. Isolation and identification of 2,5-Dimethyl-4-hydroxy-3(2H)-furanone glucoside from tomatoes. Journal of Agricultural and Food Chemistry 1994, 42, 1595-7. 16. Pickenhagen, W.; Velluz, A.; Passerat, J. P.; Ohloff, G. Estimation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (FURANEOL) in cultivated and wild strawberries, pineapples, and mangoes. Journal of the Science of Food and Agriculture 1981, 32, 1132-4. 17. Mayerl, F.; Noef, R.; Thomas, A. F. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone glucoside: isolation from strawberries and synthesis. Phytochemistry 1989, 28, 631-3. 18. Tonsbeek, C. H. T.; Plancken, A. J.; Weerdhof, T. v. d. Components contributing to beef flavor. Isolation of 4-hydroxy-5-methyl-3(2H)-furanone and its 2,5-dimethyl homolog from beef broth. Journal of Agricultural and Food Chemistry 1968, 16, 1016-21. 19. Lee, H. S.; Nagy, S. High performance liquid chromatography of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in pineapple and grapefruit juices. Journal of Food Science 1987, 52, 163-5. 20. Pyysalo, T.; Honkanen, E.; Hirvi, T. Volatiles of wild strawberries, Fragaria vesca L., compared to those of cultivated berries, Fragaria ananassa cv Senga Sengana. Journal of Agricultural and Food Chemistry 1979, 27, 19-22. 21. Walsh, M.; Rouseff, R.; Naim, M. Determination of furaneol and p-vinylguaiacol in orange juice employing differential UV wavelength and fluorescence detection with a unified solid phase extraction. Journal of Agricultural and Food Chemistry 1997, 45, 1320-1324. 22. Pisarnitskii, A. F.; Demechenko, A. G.; Egorov, I. A.; Gvelesiani, R. K. Methylpentoses are possible precursors of furanones in fruits. Applied Biochemistry and Microbiology 1992, 28, 97-100. 23. Romero, C.; Manjon, A.; Bastida, J.; Iborra, J. L. A method for assaying the rhamnosidase activity of naringinase. Analytical Biochemistry 1985, 149, 566-71.

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69 49. Kanner, J.; Fishbein, J.; Shalom, P.; Harel, S.; Ben-Gera, I. Storage stability of orange juice concentrate packaged aseptically. Journal of Food Science 1982, 47, 429-31, 436. 50. Haleva-Toledo, E.; Naim, M.; Zehavi, U.; Rouseff, R. L. Effects of L-cysteine and N-acetyl-L-cysteine on 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol), 5-(hydroxymethyl)furfural, and 5-methylfurfural formation and browning in buffer solutions containing either rhamnose or glucose and arginine. Journal of Agricultural and Food Chemistry 1999, 47, 4140-4145. 51. Shu, C. K.; Ho, C. T. Effect of pH on the volatile formation from the reaction between cysteine and 2,5-dimethyl-4-hydroxy-3(2H)-furanone. Journal of Agricultural and Food Chemistry 1988, 36, 801-3. 52. Van den Ouweland, G. A. M.; Peer, H. G. Components contributing to beef flavor. Volatile compounds produced by the reaction of 4-hydroxy-5-methyl-3(2H)-furanone and its thio analog with hydrogen sulfide. Journal of Agricultural and Food Chemistry 1975, 23, 501-5.

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BIOGRAPHICAL SKETCH Wendy Bell was born in Westmoreland, Jamaica, and graduated from the Wolmers High School for Girls in Kingston. In 1995, she moved to the United States and embarked upon her undergraduate studies at Barry University, Miami, Florida. She then transferred to the University of South Florida, Tampa, Florida, where she completed her Bachelor of Arts in Chemistry in May 1999. In pursuance of her career, Wendy gained employment at Pasco Beverage Company and Florida Department of Citrus. Wendy will receive her master's degree in December 2004 and will be joining Cadbury Adams, Morris Plains, NJ, upon graduation. 70


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Permanent Link: http://ufdc.ufl.edu/UFE0008969/00001

Material Information

Title: Examination of Aroma Volatiles Formed from Thermal Processing of Florida Reconstituted Grapefruit Juice
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008969:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008969/00001

Material Information

Title: Examination of Aroma Volatiles Formed from Thermal Processing of Florida Reconstituted Grapefruit Juice
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008969:00001


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EXAMINATION OF AROMA VOLATILES FORMED FROM THERMAL
PROCESSING OF FLORIDA RECONSTITUTED GRAPEFRUIT JUICE
















By

WENDY ANN-MARIE BELL


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


2004

































Copyright 2004

by

Wendy A. Bell

































To my Grandparents, with love.















ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my committee chair, Dr. Rouseff,

for his guidance, encouragement and support. It was a great learning experience. To my

committee members, Dr. Charlie Sims, Bruce Welt and Kevin Goodner, I would like to

extent my gratitude for their advice and assistance. I am also grateful to the Florida

Department of Citrus for providing financial support of my graduate studies.

I thank all my lab mates and Jack Smoot, for all their help. I would also like to

thank April Elston and Kristin Nelson for sniffing my samples. Thanks go to my family

for their support. To my grandmother, I would like to extend a special gratitude for her

kindness and constant support. She has always encouraged me to work hard and to put

God first in my endeavors.

Finally I would like to acknowledge my best friend, James. He was always there,

encouraging me with his kind words. I really appreciate all that he has done.
















TABLE OF CONTENTS

page

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

LIST OF TABLES ............................... ...... ...................... .. vii

LIST OF FIGURE S .................................. .. .. ...................... ............. viii

A B ST R A C T ................. .......................................................................................... x

CHAPTER

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

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

G ra p e fru it .................................................................................3
T herm al Processing .......................................................... .. ... ........ .. ........ ... 3
Possible Interactions with Stainless Steel Pasteurization Tubes .................................4
O ff-flav or ............... .................... .......................................... 5
Maillard Reaction Products .......................................................5
F u ra n s ......................................................................................... ...... . 5
Furanones .................................................. ........7
2,5 dimethyl 4-hydroxy- 3(2H) furanone (Furaneol).............. ................7
4,5 Dimethyl 3-Hydroxy- 2(5H) Furanone (Sotolone) ................................8
O their D egradation Products ............................................ .......................... 9
Instrum mental A analysis ................. .. ...... ....................9.. 9
G C T echniqu es ....................................................... 9
H PLC M methods .......... .. ......................................... .. ........ .... 10

3 MATERIALS AND METHOD ............. .... ................................. 12

R agents and Standards ............................. ..................................................... 12
Apparatus Setup ................ ......... ..... ...............12
Sam ple Preparation ......................................... .. .. .... ...... ................. 13
Sensory Analysis-Difference from Control Test ....................................... .......... 15
Instrum ental M ethods ............. .. ..... .......................... ... ............ ................17
Gas Chromatography-Flame Ionization Detector (GC-FID) ............................17
Gas Chromatography-Olfactometry (GC-O) .................... ......... ......... ...... 17
Gas Chromatography-Mass Spectrometry (GC-MS) ............... ..................18


v









HPLC Analysis of the Furans ........................... ............... 18
Sample Preparation ......... ........... ................. .............. ........ ..... 18
H PL C Instrum entation................................................ ............................. 18
Identification and Quantification................................................... 20

4 SENSORY ANALYSIS RESULTS AND DISCUSSION........................................22

Statistical Tests for Significant D differences ........................................ ....................23
Producing Heated/Cooked Flavor.. .. ........................ ...................28

5 GAS CHROMATOGRAPHIC RESULTS AND DISCUSSION............................31

G C -O .................................... ....... ............................................ 3 3
Aroma Active Compounds in Grapefruit Juice .................................. ............... 35
Aroma Active Compounds Lost during Heating..............................................39
Aroma Active Compounds That Intensified after Heating ..............................41
Caramel Aroma Compounds Formed from Sugar Degradation Reactions ...............42
Caramel Compounds Found in Grapefruit Juice...............................................45
3-M ethyl 2(5H) Furanone............... ............................ 46
Identification of Volatiles Using GC-M S .............. ........................................... 47

6 HPLC RESULTS AND DISCUSSION...... ..................... ...............53

7 C O N C L U SIO N ......... ......................................................................... ........ .. ..... .. 64

LIST OF REFEREN CE S ........................................ ........................... ............... 65

B IO G R A PH IC A L SK E TCH ..................................................................... ..................70
















LIST OF TABLES


Table page

3-1 HPLC gradient conditions with flow rate at ImL/min ................ ...............20

4-1 Difference from control sensory data from 22 panelists.......................................24

4-2 Difference from control sensory data for the second (rep 2) and third replication..25

4-3 Results of the analysis of variance for each of the three replications ...................26

4-4 LSD comparisons of the means for the three samples .................. .... ........... 28

4-5 Combination used in off-flavor duplication along with their descriptors ...............30

5-1 Preliminary identification of major grapefruit juice components based on.............33

5-2 Aroma active peaks with tentative identification ................................................36

5-3 Aroma intensities based on normalized peak heights of samples..........................38

5-4 LRI for ten sugar degradation products possessing caramel aroma.......................43

5-5 Components identified in grapefruit juice by GC-MS.........................................49

6-1 Retention time and maximum absorbance of furans standards.............................55

6-2 Concentrations of 5-HMF, furfural and Furaneol ......................................... 60
















LIST OF FIGURES


Figure page

2-1 Formation of furans by either the acidic pathway (1,2- enaminol) or by the basic
pathway (2,3-enediol)................ ......... ...... ... ...... ............

3-1 H eating apparatus s setup ............................................ ......................................... 14

3-2 Sample ballot for the grapefruit juice difference from control test using ................16

3-3 Graphical representation of the HPLC gradient conditions .................................20

5-1 Overlay of partial grapefruit juice FID chromatogram on a ZB-5 column..............32

5-2 Partial FID (top) and aromagram (bottom) overlay of grapefruit juice. .................34

5-3 Aroma intensities for the unheated reconstituted juice and glass-heated samples...37

5-4 Aroma active volatiles whose intensity diminished due to heating .......................40

5-5 Aroma active volatiles (peak numbers 8, 11, 16, 19, 29, 26, 32, & 35)...................42

5-6 Structure of 3(2H) furanones .............................................................................44

5-7 Structure of sotolone, a 5(2H) furanone............................................................. 45

5-8 Structure of the 3-methyl-2(5H) furanone ............. .................................................46

5-9 Segmented MS total ion chromatogram using ZB-5 column..............................48

5-10 Mass spectra of myrcene (top) and ao-pinene (bottom) .........................................50

5-11 Alpha-terpineol levels in the three sam ples .................................. ............... 51

5-12 Formation pathway for a-terpineol in the presence of limonene and linalool .........52

6-1 HPLC spectra of the seven standards ................................................................ 55

6-2 H om ofuraneol tautom ers........................... .... ...... ..... ................. ............... 56

6-3 Segment of HPLC chromatogram showing overlay .............................................57









6-4 Spectra of 5-HMF in standard (top) and in the grapefruit juice sample .................58

6-5 Spectra of furfural standard (top) and sample (bottom).........................................59

6-6 Linear calibration curve for 5-HMF analyzed in triplicate. ....................................60

6-7 Linear calibration curve for Furaneol at 290nm..................................................61

6-8 Linear calibration curve for furfural standard at 290nm ........................................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

EXAMINATION OF AROMA VOLATILES FORMED FROM THERMAL
PROCESSING OF FLORIDA RECONSTITUTED GRAPEFRUIT JUICE

By

Wendy Ann-Marie Bell

December 2004

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

Over fifty percent of Florida's grapefruit supply goes into juice production.

Grapefruit juice is heated after reconstitution with water to minimize spoilage

microorganisms. However, heating induces chemical changes that degrade juice flavor.

Fruit juices are traditionally heated using stainless steel surface plates or tubes. Stainless

steel contains appreciable amounts of transition metals such as nickel and chromium that

are known to catalyze chemical reactions. Acidic (pH 2.9-3.5) juice may also promote

leaching of these metals. Glass should be more inert than stainless steel and may offer a

means to produce higher quality juice. This study was undertaken to examine changes in

aroma volatiles in reconstituted grapefruit juice when heated on stainless steel and glass

surfaces.

Sensory analysis of grapefruit juices heated at 1000C for 10 minutes indicated that

both heated samples exhibited a heated, pineapple, metallic, and cooked off-flavor.

Unheated reconstituted juice had a fresh grapefruit juice character. Using data from a ten









point rating system, the "Difference from Control" test indicated that significant

differences (p<0.05) existed between the unheated juice and the two heat treatments.

Analysis of heated juices with GC-O showed appreciable reductions in aroma

intensity of compounds that are responsible for fresh grapefruit juice character.

Intensities of ca-pinene, myrcene, and P-sinensal were at least 45% lower in the heated

juices compared to the unheated juice. There was also a corresponding increase in aroma

intensity of compounds associated with flavor degradation such as 2,5-dimethyl-4-

hydroxy-3(2H) furanone (Furaneol) and methional in heated samples. Two other

Maillard reaction products, sotolone and 3-methyl-2(5H)-furanone, were detected by GC-

O. Both high processing temperatures and extended times are required to produce

cooked or heated off-flavors in grapefruit juice. Increases in 5-HMF concentration were

observed with heating, suggesting that Maillard reactions were involved.

Sensory experiments designed to induce cooked, heated off-flavor in unheated

reconstituted juice indicated that a combination of Furaneol, homofuraneol and bis (2-

methyl-3-furyl) disulfide, a thermal degradation product of thiamine (vitamin B ), could

produce a cooked off-flavor similar to that observed in the excessively heated juices.














CHAPTER 1
INTRODUCTION

Grapefruit is the second largest citrus commodity in the State of Florida. Florida

supplies over 30% of the world's grapefruit production. Over 55% of Florida grapefruit

goes into juice production while over 35% is used for fresh fruit. Grapefruit sales have

been declining in recent years due to several factors ranging from uneven flavor quality

to interactions with certain drugs. Grapefruit juice has a unique citrus flavor, but low

sugar to acid ratios and high bitterness can overwhelm the pleasant flavor aspects. Red

grapefruit is used mainly for fresh fruit or for not-from concentrate juice (NFC), while

white grapefruit juice is generally converted to "from concentrate" (FCGJ) juice.

Thermal processing alters the overall flavor of grapefruit juice but is necessary to

reduce populations of viable spoilage microorganisms, inactivate pectinesterase and,

ultimately to increase shelf life. Formation of off-flavors due to processing is highly

undesirable to consumers; therefore research is necessary to identify critical factors that

cause significant flavor alterations. Cooked and heated off-flavor was observed in both

NFC and FCOJ and had profoundly negative impacts on perceived flavor quality.

Traditional processing of grapefruit juice is achieved through the use of stainless

steel tubes or plates. Transition metals can catalyze chemical reactions known to cause

off-flavors. Glass is an inert material and should not catalyze such reactions.

Previous studies have identified several compounds that contribute negatively to

citrus juice flavor. One such class of compounds is the furans that are formed as a result

of sugar degradation. These furans impart a caramel aroma depending on the









concentration. Use of 5-HMF and furfural as markers of thermal abuse, have been

suggested by several authors. Levels of these compounds in grapefruit juice typically do

not approach their aroma threshold but as the level of these compounds increase, juice

sensory quality declines. Furaneol, 2,5-dimethyl 4 hydroxy-3(2H) furanone, imparts an

aged, pineapple like aroma to citrus juice at levels exceeding 50 [g/L. In citrus based

soft drinks, sotolone imparts a burnt spicy note at levels ranging from 1.38ng/mL to

209ng/mL.

The objectives of this study were to determine if heating grapefruit juice in contact

with glass or stainless steel resulted in distinguishable sensory flavor differences as well

determining there were differences in individual aroma volatiles. A secondary objective

was to determine what compounds were responsible for heated or cooked off flavor in

grapefruit juice.














CHAPTER 2
LITERATURE REVIEW

Grapefruit

Grapefruit is the second largest citrus commodity in the State of Florida,

accounting for over 30% of the world's grapefruit production. The entire United States

of America produces 40% of the world grapefruit production. For the 2002-2003 season,

36% of the grapefruit was used for fresh fruit, 16% for the chilled juice sector and over

40% to frozen concentrate products. With the exception of 1999-2000 season, there has

been a continuous decline in grapefruit production. Sales have been declining due to

factors ranging from poor flavor quality to medical precautions. Significant changes in

the overall flavor of processed grapefruit are of concern to the grapefruit processors as

well as the consumers.

Thermal Processing

Citrus juice generally undergoes heat treatment in order to achieve reductions in

viable spoilage organisms, to inactivate pectic enzymes, and ultimately to extend the

shelf life.

Inactivation of pectinesterase is important since it prevents the cloud separation and

associated reactions that may alter the flavor profile. Temperatures of at least 700C to

900C with hold times varying from a few seconds to 1 minute are necessary for

inactivation of most pectic enzymes. Pectin methyl esterase is a heat resistant enzyme

found in grapefruit juice and, is inactivated at temperatures exceeding 900C (1). Most

processors pasteurize not from concentrate (NFC) or chilled juice at temperatures









exceeding 900C and majority of the time juice is being heated twice. Grapefruit juice

from concentrate (FCGJ) is subjected to at least two heat treatments. The first occurs

during the evaporator concentration process where water is removed with a multi effect

evaporator. The second heat treatment occurs after the reconstitution of the juice and the

add-back of important flavor volatiles.

Possible Interactions with Stainless Steel Pasteurization Tubes

Pasteurization and concentration processes involve heating the juice in stainless

steel tubes or plates. Transition metals in food grade stainless steel tubes may catalyze

reactions throughout the heating processes. Food grade stainless steel, 316, typically has

16-18% Cr, 10-14% Ni and 2-3% Mo. Since juice contacts steel directly, leaching of

metals may be enhanced by factors such as low pH, high temperatures, and contact time.

Studies on effect of pH and temperature on chromium release from stainless steel showed

that after heating fruit juices with pH ranging from 2.5-3.0 at 950C for lhr, 31-50g/L

chromium was released (2). Residence times in tubes for NFC pasteurized juices are

typically in the range of 6-30s. The thermally accelerated short time evaporator (TASTE)

developed by Cook Machinery (Dunedin FL) is generally used to concentrate citrus

juices. This evaporator utilizes high temperature with residence times of less than one

minute (3). Although residence time is not long, high processing temperatures, combined

with the low pH may be more damaging than either factor alone.

High temperature pasteurization induces chemical changes that may affect the

quality of the juice immediately or will initiate a chain of chemical reactions that will

ultimately produce flavor degrading compounds.









Off-flavor

High heating temperatures along with prolonged storage have been shown to be

contributing factors to off-flavor development (4;5;6). Studies into heated/cooked off

flavor found in orange juice was carried out by heating orange juice from concentrate at

various time-temperature combinations and then subjecting the samples to varying

storage times. Sensory evaluation revealed that orange juice heated at 1000C for 10 min

produced a heated, cooked and over processed flavor but was not predominant. Samples

were rated either "dislike slightly" or lower by majority of the panelists (7).

Consumer testing of commercial orange juices revealed that that several had a

cooked flavor. Juices with this cooked flavor were given the lowest quality ratings. Both

chilled (NFC) and from concentrate products were evaluated (8).

Maillard Reaction Products

Off flavor formation in citrus due to thermal degradation of sugars is of high

importance. Non-enzymatic browning products may be directly or indirectly

contributory to the flavor.

Furans

In citrus juices, furans are important indicators of thermal abuse. Furfural and 5-

hydroxy methyl furfural (5-HMF) have been reported in citrus juices that have undergone

heat treatment and high storage temperatures (4;6).

Commercial canned and glass packed orange juice samples were stored at

temperatures ranging from 50C to 300C over a period of 16 weeks. Results showed that

storage temperatures above 16C increased the rate of furfural formation more rapidly

than at lower temperatures. Concentrations of furfural observed in canned orange juices

stored at 300C increased 28 times compared to juices stored at 50C. For every 5C









increase in storage temperature, the level of furfural doubled, thus demonstrating a hight

correlation between storage temperatures and furfural accumulation. Glass packed

orange juice samples showed a more rapid increase in furfural formation. In fact, a 72-

fold increase in furfural levels were noted in samples ranging from 50C 300C storage

temperatures (6).

A similar study utilizing commercially canned grapefruit juice revealed that storage

temperatures from 100C to 400C were enough to induce a 390-fold increase in furfural

levels over a 15 week storage time. Panelists were able to detect significant flavor

changes in juices in which furfural levels exceeded 0.175 ug/ml. Since its taste threshold

in orange juice was 80 [tg/mL and thus not directly flavor active, furfural can only be

used as an indicator for off flavor (9).

Amino acids can increase the rate of ascorbic acid degradation. Furfural is formed

through acid catalysis of ascorbic acid (6). Ascorbic acid is degraded more rapidly in

single strength juice than in concentrated juices (10).

Levels of 5-hydroxy methyl furfural (5-HMF) in grapefruit juice samples increased

up to 3000 times with a 400C rise in temperature over 15 week storage study (9).

Formation of 5-HMF increased slightly between 100C and 200C but rose exponentially

above 300C. Under acidic conditions, 5-HMF is formed by the 1,2 enolization and

dehydration of hexose sugars (fig.2-1). The R group in figure 2-1 is replaced by a methyl

group. It has a high threshold of 100 [tg/mL (11) and therefore is not flavor active (12).

In fact, sensory results did not correlate with 5-HMF concentration (9). Thus other

compounds must be responsible for the observed off-flavor.









H-NHR

C-OH
I undergoes series of
CHOH
HPHR / CHOH Dehydration &deamination
C -o with pentose R=H
I with hexose R= CH2 OH
CHOH 1,2-enaminol

CHOH
CHOH CHPHR H

Amdr Cundergoes series of /
Amadori C /H
Compound 11 --- --.
C-OH
S dehydration & deamination from hexose
CHOH HO

2,3-enediol
0
with pentose R=H
with hexose R= CH3
Figure 2-1 Formation of furans by either the acidic pathway (1,2- enaminol) or by the
basic pathway (2,3-enediol)

Furanones

Furanones are important flavor compounds that are found both naturally and as a

result of thermal processing in foods and beverages. Two important furanones are

Furaneol and sotolone.

2,5 dimethyl 4-hydroxy- 3(2H) furanone (Furaneol)

Furaneol gives a caramel-like, burnt pineapple aroma in high concentrations and a

sweet, strawberry like aroma at low concentrations. It is one of the major "character

impact" compounds in pineapple (13) and muscadine grapes (14). Furaneol can exist

either free or glycosidically bound in other fruits such as mango, grapes, tomato (15), and

strawberry (16;17). It is found in thermally processed foods such as beef broth (18), and

fruits such as pineapple. This furanone has been shown to mask the flavor of orange









juice, giving an aged, cooked aroma when present at 0.05 [g/mL or greater (4). In

grapefruit juice, a pineapple-like aged flavor develops with high levels of Furaneol (19).

It has a threshold of 0.03 [g/L in water (20). Orange juice samples stored at 400C had 5

times more Furaneol compared to samples stored at 40C (21). Grapefruit juice samples

stored at 500C for 15 weeks showed an increase up to 28 times that of samples stored at

200C (19). Furaneol formation in citrus juices occurs as a result of a Maillard sugar

degradation reaction.

Studies using model solutions of citrus juice showed that Furaneol is formed over a

range of pH values in the presence of rhamnose. At higher pH values Furaneol is formed

through the 2,3-enolization pathway of the Amadori compound (fig. 2-1) followed by

dehydration and molecular rearrangement (22). The amino acid arginine is also a

necessary substrate for its formation under acidic conditions. Rhamnose is a 6-deoxy

hexose sugar formed from enzymatic degradation of pectin during processing and

storage. Flavanone glycosides such as hesperidin and naringin can also degrade to form

free rhamnose (23). Acid-catalyzed fructose degradation is also a possible formation

pathway for Furaneol. In heated solutions it can decompose to smaller molecules (24) or

it can react with other groups such as thiols to produce flavorful compounds.

Like most Maillard reactions, this reaction requires only a small amount of

substrate produce potent flavors; thus; it is hard to measure the depletion of these

compounds.

4,5 Dimethyl 3-Hydroxy- 2(5H) Furanone (Sotolone)

Sotolone is a powerful aromatic compound that produces a curry-like aroma in high

concentrations, however, at low levels imparts a burnt, spicy aroma (25). Sotolone has an

aroma threshold of 0.02 [g/L in air (26) and is found in wines (25), sherry and roasted









coffee (26). Recently, it has been reported in orange essence oil (27) and was found to be

a source of off-flavor in citrus soft drinks (28) producing a spicy, burnt aroma. Its

formation was monitored in model soft drinks both with and without ascorbic acid. It

was postulated that sotolone was formed from ascorbic acid or dehydroascorbic acid in

the presence of ethanol. Oxygen and metal ions are also necessary for its formation.

Small amounts of ethanol are naturally found in citrus juices.

Other Degradation Products

Methional and 2-methyl-3-furanthiol are possible off-flavor contributors formed

from strecker and thiamine thermal degradation, respectively. Methional has a cooked

potato aroma while 2-methyl-3-furanthiol has a cooked, meaty aroma. Another thiamine

degradation product, bis (2-methyl3-furyl) disulfide, a dimer of 2-methyl-3-furanthiol

(MFT), which also produces a meaty aroma. Both MFT and its dimer have been reported

in grapefruit juice from concentrate (29). Thermal processing can also degrade 3-

carotene; producing norisoprenoids such as P-damascenone, a-ionone and P-ionone.

Instrumental Analysis

GC Techniques

Gas chromatography can separate complex mixtures of volatiles using high

resolution capillary columns. GC works on the principle that volatiles are sequentially

eluted in the general order of boiling point. Thus they are thermally desorbed from the

capillary column as the column (oven) temperature increases. Separation efficiency also

depends on polarity of both the compound and the stationary phase, which lines the

column. On a polar column such as DB-wax, polar compounds will be retained by the

stationary phase more than they would on a non-polar DB-5 column.









Gas chromatography-olfactometry (GC-O) is a powerful analytical technique that

combines an instrumental detector as well as human response. It offers the ability to

define aroma active volatiles in terms of odor and intensity (30). The human nose has an

odor detection threshold of 10-19 moles (31) compared to 10-12grams for a mass

spectrometer (32). Isolation of the compounds of interest from the food matrix is very

important (31). GCO analysis can involve flavor dilution techniques such as Charm or

aroma extraction dilution analysis. These techniques are useful in determining flavor

threshold of important aroma active compounds. Another technique, OSME or time-

intensity, is based on evaluating intensities of aroma over the course of the GC analysis

and in most cases is not as time consuming as the flavor dilution techniques. Drawbacks

to sniffing include fatigue, saturation and adaptation (31) therefore assessors should limit

repetitions that are done in a single day and also lengthen the time between sniffs.

HPLC Methods

HPLC is an excellent alternative method of analysis. It is a non-thermal

chromatographic technique, which allows the separation of both volatiles and non-

volatiles. Reversed phase chromatography has been one of the choice methods for furan

analysis in citrus products (19;21;28;33). Grapefruit juice is a complex mixture and

efficient separation of its components lies in sample preparation and chromatographic

conditions. There have been several sample preparation procedures for furan analysis in

citrus juices. Use of the Carrez clarifying reagent was found to be effective in removing

pulp, fat, protein and carotenoids that may co-elute with the furans (34). The same author

(9) employed a simplified method of centrifuging the juice to remove the pulp layer. In

both cases passing the remaining solution through a C-18 cartridge to selectively remove

the desired polar compounds (34). Previous studies used mobile phases of consisting of









acetonitrile-water (85:15), (34), acetonitrile-glacial acetic acid-water (9) to effectively

isolate 5-HMF and furfural. Methanol and sodium acetate buffer were found to

satisfactorily isolate Furaneol. Initial efforts to quantify Furaneol in grapefruit juice

using aqueous methanol (30%) or phosphate buffer with 30% methanol were

unsuccessful due to co-elution with another compound (19). A linear gradient and

mobile phase consisting of methanol, acetonitrile and pH4 acetate buffer provided good

separation of Furaneol (21). However, in citrus juices another compound will co-elute

with Furaneol. The technique of creating difference chromatograms by subtraction of

two detection wavelengths (35) has been employed to isolate Furaneol. Furaneol has

minimal absorbance at 335nm and appreciable absorbance at 292nm whereas the

interfering compound had an absorbance maximum at 335 nm. Thus, the difference

chromatogram should provide a peak which is due almost entirely to Furaneol.

Sotolone has previously been isolated using both reversed and normal phase

chromatography. Reversed phase chromatography using a mobile phase consisting of

acetonitrile and water adjusted to pH 2.5 with sulfuric acid was used to quantify sotolone

in citrus soft drinks (28). This compound was monitored at 235nm, a wavelength close to

its maximum absorbance. Normal phase chromatography has been the predominant

method of isolating or analyzing sotolone. The sotolone content in wines was determined

with a detection limit of 10g/L using a diol column and hexane/ dichloromethane as the

mobile phase (25). HPLC can also be used as a preparative step by collecting fractions of

sotolone and then using GC-MS to identify and quantify sotolone (36).














CHAPTER 3
MATERIALS AND METHOD

Reagents and Standards

Pentane, an organic solvent used in the gas chromatographic analysis was obtained

from Fisher Scientific (Pittsburgh, PA). Acros Organics (New Jersey) supplied diethyl

ether used for solvent extraction, and the methanol used in HPLC solvents and for

preparation of standards. Standards of 3-methyl-2(5H) furanone, 4-hydroxy-5-methyl-

3(2H) furanone, 2,5-dimethyl-4-hydroxy-3(2H) furanone, 2,5-dimethyl-4-methoxy-3(2H)

furanone, 2-ethyl-4-hydroxy-5-methyl-3(2H) furanone, 5-hydroxy methyl furfural, 4,5-

dimethyl-3-hydroxy-5(2H) furanone, E-2-decenal, E,E-2,4-decadienal, methional, E,Z-

2,4-decadienal, E,E-2,4-nonadienal, E,Z-2,6-nonadienal, 1-octen-3-one, maltol and 2-

methyl-3-furanthiol were obtained from Aldrich Chemical Co. (Milwaukee, WI).

Vanillin was obtained from Sigma Chemical Co (St. Louis, MO). Linalool, octanal,

decanal, nootkatone, P-sinensal, a-terpineol, myrcene, and a-pinene Sodium phosphate

was obtained from Sigma while the phosphoric acid from Fisher Scientific.

Apparatus Setup

Figure 3-1 illustrates the heating apparatus setup. An oil bath consisting of light

mineral oil (Fisher Scientific) was heated using a hot plate. A condenser with chilled (-

5C) 50:50, ethylene glycol: water, was attached to the round bottom flask to allow for

refluxing of volatiles through condensation. The mouth of the condenser was also

covered with aluminum foil. For each heating in the stainless steel and the glass

containers, temperatures of both the oil bath and the juice samples were monitored.









Sample Preparation

Mid Season White Grapefruit Concentrate was obtained from a local processor.

The concentrate was reconstituted to 10Brix using deionized water. 300ml of the

reconstituted grapefruit juice was placed in a 1000ml stainless steel round bottom flask.

This was then submerged into a pre-heated oil bath. A hold time of 10 min was

maintained once the sample reached a temperature of 100C. After heating, samples were

placed in a pre-sterilized 10oz glass flint bottle (All American Container, Tampa, FL),

and immediately cooled on ice. The heating was repeated using a glass round bottom

flask.

Sample preparation for GC analysis was carried out using liquid-liquid extraction.

20 ml of grapefruit juice were added to 10 ml of 1:1 pentane and diethyl ether for

extraction. A Mixxor-like apparatus consisting of two 50 ml syringes connected via a

stainless steel luer-lock connector was used to extract the volatiles. Extraction was

facilitated by moving syringes in a back and forth motion twenty times. The mixture was

then centrifuged at 4000 rpm for 10 minutes to break the emulsion. The solvent layer

was removed and retained, while the aqueous portion was extracted a second time. After

the second extraction, both solvent fractions were combined. The extract was dried with

sodium sulfate.








































Figure 3-1 Heating apparatus setup

A 50 [tl volume of 2000 [g/mL ethyl valerate and 2-heptadecanone were added as

internal standards. The solvent layer was concentrated to approximately 100[l using a

stream of high purity nitrogen and placed in a 100l1 limited volume insert, placed in a

1.8 mL sampling vial and sealed with a screw top lid. Each sample was refrigerated until

GC-O/GC-FID (GC) and GC-MS analysis. Each sample was analyzed four times for

GC-O analysis and twice for GC-MS.









Sensory Analysis-Difference from Control Test

Six batches of grapefruit juice were heated in stainless steel and glass containers.

The batches were then combined so as to present a uniform mixture to panelists.

Twenty-one to twenty-five untrained panelists from the Citrus Research and

Education Center were recruited for the study. A control and three samples, each coded

with a randomly selected 3-digit number were presented to the panelists. Order of

presentation randomized for each panelists based on six orders of presentation (ABC,

ACB, BCA, BAC, CBA, BAC). Panelists were then asked to taste the control then to

taste the sample and to rate the degree of difference between them. They were also told

that a hidden control was present among the samples. A sample ballot is shown in figure

3-2. The control was unheated reconstituted grapefruit juice.

Panelists were also asked to cleanse their palettes by eating a piece of unsalted

cracker and drinking a sip of water between samples to prevent any carryover from the

previous sample. A numerical ten-point scale similar to the hedonic scale was used, with

scale ranging from no difference to extreme difference. Color changes during the heating

process prompted the use of red lighting so as to mask the color difference. The above

procedure was repeated two additional times yielding a total of the three sensory

evaluation sessions.






16


DIFFERENCE FROM CONTROL TEST

Name: Date:

Today you will be tasting grapefruit juice.
Please eat a piece of cracker and drink a sip of water in between each sample to
cleanse your palette.

Three samples and one control are presented to you. Please circle the degree of
difference against the control for each sample.

It is possible that there may be no difference between the control and one of the
samples.

Control vs. # Control vs. # Control vs. #
0 =No difference 0 =No difference 0 =No difference
1= slight difference 1= slight difference 1= slight difference
2 2 2
3 = mod difference 3 = mod difference 3 = mod difference
4 4 4
5 = large difference 5 = large difference 5 = large difference
6 6 6
7 = very large difference 7 = very large difference 7 = very large difference
8 8 8
9 = extreme difference 9 = extreme difference 9 = extreme difference

Comments


Figure 3-2 Sample ballot for the grapefruit juice difference from control test using a ten-
point rating scale









Instrumental Methods

Gas Chromatography-Flame Ionization Detector (GC-FID)

Volatiles from grapefruit juice extracts were separated using an HP 5890 gas

chromatograph (Hewlett Packard, Palo Alto, Ca, USA) equipped with a flame ionization

detector. One-half micro liter sample was injected onto a ZB-5 or DB-Wax capillary

column (30m x 0.32mm I.D., 0.5Oim film thickness) (J&W Scientific, Folsom, CA).

Oven temperature was programmed with an initial temperature of 400C and ramped up to

2400C for wax column and 2650C for ZB-5 column, at 70/min with a final hold time of 5

minutes. Samples were injected using splitless mode with an injector port temperature of

2200C and the detector at 2500C. The effluent was split 1:2 between a flame ionization

detector and a sniff port (Datu, Geneva, NY). Data were collected and integrated using

the ChromPerfect v. 5.0software (Justice Innovations, Denville, NJ).

Gas Chromatography-Olfactometry (GC-O)

A sniff port was attached to the GC in order to facilitate the identification of aroma

active volatiles. The GC temperature program was the same as that for the GC-FID.

Two assessors were used to evaluate the aroma quality of the samples. Each

assessor analyzed the samples in duplicate accounting for four sniffs per sample. Only

those odors that were detected in 50% of the sniffs were considered to be aroma active.

Sniffing began after the elution of the solvent peak, approximately three minutes into

each run. A sliding scale ranging from none to extreme was used to indicate the aroma

intensity. Assessor's responses were captured using a variable potentiometer, which was

collected on the ChromPerfect software. Sniffing time for each run was 25 and 29

minutes for the ZB-5 and DB-wax column respectively. Aroma quality of each sample

was recorded.









Gas Chromatography-Mass Spectrometry (GC-MS)

Data were collected using a Finnigan GCQ Plus GC-MS system (Thermo Electron,

San Jose, CA) using a 99.999% pure helium as the carrier gas. A DB-5 capillary column

(60m x 0.25 mm id, 0.25[im film thickness column (J&W Scientific) was used. Oven

temperature was programmed from 400C to 2500C at a rate of 70C/min. Samples were

injected using split less mode with an injector port temperature of 2200C. Electron

impact (EI) mode was used with ionization energy of 70eV. Scans were obtained after 7

minutes to avoid the solvent peak and were measured from 40-300 m/z. Data were

analyzed with XCalibur v. 1.3 software (Thermo Electron, San Jose, CA).

HPLC Analysis of the Furans

Sample Preparation

Solid Phase Extraction using a method modified by Walsh was employed (21).

10ml of grapefruit juice were centrifuged for 5 min at 3000 rpm. Three milliliters

supernatant were passed thru a C-18 cartridge (J & W Scientific, SPE Accubond

500mg/6ml, Folsom, CA) that had been pre-conditioned with 2.5 ml methanol and

washed with 6 ml water. The cartridge was then washed with 2 ml water to remove the

sugars and eluted with 2 ml methanol at a rate of 1 drop per 10s. Samples were filtered

thru a 0.45[i nylon filter (Fisher Scientific) and stored in a 1.8 ml amber vial until

injected into the HPLC.

HPLC Instrumentation

Separation was achieved by injecting 20 [tl of the juice extract onto a Phenomenex

5[ C-18 column (4.6mm id x 250mm) (Phenomenex, Torrance, CA). A 10ul injection

volume was used for standards. Column temperature was kept at 250C.

Chromatographic analysis was carried out using Surveyor HPLC equipped with a PDA









detector. Versatility of the PDA detector enabled the monitoring of three individual

wavelengths: 235nm, 290nm and 335nm, while obtaining a spectral scan 220-380nm.

Data was collected and analyzed using Atlas software v.1.0 at an acquisition rate of 10Hz

(Thermo Electron) over a 40 minute run time.

The mobile phase consisted of methanol and phosphate buffer. Phosphate buffer

(pH4.1 +1) was made using a 0.05M sodium phosphate solution and phosphoric acid.

Linear gradient conditions are summarized in table 3-1 while figure 3-3 shows the

graphical gradient conditions. A flow rate of Iml/min was maintained and a re-

equilibration time of 10 min was applied.









Table 3-1 HPLC gradient conditions with flow rate at ImL/min
Time % Methanol % phosphate
0 5 95
10 15 85
27 30 70
33 40 60
40 5 95
50 5 95


100

S80-

5. 60
0

S40

S20-

0
'0 0


20 30
Time (min)


Figure 3-3 Graphical representation of the HPLC gradient conditions

Identification and Quantification

Identification of aroma active components was achieved by comparing linear

retention indices and aroma descriptors with those from a database or published values.

Confirmation was achieved by matching fragmentation pattern of the sample with that of

standards or library. A series of alkanes (C5-C25) analyzed under the same GC conditions


- -%methanol buffer


--0 S









were used to calculate linear retention indices (37). A scatter plot of retention time along

with retention indices of alkane standards were graphed on Excel. Retention indices for

the standards were obtained by multiplying the carbon number by a factor 100. A

polynomial was obtained from a regression. This equation was then used to calculate

linear retention indices for individual components in grapefruit juice. Alkane standards

were analyzed for both DB-wax and ZB-5 columns, as well as for GC-MS.

Components from HPLC analysis were identified using a combination of

comparing retention times and spectra of standards with that of sample. Quantification

was done using external standard method. Sets of standards with known concentrations

were analyzed in triplicate. Areas of those standards were plotted against the amounts

and a linear regression was obtained along with a linear equation. Responses from

unknown components were then substituted in the linear equation in order to find the

amounts and ultimately the concentration of components in juice samples.














CHAPTER 4
SENSORY ANALYSIS RESULTS AND DISCUSSION

There are several sensory analysis tools that are used in difference testing of foods.

"Difference from Control" test was used in this study as it enabled the evaluation of three

treatments in one sitting. There are two objectives of the difference test. First, it

determines if a difference exists between samples and the control. Secondly, it estimates

the magnitude of the difference (38).

The three samples that were evaluated were

Unheated unheated reconstituted grapefruit juice (as well as hidden control)

Glass FCGJ heated 10 min. on glass surface

Metal FCGJ heated 10 min. on stainless steel surface

The sensory evaluation data for each replicate are shown in tables 4-1 and 4-2. Except

for one (panelist #12, table 4-2 rep 3), all panelists were able to identify the hidden

control, as having little or no difference from the control. Although overall scores (table

4-2 rep3) ranged from no difference (0) to very large or extreme (8) difference, only 68%

of the panelists were able to distinguish differences between the control and the juice

heated in metal. A difference score of three and greater was considered to show

difference, difference scores of less than three was not considered to be different from the

control. Scores for juice heated in glass ranged from moderate difference to very large

difference (7) for over 86% of the panelists. Seven of the twenty-two panelists rated the

juice heated in metal with a score of two or less compared to only three for the juice

heated in glass. Perhaps, these panelists could not distinguish the differences due to their









low bitterness threshold and hence high sensitivity. This sensitivity incapacitates

panelists from being able to distinguish flavor differences among samples. An example

of this is panelist number 15 (table 4-1 rep 1) and panelist # 4 9table 4-2 rep 2) who was

unable to detect much difference and commented that all three samples had high

bitterness levels. Also they could be anosmic or unable to detect heated off-flavors.

Although panelists' scores improved for the second and third evaluations, some panelists

were still unable to detect flavor differences in the juices. Average difference score for

the unheated reconstituted juice ranged from 1.96 in rep 1 to 0.45 in rep 3 suggesting an

improvement in identifying the hidden control. Most panelists had difficulty detecting

differences between the two heated samples e.g. panelists #1, 2, 5, 6, 8 & 10 from table

4-1 reply indicated at most, a one-point difference between the glass and metal samples.

Although there may have been a difference in overall flavor between the two heated

sample types, the severe heating produced such a strong off flavor that it was difficult for

panelists judge the degree of difference. Both samples had very strong heated/ cooked

off-flavors.

Statistical Tests for Significant Differences

Statistical differences between samples were determined by using a two-way analysis of

variance (ANOVA) test, at the 95% confidence level, as it allows for the comparisons of

more than two samples (table 4-3). The advantage of using a two-way ANOVA is that

any error due to the panelists can be distinguished.










Table 4-1 Difference from control sensory data from 22 panelists between a control and
three samples, a hidden control (unheated reconstituted), grapefruit juice
heated in glass, and juice heated in metal using a 10-point scale.
Rep 1
Unheated
Panelists reconstituted Glass Metal

1 1 5 5
2 0 6 5
3 4 5 1
4 0 5 3
5 5 8 7
6 2 6 5
7 2 3 5
8 1 6 7
9 0 3 3
10 0 5 4
11 0 3 5
12 1 0 3
13 4 6 4
14 1 2 5
15 0 0 0
16 4 3 2
17 3 5 3
18 4 7 2
19 3 5 1
20 2 2 4
21 2 5 7
22 1 7 7
23 5 7 7
24 3 9 1
25 1 1 3
Average 1.96 4.56 3.96
Median 2 5 4
St Dev 1.62 2.46 2.09
Min 0 0 0
Max 5 9 7










Table 4-2 Difference from control sensory data for the second (rep 2) and third
replication (rep 3).
Rep 2 Rep 3
Unheated Unheated
Panelist No. reconstituted Glass Metal reconstituted Glass Metal
1 0 5 3 0 5 5
2 4 9 6 0 7 3
3 1 3 1 0 3 1
4 1 0 0 1 1 3
5 1 4 6 0 3 3
6 0 3 5 1 6 0
7 1 3 3 1 3 1
8 0 5 8 0 4 5
9 0 7 5 1 4 7
10 1 3 5 0 1 2
11 3 5 7 1 7 7
12 2 1 3 2 7 8
13 1 3 3 1 5 8
14 1 6 5 0 3 4
15 0 5 5 1 5 5
16 0 5 3 0 5 1
17 1 3 0 0 5 6
18 1 3 1 0 5 0
19 1 0 5 0 7 3
20 3 5 1 0 5 5
21 3 3 3 0 3 1
22 1 1 3
Average 1.19 3.86 3.71 0.45 4.32 3.68
Median 1 3 3 0 5 3
St Dev 1.14 2.10 2.21 0.58 1.87 2.46
Min 0 0 0 0 1 0
Max 4 9 8 2 7 8










Table 4-3 Results of the analysis of variance for each of the three replications. Repl-
table 4-1, reps 2 and 3 data are shown in table 4-2.
Repl


The F test is a statistical technique that was utilized to compare multiple means and

to test for variability among the means for the three samples (treatments) against the

variability of the observations (error). In this study the null hypothesis assumed that the

treatment means were the same, however, if the mean for the treatments were different

then the null hypothesis would be rejected. The maximum ratio between variations

among the treatments was measured using the Fcrit value. If the calculated F value were

greater than the Fcrit then there would be significant variations, which would lead to

differences among the sample means.


Source of Variation SS df MS F P-value F cnt
Panelists 158.08 24 6.59 2.08 0.015 1.75
Treatments 92.67 2 46.33 14.63 1.09E-05 3.19
Error 152 48 3.17


Total 402.747 74


Rep2
Source of Variation SS df MS F P-value F crit
Panelists 107.94 20 5.40 1.89 0.043 1.84
Treatments 94.51 2 47.25 16.56 5.77E-06 3.23
Error 114.16 40 2.85


Total 316.603 62


Rep3
Source of Variation SS df MS F P-value F crit
Panelists 105.15 21 5.01 1.88 0.040 1.81
Treatments 188.82 2 94.41 35.45 9.58E-10 3.22
Error 111.85 42 2.66


Total 405.82 65









From table 4-3 it can be seen that the Fcacl value is greater than the Fcrit, in all three

replicates, therefore we reject the null hypothesis and conclude that at least one of the

treatments means is different. Critical F values were approximately 3 compared to

calculated F values of 15, 17 and 35 respectively for reps 1, 2, & 3.

Since the analysis of variance test only suggests that a difference exist among the

populations, a multiple comparison test was used to rank the means and identify the

means that were different. Fisher LSD multiple comparison test was applied to the

means because it is the least conservative test in comparison to the Tukey and Duncans

test and should produce the most difference. The LSD test is defined as any observed

difference necessary to declare the corresponding population means different"(39). The

following equation was used to calculate the LSD:

LSD = t,/2~(2*EMS)/n

where t /2 was found at the degree of freedom of the error and an alpha level of 0.025

from a t-table, EMS, error mean square was taken from the ANOVA results and n was

the number of panelists.

Means for each population were subtracted from each other. Those with a difference

greater than the LSD value, were considered to be significantly different, while those that

were similar or less than the LSD value were not significantly different. From the data in

table 4-4 rep3, the difference between the glass and the metal was only 0.64 while the

difference between the unheated reconstituted juice and glass was 3.87. The calculated

LSD value was 0.99.









Table 4-4 LSD comparisons of the means for the three samples
Unheated Glass Metal LSD

reconstituted

Means Rep 1 1.96a 4.56b 3.96b 1.02

Means Rep 2 1.19a 3.86b 3.71b 0.50

Means Rep 3 0.45a 4.32b 3.68b 0.99


Since the difference between the unheated reconstituted juice and two heat treatments in

all three replications were greater than LSD values, it can be concluded that a significant

difference existed between unheated reconstituted and heated samples. However the

difference of means for two heated samples were less than the LSD value, therefore they

were not statistically different.

Cooked, heated, processed, pineapple flavor were some comments recorded by

panelists for heat-treated samples while the unheated reconstituted sample was identified

as having good grapefruit flavor. Panelists who had difficulty recognizing differences

between the control and two heat-treated samples recorded comments such a high

bitterness, acidity.

Producing Heated/Cooked Flavor

Attempts to reproduce the heated cooked off-flavor were carried out using three

furans known to produce a caramel aroma, Furaneol, sotolone, homofuraneol. In

addition, bis (2-methyl-3-furyl) disulfide, a thiamine degradation product sometimes

found in thermally abused citrus juices, was also added. Table 4-5 shows the

combination of the compounds used as well as their sensorial characteristics when added

to grapefruit juice based on a 6-member panel. It has been previously reported that









concentration greater than 0.05 [g/mL Furaneol masked the flavor of grapefruit juice.

This was seen in the combinations that had 1 lg/mL Furaneol. Little or no grapefruit

juice character detected by the panelists. However, in the combination containing 0.5

[g/mL, a slight grapefruit juice character was noticed, but caramel and sweet flavor

impression were more prominent. The combination of homofuraneol (1 [g/mL ),

Furaneol (1 lg/mL) and bis (2-methyl3-furyl) disulfide (2[g/mL) produced a cooked

off-flavor.

Extremely low concentrations of sotolone (0.01 g/mL ) also contributed to over-

processed flavor. Sotolone is a very potent aroma active compound and would not

require a high concentration to produce a pronounced off-flavor.









Table 4-5 Combination used in off-flavor duplication along with their descriptors
Combination
No. Concentration Flavor Descriptor
1 Cg/mL homofuraneol + Sl cooked fl, caramel
1 1 Cg/mL Furaneol + 1 Cg/L aroma, no gfj character,
MFT-MFT sweet, less acid
1 Lg/mL homofuraneol More cooked/caramel
2 +1 g/mL Furaneol + 2Lg/mL aroma, less caramel aroma,
MFT-MFT less tart, sl gfj character
0.5Lg/mL Furaneol + 1 tg/mL Less cooked aroma, more
Less cooked aroma, more
3 homofuraneol +2pg/L MFT-
Sh r l gfj character, more sweet
MFT
+ Spicy, strong caramel,
4 0.5Lg/mL sotolone +
combination #1 spoiled juice, no gfj
combination #1 character
character

5 0.01 Lg/mL sotolone Processed, spicy aroma














CHAPTER 5
GAS CHROMATOGRAPHIC RESULTS AND DISCUSSION

Gas Chromatography is a useful method of separating volatiles based on several

factors including polarity and boiling point. In this study, juice volatiles were separated

using high-resolution capillary gas chromatography. The use of at least two dissimilar

chromatographic phases is advantageous in that compounds that are not well separated

on a polar phase column can often be better separated on a non-polar column and the

reverse. Polar compounds tend to remain on a polar column longer and thus would be

better separated. Since GC is a separation technique, these components cannot be

identified without additional information. From the overlay of the FID chromatograms

(fig. 5-1) obtained from the three sample types, it was seen that some peaks were

diminished due to heating while others increased.

Peaks I, II and III showed a higher FID response for the unheated reconstituted

juice compared to that of the two heated treatments. In contrast, peak VII had a higher

response in the two unheated reconstituted samples. Table 5-1 lists some of the

volatiles that were tentatively identified using the linear retention indices on two

columns.






























1)
e-
OIII
0



LL










6 8 10 12 14 16 18
Time (min)
Figure 5-1 Overlay of partial grapefruit juice FID chromatogram
glass, C-juice heated in metal.


20 22 24 26 28


on a ZB-5 column. A-unheated reconstituted juice, B-juice heated in









Table 5-1 Preliminary identification of major grapefruit juice components based on
standardized retention times using FID responses from both polar (wax) and
non-polar (ZB-5) columns.
Peak RT LRI Lit LRI Preliminary LRI (wax) Lit LRI
(min) (ZB-5) (ZB-5) Identification (wax)

i 4.61 847 846 Ethyl 2-methyl 1022 1041
ii 6.39 939 939 a-pinene 978 1011
iii 7.49 989 989 myrcene 1172 1168
iv 8.5 1032 1025 limonene 1214 1205
v 9.82 1087 1074 cis-linalool oxide 1480 1448
vi 10.16 1100 1100 linalool 1552 1552
vii 12.37 1192 1207 a-terpineol 1713 1713
viii 14.75 1315 1376 a-copaene 1480 1472
ix 16.97 1432 1418 P-caryophyllene 1644 1624
x 21.38 1677 1706 p-sinensal 2254 2251
xi 23.81 1834 1814 nootkatone 2587 2595


GC-O

Since the invention of GC-O as a tool to evaluate flavor volatiles, it has been

adapted by the food, environmental, and flavor industry as a major tool used to identify

those compounds that are aroma active. The selectivity and sensitivity of the human

nose combined with the resolving power of the GC provides useful information about

those volatiles that produce any aroma sensation. This is a particularly useful tool for

those potent aroma compounds that have extremely low thresholds. It can be seen that

little or no FID peaks were detected for the two peaks, 1-octen-3-one and 1-octen-3-ol,

in the region of 7-8 minutes (fig. 5-2). Their analytical concentrations were below

detection levels of the FID detector, yet their concentrations exceeded their aroma

thresholds so they were aroma active.














) .limonene

rC
0


S5 linalool oxide


a ophyllene










6 8





Figure 5-2 Partial FID (top) and aromagram (bottom) overlay of grapefruit juice analyzed on a ZB-5 column.









Similarly, not all volatiles found in relatively high concentrations or large FID

peaks are aroma active. Large FID peaks such as limonene and P-caryophyllene shown

in fig. 5-2 had little to no aroma activity. In the region between 24-28 min there were

several prominent FID peaks that did not produce detectable aroma activity.

Aroma Active Compounds in Grapefruit Juice

GC-O is particularly useful in explaining sensory data. However, not everyone

has the same sensitivity to all aroma compounds and there will be variability between

individuals in assessing these aromas. To compensate for individual sensory variability,

each sample was sniffed at least four times, twice per assessor. A compound was

considered aroma active only if it was detected in 50% of the sniff runs. By using two

panelists, effects of individual hypersensitivities and specific anosmias are minimized.

If one assessor is anosmic to a certain class of compounds and the other isn't, then there

is a better chance of detecting these compounds in the averaged assessor results.

GC-FID and GC-O were carried out on two dissimilar columns. The advantage to

this method is that volatiles are eluted in a different order with each column type.

Tentative identification is based upon cross-referencing calculated retention indices

with those from a library, published results or with standards.

Over forty aroma active grapefruit juice volatiles were detected using both wax

and ZB-5 columns. Shown in table 5-2 are twenty-six aroma active compounds that

were identified by matching retention indices along with aroma descriptors of

components on both columns. Standards were run under similar conditions to confirm

retention values and odorant descriptors.










Aroma intensities were normalized for each assessor to minimize differences

between assessor uses of the intensity scale. Aroma intensities detected on wax column

were normalized based on vanillin, which had the highest peak height among all the

volatiles (see table 5-3 and fig. 5-3).

Table 5-2 Aroma active peaks with tentative identification based on aroma attributes
and retention characteristics for grapefruit juice samples as detected by GC-


O on polar and non-polar columns.
ZB-5 Component Name
939 a-pinene
800 ethyl butyrate
847 ethyl-2-methyl butyrate
989 myrcene
1032 limonene impurity
1003 octanal
978 1-octen-3-one
943 4-mercapto-4-methyl-pentanone
906 methional
1206 decanal
1095 Z-2-nonenal
1544 Z-4-decenal
1103 linalool
1194 E,Z-2,6-nonadienal
820 butanoic acid
1232 citronellol
1321 E,E-2,4-decadienal
1393 P-damascenone
1264 geraniol
1383 4,5-epoxy-(E)-2-decenal
1062 Furaneol
1325 4-vinyl guaiacol
1677 3 -sinensal
1470 wine lactone
1834 nootkatone
1418 vanillin


Descriptor
piney
Sweet, fruity
fruity
green, earthy, metallic
minty, licorice
green, fresh, lemony
mushroom
cat litter
Cooked potato
green, fatty, lemony
metallic, green
fresh, floral, fatty
floral, orange, greenish
faint cucumber, green
skunky, rotten, fresh
sweet, roasted
fatty, sl orange
tobacco(faint), piney
rose, fresh, lemon pledge
metallic, cooked, burnt
caramel
spice
grapefruit, citrus
nutty, musty
tropical, sour
vanillin


DB-Wax
978
1001
1022
1172
1214
1306
1315
1390
1470
1509
1514
1544
1552
1596
1633
1773
1828
1840
1861
2017
2043
2201
2254
2259
2587
2608







GC-O Aromagram


Unheated


1 4
5


0










-10
-Ic


7 8 28 30
7 |18 22 31 37


S1 16 41
I || 4


14 17 19
Retention Time (min)


22 24


Figure 5-3 Aroma intensities for the unheated reconstituted juice and glass-heated samples analyzed on wax column. Intensities were
normalized peak height based on vanillin, which had the highest peak height. Peak numbers correspond to those listed in
table 3.


Glass










Table 5-3 Aroma intensities based on normalized peak heights of samples analyzed
using wax column. Descriptors are listed in ascending order of LRI.
Normalized Aroma Intensities
Component Calculated Unheated
No LRI Descriptors reconstituted Glass Metal
1 978 piney 6.06 2.91 2.36
2 1001 sweet, fruity 2.24
3 1022 fruity 4.94 5.22 6.88
4 1098 sulfur, stale 6.26 5.81
5 1172 green, earthy, metallic 6.09 1.88 0.83
6 1214 minty, licorice 5.45 6.52 3.83
7 1306 green, fresh, lemony 3.91 5.84 5.38
8 1315 mushroom 2.70 5.74 5.65
9 1381 green, grassy, piney 6.85 8.66 8.40
10 1390 cat litter 6.11
11 1470 cooked potato 0.65 9.01 8.12
12 1509 green, fatty, lemony 6.98 5.58 7.17
13 1514 metallic, green 2.53 6.91 2.81
14 1544 fresh, floral, fatty 4.24 3.41 4.06
15 1552 floral, orange, greenish 8.37 9.32 4.09
16 1596 faint cucumber, green, sweet 2.06 4.30 3.59
17 1633 skunky, rotten, fresh 3.72 3.31 3.06
18 1773 sweet, roasted 3.43
19 1828 fatty, sl orange 3.27 7.95 6.55
20 1840 tobacco(faint), piney 4.37 2.87 1.53
21 1861 rose, fresh, lemon pledge 2.88 7.52 6.30
22 1868 bug spray,spice 3.64 4.36 5.92
23 1875 caramel, sweet, fresh 3.34 3.57 3.90
24 1891 fresh cucumber,floral, fresh 3.18 2.36
25 1940 metallic 3.16
26 2000 burnt, spicy 5.85 2.72
27 2017 metallic,cooked, burnt 3.95 3.16 3.30
28 2038 spice 3.92
29 2043 caramel 4.35 8.00 8.61
30 2059 spicy(strong), curry 3.93 5.48
31 2154 sweet caramel 3.90 1.48
32 2163 sweet, caramel 7.35 7.84
33 2179 charred, sweet 5.07
34 2188 grapefruit, spice(lingering) 3.14 7.94 5.82
35 2201 spice 4.68 5.11 8.78
36 2207 sweet, burnt wood 2.85
37 2243 rancid fruit 3.27
38 2254 grapefruit, citrus 7.55 4.37 4.87
39 2259 nutty, musty, crayons 1.05
40 2298 grapefruit 5.86 2.37
41 2587 tropical, sour 5.20 2.89
42 2608 vanillin 9.73 4.26 10.00









Aroma Active Compounds Lost during Heating

Many of the components listed in table 2 have been previously reported in fresh

grapefruit juice (40). However, their concentrations have probably been altered due to

the heating process. During the evaporation process when making concentrate, most

volatiles are lost along with water. Although it has been reported that over 95% of the

volatiles in the fresh juice are lost in the process of concentrating grapefruit juice, over

57% of the total aroma activity remains (29). Typically, fresh juice flavor

characteristics are restored to the concentrated juice by the addition of juice essence oil,

aqueous essence and peel oil. The juice concentrate used in this study had 0.012% cold

pressed grapefruit oil added to it. This oil was the source for most of the volatiles and

many aroma active volatiles. Since essence oil and aqueous essence are obtained from

the condensate of the evaporation process, they will contain volatile heat reaction

products and thus were not used for this study. Therefore, any heated, cooked, caramel

aromas were produced during the specific heating conditions in this study. Normalized

intensities of aroma active volatiles (table 5-3) present in both the unheated

reconstituted juice as well as the two heat-treated juice samples were compared in order

to identify aroma differences due to heat treatment and contact surface.

Three aroma active compounds, c-pinene (1), myrcene (5) and P-sinensal (38),

exhibited greater aroma intensities in the unheated reconstituted juice compared to that

of the two heated juice samples (fig. 5-4). Another aroma active compound, ethyl

butyrate (2), was detected only in the unheated reconstituted juice with a normalized

intensity of 2.24 on a 10-pt scale. Ethyl butyrate imparts a sweet, fruity aroma and is

considered to be a positive contributor to grapefruit juice aroma (40). It is a low






40


molecular weight compound and is highly volatile, thus it likely to be degraded during

extended heating times.

10





C 1







Retention Time (min)

Figure 5-4 Aroma active volatiles whose intensity diminished due to heating

As seen in table 5-3, alpha-pinene (1) and myrcene (5) are two early eluting, low

boiling point compounds, which had higher aroma intensities in the unheated

reconstituted juice compared to the two heated juice treatments. In both heated

samples, the response for a-pinene was less than 45% that of the unheated reconstituted

juice while that for myrcene was less than one-third of the intensity in the unheated

reconstituted juice. A piney aroma was elicited by a-pinene while myrcene imparted a

green, metallic, earthy aroma. Both these compounds are positively correlated with

citrus juice flavor (41). They are responsible for the green notes in citrus juices.

Beta sinensal (38) is a rather high boiling point volatile, which was reduced

through heating. The aroma intensity of this aliphatic aldehyde was 42% higher in the

unheated reconstituted juice compared to that of the juice heated in glass and 36%

higher than the juice heated in metal. Beta sinensal possesses a grapefruity, citrus
(0 } iiiiiiiiiii,





0- --- In __





























higher than the juice heated in metal. Beta sinensal possesses a grapefruity, citrusy









aroma and is one of the important compounds responsible for the fresh grapefruit juice

flavor.

Aroma Active Compounds That Intensified after Heating

Eight aroma active compounds (1-octen-3-one (8), methional (11), (E,Z)-2,4-

nonadienal (16), (E,E)-2,4-decadienal (19), Furaneol (29), and 4-vinyl guiaicol (35) and

two unknowns (26 & 32) were present in intensities greater than 50% in comparison to

the unheated reconstituted juice (fig 5-5). Both Furaneol (29)(discussed later) and

methional (11) are thermally generated. Furaneol is formed from the reaction of

rhamnose and an amino acid in the presence of heat. Methional is believed to be a

Strecker degradation product of methionine in the presence of ascorbic acid. Its

normalized intensity in the two heated samples was 8 and 10 compared to 0.65 in the

unheated reconstituted juice. (E,Z)-2, 6-nonadienal (16) and (E,E)-2,4-decadienal (19)

are unsaturated aldehydes and are likely to be formed from fatty acid degradation (42).

Both of these aldehydes are essential flavor components of citrus oils. A faint

cucumber, green aroma was detected from (E,Z) 2,6 nonadienal while the aroma of

(E,E)-2,4-decadienal was described as fatty with a slight orange character. The two

unknown compounds (26 & 32) have a spicy and sweet, caramel aroma respectively.

Neither of the two unknowns was detected in the unheated reconstituted juice.

However the intensity of unknown # 26 in the juice heated in glass was 54% higher than

the juice heated in the metal. Unknown # 32 had similar aroma intensity in both the

heated samples. Their linear retention index values on wax were 2000 and 2163

respectively. Maltol, a Maillard reaction product elutes at an LRI close to 2000

possesses a caramel aroma and could be a possible identity. Identification of this

compound would be satisfied through the use of GC-MS and by matching the spectra of










the compound alongside that of the pure standard. However, maltol does not have a

unique mass spectrum and may be present in concentrations lower than the detection

threshold of the GC-FID or GC-MS, but not of the human nose.

10




SGI




I ,


-










Retention Time (min)

Figure 5-5 Aroma active volatiles (peak numbers 8, 11, 16, 19, 29, 26, 32, & 35) whose
intensity increased after heat treatment was applied.

Caramel Aroma Compounds Formed from Sugar Degradation Reactions

From table 5-3, it can be noted that caramel burnt aroma was detected several

times throughout the sniff run. In some cases the volatiles lingered for up to 20 sec. In

order to identify these compounds, several standards that are known to impart caramel

aromas were analyzed under similar conditions on both the wax and ZB-5 columns.

Table 5-4 lists the furans and a pyranone standard that were examined to identify the

unknown compounds responsible for the caramel aroma.










Table 5-4 LRI for ten sugar degradation products possessing caramel aroma analyzed
by GC on both polar (wax) and non-polar (ZB-5) columns.

Compounds LRI (ZB-5) Present (ZB-5) Present (Wax) LRI (DB-Wax)
Furfural 850 + 1215

5-methyl furfural 967 1560

3-methyl- 2(5H) furanone1 989 + + 2153

Furaneol1 1064 + + 2096

Norfuraneol 1054 2124

Mesifurane 1064 1602

Maltol 1114 -- 2002

Sotolone' 1118 + 2203

Homofuraneol 1145 2045

5-HMF' 1236 + 25261

1 Identified in all three samples

2 found in unheated reconstituted only

All the furans listed are oxygenated. Furaneol, mesifurane and norfuraneol elute in

close proximity on the ZB-5 column. Both Furaneol and mesifurane had identical

retention indexes values (1064) while norfuraneol had a linear retention index value of

1054. The ease of volatilization of a compound is one of several factors influencing GC

separation. Most of the furans are structurally similar and, would be expected to

volatilize easily. Also, column chemistry plays a key role in separation. Polar

compounds elute more quickly on a non-polar column but will be better retained on a

polar column. The rule "like attracts like" applies. On the ZB-5 column it was seen

that the 3(2H) furanones elute in close proximity to each other e.g. Furaneol and

mesifurane eluted at 1064, and norfuraneol eluted at 1054. The structures of these

compounds are shown below (fig. 5-6).











R3 0 R1



R2 0

3(2H) furanone


H3 O CH3



HO 0


Furaneol
2,5 dimethyl-4-hydroxy-3(2H) furanone


H3C O CH3



MeO 0
Mesifurane
4-methoxy-2,5- dimethyl-3(2H) furanone


H3C O CH3CH2



HO 0
Homofuraneol
2 ethyl 4-hydroxy 5 methyl-3(2H) furanone


H3C 0



HO O
Norfuraneol
4 hydroxy-5-methyl-3(2H) furanone


Figure 5-6 Structure of 3(2H) furanones

Homofuraneol has an ethyl group as one of the side groups and this additional

increase in molecular weight and boiling point is probably the reason for its later elution

compared to the other furanones.

Sotolone (fig.5-7), a 2(5H) furanone, also has a molecular weight of 128 and

combined with the different position of the R groups could be responsible for the

decrease in its volatility.









H3C 0 0



H3C OH

Sotolone
Figure 5-7 Structure of sotolone, a 5(2H) furanone

The use of a dissimilar column such as the wax column is an excellent choice in

situations like these, as the order of elution will be different. By applying the "like

attracts like" principle, polar compounds would be retained for a longer time on the

polar wax column.

Caramel Compounds Found in Grapefruit Juice

Furaneol and sotolone were detected by GC-O while 5-HMF was detected by GC-

FID. Furaneol has previously been reported in grapefruit juice and has been known to

mask the flavor of orange juice (4;11). Its intensity was over 50% higher in the two

heat treated samples as opposed to the unheated reconstituted juice (table 5-3).

Furaneol is formed from rhamnose through the 2,3-enolization pathway of the Amadori

compound followed by dehydration and molecular rearrangement (22). The amino acid

arginine has been shown to be a necessary substrate for its formation under acidic

conditions. Sotolone has recently been reported as an off-flavor to citrus sodas

containing ethanol and ascorbic acid (28). Sotolone imparted a caramel, cooked flavor

to grapefruit juice. The 5-HMF has been reported in citrus juices and has been

suggested as a marker for thermal abuse. This compound is formed from the 1,2-

enolization pathway and can be polymerized to form colored pigments or other flavor

compounds. Unlike the other furanones, 5-HMF has a high threshold and consequently

does not have a direct aroma impact. However, it produced substantial FID response.









Furfural was detected by GC-MS. It has a unique fragmentation pattern with the major

ion found at mass 95, indicating the cleavage of a hydrogen atom. By using this

knowledge as well as selecting mass peaks 96 and 95, the spectra was deduced and then

matched with that of the standard at the expected retention time.

3-Methyl 2(5H) Furanone

A maltol degradation product, 3-methyl-2(5H) furanone (fig. 5-8), (43) not

previously reported in grapefruit juice was tentatively identified on both polar and non-

polar columns. Its LRI on a the non-polar column was 982 on and 2153 on the polar

column. It had a weak-moderate caramel aroma. It has been reported in mammee

apples (44).





CH3

3-methy 2(5H) furanone

Figure 5-8 Structure of the 3-methyl-2(5H) furanone

When the standard was analyzed on GC-MS, the retention time and linear

retention index suggested that it eluted with peak # II (fig. 5-9).

Commercial FCGJ undergoes at least two heat treatments, once during the

evaporation step and second after reconstitution. However, the untreated juice, which

was reconstituted grapefruit juice, underwent only the first heat treatment, which

involves heating temperatures as high as 93C. A high temperature such as this is

sufficient to induce the formation of these furanones. The heating of the juice in both

the glass and stainless steel containers may have increased the formation or









concentration of these compounds, but due to their extremely low threshold the aroma

remains quite intense.

The Maillard reaction is initiated by high temperature treatment of foods, and its

products are increased with extreme heat treatment and long-term storage. The first

step involves the reaction of a sugar with an amine and subsequent loss of water. There

is only a slight loss of reactants since sugars are relatively plentiful in grapefruit juice.

Identification of Volatiles Using GC-MS

Since MS is an identification tool, it was used to identify the volatiles based on

their specific fragmentation spectra as well as their linear retention indices. Most

compounds fragment in a unique pattern and by comparing this pattern with standards

analyzed under the same conditions or by comparing to library databases with known

spectra, compounds can usually be identified. Fig. 5-9 shows the total ion

chromatograph of grapefruit juice from 10-33 minutes while table 5-5 lists the identities

of a few of the major components in grapefruit juice. The areas were normalized to

internal standard #2.

Matching linear retention indices was used for a secondary confirmation. This is

particularly important in the detection of terpenes. Terpenes are C10H16 hydrocarbons

and have a molecular weight of 136. It is very difficult to identify these compounds by

solely matching mass spectra as they tend to exhibit similar fragmentation patterns

particularly for the major mass peaks.














Total Ion Chromatogram
for
Grapefruit Juice


XIII
E
c
C
_c


v. 11 IX

o V
0cXV











10 16 20 24 28 32
Retention Time (min)


Figure 5-9 Segmented MS total ion chromatogram using ZB-5 column for unheated reconstituted grapefruit juice (A), juice heated in
glass (B) and juice heated in stainless steel (C).
glass (B) and juice heated in stainless steel (C).









Table 5-5


Components identified in grapefruit juice by GC-MS
column.


and analyzed on a DB-5


Peak Retention LRI Component Unheated Glass Stainless
Number Time Name reconstituted Steel
(min)
%Area %Area %Area
I 12.47 941 a-pinene 15.68 2.63 0.74
II 13.68 987 myrcene 45.93 7.52 4.66
III 14.93 1035 limonene 2512.60 611.60 364.36
IV 15.14 1043 E-ocimene 2.25 0.74 0.73
V 15.92 1073 linalool oxide 6.72 27.23 18.83
VI 16.31 1089 linalool 3.79 16.42 11.51
VII 19.05 1201 c-terpineol 10.57 20.67 11.74
VIII 23.19 1385 a-copaene 6.80 0.32 3.39
IX 24.24 1435 P- 30.21 1.73 12.61
caryophyllene
X 25.55 1499 a-humulene 12.40 7.43 7.75
XI 26.00 1522 bisabolene 11.22 1.02 6.49
XII 26.14 1529 y-cadinene 1.43 1.77 0.96
XIII 31.44 1823 nootkatone 80.76 43.69 41.95


An example of this can be seen in peaks number I and III, which were identified as a-

pinene and myrcene respectively. Both these compounds are terpenes having piney and

musty aroma respectively. From the spectra of these two compounds, fragments 121,

91 and 77 (fig. 5-10) were common to both and appeared to have the same intensity.

Mass peaks at 92, 93 and 79 had slightly different intensities and may be used as

distinguishing fragments. However, by using the linear retention indices of 939 and

989 respectively, these two compounds could be easily distinguished.









91.2


myrcene


77.4


m/z

107.1
I f


121.2
I


105.1
109.2 121.2
I,~ .. .. I l Il


40 60 80 100 120 14(
Mass/Charge (m/z) ratio
Figure 5-10 Mass spectra of myrcene (top) and a-pinene (bottom) taken from a sample
analyzed on a DB-5 column.

Limonene (peak III) was present in the highest amount as expected. Limonene is the

most abundant terpene present in citrus juices. Peak VII was identified as a-terpineol.

Figure 5-11 is an expanded view of the chromatogram indicating the differences among

the three samples. Alpha terpineol content of juice heated in glass was higher than both

the stainless steel (metal) and unheated reconstituted from concentrate juice. It 63%


0


67.3
65.3


79.3

S0.3


135.2


41.2


51.4


a-
pinene


51.2
40.9
, I J I ,i


80





77.2

79.3


65.3


81.2


137.2
1









higher than the unheated reconstituted juice and 43% higher than the juice heated in

metal.


a-terpineol levels in
Grapefruit Juice


Glass






_ Metal

i- A


18.95 19.05 19.15 19.25
Retention Time (min)

Figure 5-11 Alpha-terpineol levels in the three samples, unheated reconstituted juice,
juice heated in glass and juice heated in metal

Alpha-terpineol can be formed from either (or both) the acid-catalyzed reaction of

limonene or linalool (fig. 5-12) in the presence of heat (45). Alpha terpineol has also

been shown to negatively correlate with the flavor of citrus juices (4) and has been

suggested as an indicator for over-processed or stored orange juices.









H+, -HOH
-


d-limonene


a-terpineol


I


H*, -HOH


Linalool


Figure 5-12 Formation pathway for a-terpineol in the presence of limonene and linalool
in grapefruit juice


H3C,














CHAPTER 6
HPLC RESULTS AND DISCUSSION

Unlike GC, HPLC is a non-thermal method of analysis used in the identification of

both volatiles and non-volatiles. HPLC is useful in situations where polar volatiles have

strong UV chromaphores and are difficult to extract using typical GC extraction

procedures. For example, Furaneol in grapefruit juice is usually found at levels below the

detection threshold of the flame ionization detector, making detection and quantification

difficult. Solid phase extraction employing C-18 chemically bonded silica, can trap polar

volatiles onto the modified silica for later analysis on reversed phase HPLC.

Since furans are thought to be responsible for heated off-flavor, HPLC was used to

identify and measure furans in grapefruit juice. Over the years reversed phase HPLC has

been favored in the isolation of furans in several fruit juices (19;21;46). Reversed phase

LC is the most popular liquid chromatographic method used today (47). Compounds

with increasing hydrophobicity or non-polar behavior will be retained on the column

more strongly. Water is the weak solvent and a methanol or acetonitrile gradient is

employed to selectively elute the materials off a reverse phase column.

In citrus juices several mobile phase compositions have been used ranging from

methanol or acetonitrile/water, acetonitrile/methanol/buffer, methanol/acetate buffer.

These solvent systems either did not produce a good separation or did not exhibit good

reproducibility. Acetate buffer has an absorbance maximum at 235nm, which interferes

with the spectra of sotolone whose maximum absorbance is 237nm. Although previous

studies (19) reported that methanol/phosphate buffer condition could not resolve furfural









and Furaneol, this mobile phase composition was re-examined since the phosphate buffer

had a lower UV absorbance background. It had been reported that 30%

methanol/phosphate buffer could not resolve furfural and Furaneol. Thus several

gradients were evaluated until a suitable separation was between 5-HMF, furfural, and

Furaneol was achieved. These three compounds had been previously been identified in

grapefruit juice using RP-HPLC (19). In order to increase retention time and promote

resolution, the amount of modifier, methanol was decreased to 5% with a slow increase

up to 30% in 27 minutes. It was found that at approximately 20% methanol Furfural was

eluted and Furaneol close to 24% methanol. Additionally, the temperature of the column

was kept at 250C and the sample 240C. Since the three compounds were completely

resolved, other Maillard reaction products, (mesifurane, homofuraneol, sotolone, and

maltol) were evaluated under similar conditions and the result shown in fig. 6-1. The

small humps at the end of the late eluting peaks were apparently due to a small void at the

head of the HPLC column. These standards are all products of the Maillard reaction and

possess caramel or burnt sugar aroma that could possibly be formed in grapefruit juice

during heating. These seven compounds along with their retention times and maximum

absorbance are listed in table 6-1.










Table 6-1 Retention time and maximum absorbance of furans standards on a C-18
column and using a methanol/phosphate buffer at flow rate of ImL/min
Furan Standards Retention Time Max Wavelength
(min) (nm)

5-HMF 13.04 283
Furfural 14.95 276
Maltol 17.41 274
Furaneol 18.86 287
Sotolone 20.24 235
Mesifurane 27.63 278
Homofuraneol A 30.40 287

Homofuraneol B 31.80 287


276nm


3

284nm c


234nm


<11

278nm a p
1 0

287nm

287nm m


15 20 25 30


Retention Time (min)


Figure 6-1 HPLC spectra of the seven standards (5-HMF, furfural, Furaneol, sotolone,
mesifurane and homofuraneol) monitored from 230-380nm.

Homofuraneol has been known to exist in two tautomeric forms accounting for the

presence of an A and B form (48) and produces two distinct HPLC peaks. As can be








seen from fig. 6-2, homofuraneol has an ethyl group that can be interchanged with the

methyl group so that it can be either in the 2 or 5 position.

H3C 0 CH3CHCH CH3CH2

I I
HO HO

A B

Figure 6-2 Homofuraneol tautomers, A- 2-ethyl-4-hydroxy-5-methyl-3(2H) furanone; B-
5-ethyl-4-hydroxy-2-methyl-3(2H) furanone

The value of the PDA detector lies in its ability to distinguish compounds based on

their spectra as well as retention time. This feature can be useful in identifying unknown

peaks and determining if there are co-eluted compounds in a single HPLC peak. With the

exception of sotolone, which has a maximum absorbance at 235nm, most furans exhibit

maximum wavelength absorbance between 270-290nm. For this reason dual wavelength

monitoring at 235nm and 280nm was carried out. Additionally, monitoring was done at

335nm as these furans had minimum absorbance at that wavelength. This would be

useful in distinguishing furans from other co-eluting compounds. A comparison of the

spectra and the retention times of components in the sample with those of standards,

suggest the presence of 5-HMF, furfural, and Furaneol in the grapefruit juice (fig. 6-3).

As discussed earlier, these three compounds are heat induced hexose sugar degradation

products that have been previously identified in citrus juices.


























-- Metal
-Glass
....unheated
I I I I I I
13 14 15 16 17 18 19

Retention Time (min)

Figure 6-3 Segment of HPLC chromatogram showing overlay of unheated, juice heated
in glass and juice heated in metal and monitored at 290nm

By looking at the spectra of each of these compounds in the sample and comparing

it with that of the standard, it was found that 5-HMF was the only compound of interest

that was well resolved from other grapefruit juice components (fig. 6-4).












250000-


200000-


150000-


100000-


50000-

I I CO CO
0-
220 240 260 280 300 320 340 360
Waveler



m25000-
C

20000-

15000-

10000- --

5000- 0 g


'-' I I I '_ I I I0 I
220 240 260 280 300 320 340 360
Waveler


Figure 6-4 Spectra of 5-HMF in standard (top) and in the grapefruit juice sample
(bottom) analyzed on C-18 column and monitored at 290nm.


Spectral data for both Furaneol and furfural peaks suggested the presence of


coeluted interfering compounds. Figure 6-5 shows the spectra of furfural and it can be


seen that the peak characteristics are different from the standard. An absorbance


maximum of 276nm was seen for the standard while a maximum of 326nm was seen for


the sample. Between 260 nm and 300nm, there appears to be another peak indicating that


co-elution might have occurred. It was not possible to obtain an absorbance "difference"


chromatogram using output from the two wavelengths (326nm and 278nm) at this time.


Previous studies had used this technique to identify and characterize Furaneol in a


grapefruit juice matrix (21).








59





1200000-


150000-


100000-


50000-

0-

240 260 280 300 320
Wavelength (r
I I

















Wavelength (r
025000

S20000 / \

15000\

10000 _

5000 \

0-

220 240 260 280 300 320 340 360
Wavelength (r



Figure 6-5 Spectra of furfural standard (top) and sample (bottom) showing a difference in
the wavelength maxima at 15.15 min


Grapefruit juice furan and furanones were quantified using the external standard


method, wherein different concentrations of standards were injected onto the LC in


triplicate. Area responses were then plotted against the amounts injected and linear


responses were obtained. The 5-HMF amounts ranged from 1.07ng to 21.4ng to ensure


that the range found in samples was covered. From calibration plot (fig. 6-6), the r2 value


which shows how well the data fit a linear regression was 0.9993, indicating good


linearity.











Calibration Curve for 5-HMF


600000

500000

a 400000

S300000

S200000

100000

0
0 5 10 15 20 25
Amount (ng)


Figure 6-6 Linear calibration curve for 5-HMF analyzed in triplicate.

Based on the linear equation, the amount of the each furan in grapefruit juice samples

could be calculated. Juice heated in the presence of stainless steel had a 5-HMF

concentration of 0.47ng/ul, while the same juice heated in glass had 0.34ng/ul (table 6-2).

Unheated juice contained the least amount of 5-HMF (0.03ng/ul).

Table 6-2 Concentrations of 5-HMF, furfural and Furaneol in the three grapefruit juice
samples
5-HMF (ng/ul) Furfural (ng/ul) Furaneol (ng/ul)

Unheated 0.03 0.40 0.22

Glass 0.34 0.25 0.08

Metal 0.47 0.25 0.08









A similar plot (fig.6- 7) was conducted for Furaneol with amounts ranging between 1.8ng

to 18ng and an r2 value of 0.92 was found. Unheated reconstituted juice had a

concentration of 0.22ng/ul while the two heated juice samples had levels of 0.08ng/ul.


Furaneol Calibration curve


1000000


750000


500000


250000


0


5 10 15 20
Amount (ng)


Figure 6-7 Linear calibration curve for Furaneol at 290nm.

The standard curve (fig. 6-8) for furfural also had a good fit with an r2 value of 0.99

using a range of 2ng to 200ng. Concentration of furfural in the unheated reconstituted

juice was 0.40ng/ul while juices heated in glass and metal had 0.25ng/ul.











Furfural Calibration Curve


4000000

3000000

2000000

1000000- R-

0 -
0 50 100 150 200 250
Amount (ng)


Figure 6-8 Linear calibration curve for furfural standard at 290nm.

All three compounds, 5-HMF, furfural and Furaneol are sugar degradation

products. Formation of 5-HMF occurs through the 1,2 enolization at low pH values in

the presence of a hexose sugar. The pH of grapefruit juice is usually between 2.9- 3.2,

which is highly acidic, and provides an explanation for the increase in the 5-HMF

formation in the two heated samples. Studies have shown when grapefruit juice that

under similar conditions the rate of formation of 5-HMF was far greater than that of

furfural (9). Furfural can be formed from acid catalysis of ascorbic acid in the presence

of an amine. Since it can be formed from ascorbic acid, which is known to degrade with

heating, it was expected that this compound would have higher concentrations in the two

heated samples. However, it is a very reactive compound and, in the presence of acids

can react with aldehydes, ketones and amino acids (49). This could possibly be a reason

for the lower levels in the heated samples.

Furaneol is also a sugar degradation product and its concentration was expected to

increase as a result of the heating. Its formation is favored at high pH and goes through









the 2,3-enolization pathway, however at low pH values it is formed in the presence of

alanine. Studies have shown that Furaneol is unstable and can be degraded at high

temperatures (24) to form highly reactive dicarbonyls, ketones and alcohols. In

equilibrium, Furaneol can exist in its open chain form, thereby facilitating attack of sulfur

groups such as thiols on the carbonyl (50). It can also react with sulfur compounds, such

as cysteine at low pH, and hydrogen sulfide during thermal processing (51;52).

Consequently, at high temperatures, the concentration of Furaneol would be lower than

expected.














CHAPTER 7
CONCLUSION

No significant flavor difference was detected between juices heated 10 min in

contact with stainless steel and the same juice heated in glass. However, this excessive

heating induced a flavor change that was significantly different from the unheated

reconstituted grapefruit juice at the 95% confidence interval. Thermal processing

produced heated, cooked, pineapple and metallic off-flavors.

Similar volatiles were detected in both heat-treated samples, suggesting that high

processing temperatures and long times appeared to be major contributing factors for

cooked off flavor formation. The quality of the juice heated on glass surface was not of

superior quality to that of metal surface, therefore, the current practice of using stainless

steel is agreeable.

Intensities of compounds such as a-pinene and myrcene that are known to

positively contribute to fresh citrus juice character were diminished during the heating

process, while aroma intensity of heat induced compounds such a methional and Furaneol

increased during heating. Sugar degradation products such as sotolone, and 3-methyl-2

(5H) furanone were detected by GCO. The 3-methyl-2 (5H) furanone was tentatively

identified in grapefruit juice for the first time. Concentrations of 5-hydroxy methyl

furfural, another sugar degradation product, increased with heating.
















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BIOGRAPHICAL SKETCH

Wendy Bell was born in Westmoreland, Jamaica, and graduated from the Wolmer's

High School for Girls in Kingston. In 1995, she moved to the United States and

embarked upon her undergraduate studies at Barry University, Miami, Florida. She then

transferred to the University of South Florida, Tampa, Florida, where she completed her

Bachelor of Arts in Chemistry in May 1999. In pursuance of her career, Wendy gained

employment at Pasco Beverage Company and Florida Department of Citrus.

Wendy will receive her master's degree in December 2004 and will be joining

Cadbury Adams, Morris Plains, NJ, upon graduation.