FLAVOR STABILITY AND OFF-FLAVORS IN THERMALLY PROCESSED ORANGE JUICE By J. GLEN DREHER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1
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FLAVOR STABILITY AND OFF-FLAVORS IN THERMALLY PROCESSED ORANGE JUICE
By
J. GLEN DREHER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
2 REVIEW OF LITERATURE.................................................................................................15
Orange Juice ...........................................................................................................................15 Orange Juice Flavor and Processing.......................................................................................16 Flavor Production ...................................................................................................................18
Gas Chromatography-Olfactometry .......................................................................................25 Extraction Methods.................................................................................................................29 Thiamin as a Source of Potent Sulfur Aroma Compounds.....................................................30
2-methyl-3-furanthiol ......................................................................................................30 Bis(2-methyl-3-furyl) disulfide .......................................................................................31 Thiamin Degradation Pathway ........................................................................................31 Alternate Pathways for the Production of 2-Methyl-3-furanthiol ...................................32
3 AN AROMA COMPARISON BETWEEN ORANGE JUICES OF DIFFERING ORGANOLEPTIC QUALITIES............................................................................................35
Introduction.............................................................................................................................35 Materials and Methods ...........................................................................................................36
Survey of Commercial orange juice................................................................................36 Chemicals ........................................................................................................................37 Sample Preparation..........................................................................................................37 Gas Chromatography-olfactometry Conditions ..............................................................38 Time-intensity Analysis...................................................................................................39 Sulfur Analysis ................................................................................................................39
Results and Discussion ...........................................................................................................39 α-Terpineol, Furaneol, and 4-vinylguaiacol....................................................................41 α-Terpineol......................................................................................................................42 4-Vinylguaiacol ...............................................................................................................44
4 ORANGE JUICE FLAVOR STORAGE STUDY: DIFFERENCES BETWEEN GLASS AND PET PACKAGING OVER TIME AND TEMPERATURE...........................54
Introduction.............................................................................................................................54 Materials and Methods ...........................................................................................................56
Chemicals ........................................................................................................................56 Orange Juice.....................................................................................................................57 Visual and Organoleptic Evaluation ................................................................................57 Sample Preparation..........................................................................................................57 Gas chromatography-olfactometry Cnditions .................................................................58 GC-olfactometry..............................................................................................................58 Gas Chromatography-mass spectrometry (GC-MS) .......................................................59
Results and Discussion ...........................................................................................................60 Aroma Changes over time...............................................................................................61 Off-Flavor Compounds ...................................................................................................61
Methional .................................................................................................................62 Furaneol and 4-vinylguaiacol...................................................................................62 2-Methyl-3-furanthiol and bis(2-methyl-3-furyl) disulfide......................................62 M-cresol ...................................................................................................................63 Sulfur Compounds ...................................................................................................63 Carvone ....................................................................................................................64 Vanillin.....................................................................................................................64
Changes in Fresh Juice Compounds................................................................................65 (Z)-3-Hexenal...........................................................................................................65 Linalool ....................................................................................................................65 Ethyl butyrate ...........................................................................................................65 Octanal .....................................................................................................................66 Acetic and butanoic acids.........................................................................................66 Trans-4,5-epoxy-(E)-2-decenal ................................................................................67
5 GC-OLFACTOMETRIC CHARACTERIZATION OF AROMA VOLATILES FROM THE THERMAL DEGRADATION OF THIAMIN IN MODEL ORANGE JUICE............78
Introduction.............................................................................................................................78 Materials and Methods ...........................................................................................................79
Preparation of Model orange juice solutions ................................................................. .80 Sample Preparation..........................................................................................................80 Gas Chromatography-pulse flame photometric detector (GC-PFPD) .............................80 Quantitative Analysis .......................................................................................................81 Gas Chromatography........................................................................................................81 GC-olfactometry...............................................................................................................81 Gas Chromatography-mass Spectrometry (GC-MS) .......................................................82
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Injector Decomposition Study .........................................................................................83 Microbiological Analysis ................................................................................................83
Results and Discussion ...........................................................................................................83 Day 7 and 42 Aromagrams..............................................................................................84 Aroma Volatile identifications ........................................................................................85 Quantification of MFT and MFT-MFT...........................................................................88 Thiamin as a Source of MFT and MFT-MFT in Citrus Juices........................................89 Possible GC Injector Thermal Artifacts..........................................................................90 Possible Microbiological Artifacts..................................................................................91
Table page 3-1 Summary of aroma active compounds found in good and poor quality juice........................47
4-1 Aroma active compounds in orange juice stored at 4 and 35°C over 112 days. ....................69
4-2 Comparison of total overall aroma intensity under various package, time and temperature conditions.......................................................................................................73
5-1 Aroma active compounds detected in model orange juice solution .......................................93
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LIST OF FIGURES
Figure page 2-1 Pathways for α-terpineol formation from linalool and (+)-limonene ....................................34
2-2 Thiamin thermal degradation pathways A =thiamin hydrochloride, B = pyrimidine moiety, C = thiazoles moiety, D = diaminopyrimidine, E = formic acid, and F = 5-hydroxy-3-mercapto-2-pentanone......................................................................................34
3-1 Normalized aroma peak intensity comparison of good and poor quality orange juice. .........49
3-2 Aldehyde comparison between good and poor quality orange juice......................................50
3-3 Comparison of known off-flavor components in orange juice...............................................51
3-4 Possible pathway formations of α-terpineol...........................................................................51
3-5 Individual response chromatogram of α-terpineol GC/FID aromagram overlay...................52
3-6 GC-O aroma threshold determination of α-terpineol. ............................................................52
3-7 Methional formation through Strecker degradation of methionine ........................................53
4-1 Aroma comparison of day 0 and 112 (35°C) in glass packaging. ..........................................73
4-2 Aroma comparison of day 0 and 112 (35°C) in polyethylene terepthalate packaging...........74
4-4 Aroma comparison of orange juice stored at 4 and 35° for 112 days in polyethylene terepthalate.........................................................................................................................76
5-1 SPME headspace samples of GC-O aromagrams comparing day 7 and 42, where peak intensities were inverted for day 42 data. ..........................................................................93
5-2 Structures of select aroma active sulfur compounds detected in the model orange juice solution...............................................................................................................................94
5-3 Comparison between PFPD chromatogram and corresponding aromagram from a model orange juice solution stored for 7 days at 35°C. ...............................................................95
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
FLAVOR STABILITY AND OFF-FLAVORS IN THERMALLY PROCESSED ORANGE JUICE
By
J. Glen Dreher December 2007
Chair: Russell Rouseff Major: Food Science and Human Nutrition
The aroma active components of thermally processed orange juice were determined and
compared between orange juices of above and below average quality. A loss of aldehydes
including hexanal, heptanal and octanal; imparting aromas such as floral, green and citrus
coupled with the occurrence of potent off-flavor compounds 4-vinylguaiacol and methional
contributed to the differences seen between the above and below average quality juices. Of
significance, the widely reported orange juice storage off-flavor compound α-terpineol was
found in greater concentration than previously reported but without aroma activity.
The aroma active components of orange juice were noted to change over time during
storage at 35°C. Difference in aroma active compounds at 4°C and 35°C were seen, with a loss
and/or diminishing impact of aroma active compounds that contribute to good quality orange
juice flavor including (Z)-3-hexenal, octanal, (Z)-4-octenal and (E)-2-octenal. Qualitative
differences were noted between glass and PET containers, with orange juice stored in PET
forming off-flavor compounds including eugenol, sotolon, 4-mercapto-4-methyl-2-pentanone, 2-
methyl-3-furanthiol as well as higher aroma intensities of the well documented storage off-flavor
4-vinylguaiacol.
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Through a model orange juice solution, thiamin, the second most abundant water-soluble
vitamin in orange juice, was determined to be the precursor for the off-flavor compound 2-
methyl-3-furanthiol (MFT) and its very potent dimer, bis(2-methyl-3-furyl) disulfide (MFT-
MFT). Both MFT and MFT-MFT impart a meaty aroma have recently been documented as off-
flavors in stored orange juice. MFT and its dimer increased in concentration over time at storage
conditions of 35°C.
The results of this study show the importance of balance in flavor composition and how
packaging and storage can affect the quality of orange juice. Producers can take steps to add
back the specific fresh aroma active compounds lost during processing, while designing the
packaging to minimize storage off-flavors and limiting off-flavor compounds through
fortification.
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CHAPTER 1 INTRODUCTION
Orange production has an enormous impact on the world and U.S. economy both as fresh
fruit and juice. The total dollar amount spent in the US in 1999 was approximately $1.7 billion
on fresh orange and juice combined (2007). Citrus is valued for its balance of sweet and sour
tastes as well as distinctive aroma. Although the orange has its highest monetary value when
sold as fresh fruit, over 90 percent of orange production in Florida is for juice processing
(Chadwell et al., 2006).
The flavor of orange juice is complex and the difference between a good and poor quality
juice starts with the initial flavor quality of the orange. The ripening process for an orange is
non-climacteric, ripening only occurs while on the tree (Alonso et al., 1995). During non-
climacteric maturation, respiration remains level, decay is rapid and no definitive abscission time
exists; whereas climacteric fruit such as bananas have an increased respiration during maturation
and a definitive abscission time. For this reason, oranges are picked for the optimal °Brix
(primarily sugars) to acid ratio. As the orange matures, the acidity decreases while the °Brix, or
soluble solids, increases. Although citrus is a non-climacteric fruit, peel color may be altered
after picking through controlled atmosphere storage. Stewart and Wheaton (1972) found
carotenoid accumulation in Robinson tangerine to increase in the presence of ethylene at 10
µg/mL, with degreening occurring after 1 week followed by carotenoid development from
yellow to orange in weeks 2 and 3. The study also reported that carotenoid development is best
at lower degreening temperatures and is inhibited at temperatures above 30°C.
The proximate analysis of orange juice is 11.27 °Brix, 0.67% citric acid, 12% pulp
(volume by centrifuge) and 0.0123% oil (v/v) (Balaban et al., 1991). As with most foods, the
smallest component of the total, oils/aromas, contributes the most impact to the overall flavor of
the fruit. The °Brix/acid ratio is important, but the aroma composition can profoundly impact
juice quality because much of what humans perceive as flavor is really produced from aroma
components. Aroma active volatiles are secondary metabolites formed during maturation and
are concentrated in the oil glands in the peel as well as in the juice vesicles.
Orange juice flavor is not only produced during fresh fruit maturation but is also affected
by subsequent processing and storage of the finished juice. The main factor which alters flavors
during processing is heat. Thermal processing is necessary to create a stable product; however,
heat can also alter the volatile composition by reducing some of the initial flavor volatiles
through reactions as well as produce off-flavors from non volatile precursors. Aroma
composition will continue to change during storage because of certain chemical reactions. The
extent of these chemical changes will be dependent on storage time and temperature. Packaging
material can also affect juice flavor. Materials such as low and high density polyethylene and
polyethylene terephthalate can cause flavor scalping or addition of compounds to the juice
through migration especially with the major orange juice volatile (+)-limonene (Kutty et al.,
1994; Lune et al., 1997; van Willige et al., 2003; Fauconnier et al., 2001).
There were three objectives in this study. The first objective was a comparison between
orange juices of differing qualities, determining differences in volatile compound composition
and concentrations to identify which components correlate with good quality and which
components correlate with poor quality. Secondly, orange juice aroma impact compounds were
determined in a time/temperature/packaging study to determine the effects of storage time and
temperature as well as packaging materials. Finally, a model orange juice system was employed
to determine possible formation pathways of the off-flavor aroma compounds 2-methyl-3-
furanthiol and bis(2-methyl-3-furyl) disulfide that were detected in the first two studies.
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By determining the difference between a poor and good quality orange juice as well as
flavor changes associated with different packaging materials during storage, a processor can
tailor the add back flavor package or alter packaging material to improve juice quality. A real
world application of my final model orange juice study solution would be the confirmation of the
source of a potent off-flavor and the information necessary to alter processing, packaging or
storage so as to provide the highest quality of orange juice to the consumer.
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CHAPTER 2 REVIEW OF LITERATURE
Orange Juice
Sweet oranges, Citrus sinensis, have long been prized as a fresh fruit and as juice. As a
fresh fruit, the orange ranks third behind bananas and apples in consumption per year in the U.S.
(USDA, 2006a). As a juice, oranges rank number one, with American’s drinking 2.5 times more
orange juice than the second-ranked apple juice (Pollack et al., 2003). An 8oz serving of orange
juice contains 100% of the daily value (d.v) of Vitamin C, 20% of the d.v. for folic acid, 15% of
the d.v. for potassium and 10% of the d.v. for thiamin.
Oranges are the most important fruit in the citrus family, comprising roughly 65% of the
world’s estimated citrus crop. Prior to the 2004/2005 season, the United States has been
traditionally the second largest producer of citrus behind Brazil. Due to hurricane damage, the
United States is currently the third largest citrus producer behind Brazil and China.
Approximately 68% of citrus produced in the United States is processed into juice, but 95 – 96%
of Florida’s orange crop in used for juice (USDA, 2006b).
The different cultivars of oranges are split into three categories by the ripening season:
early, mid, and late. Early cultivars reach maturity before December and include the “Hamlin,”
“Parson Brown” and navel oranges. Mid-season cultivars reach maturity between December and
March and include “Pineapple,” “Queen,” Sunstar,” “Gardner” and “Midsweet” cultivars. The
late season fruit peak from March to June, with the main cultivar being “Valencia.” The navel
orange is prized for fresh fruit consumption as they can develop a bitter note when processed into
juice. The “Valencia” is the primary sweet orange cultivar grown in Florida and the world and is
mainly processed into juice (Williamson and Jackson, 1993).
Orange Juice Flavor and Processing
There are four main categories in which orange juice can exist: fresh squeezed orange
juice, frozen concentrate orange juice (FCOJ), not-from concentrate orange juice (NFC) and
orange juice from concentrate (RECON). The first group, fresh squeezed, is highly valued for its
fresh flavor and natural quality. The lack of heat treatment sets this group apart from the others
(Schmidt et al., 2005). However, because the juice does not have any heat treatment, its shelf-
life is limited to a few days. Fresh squeezed juice is an important part of the European market
(2006a).
Frozen concentrate orange juice is concentrated by thermal processing, during which
water and volatile flavors are removed. The flavor vapors are cooled and reclaimed in one of the
first stage condensers and fractionated into oil and aqueous phases. A flavor system comprised
of portions of the captured essence is then added back to the concentrated juice to restore some
of the lost flavor.
Not-from concentrate orange juice comprises the largest single segment in the United
States, as it was responsible for 49% of the total orange juice market in the 2004-2005 season
(2006b). NFC is pasteurized but not concentrated or frozen. NFC is the closest thermally treated
juice to fresh squeezed in terms of flavor.
Orange juice from concentrate is FCOJ that has been commercially reconstituted to single
strength orange juice. The advantage of reconstituting FCOJ commercially is reduction in
transportation cost to the producer. However, the main disadvantage from a flavor standpoint is
that RECON receives a second heat treatment when it is repackaged, causing more flavor loss
and degradation. From a flavor standpoint, RECON is the furthest away from the fresh squeezed
juice that is prized for its flavor.
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Of the four types of processed juice, the two largest groups consist of NFC and FCOJ.
The standards of identity for these types of juices are set in the Code of Federal Regulation
(CFR) Title 21. The USDA has set standards for grading orange juice within the 47 Federal
Register (FR) (USDA, 1983). The orange juice is separated into grades A, B and substandard
within the types of orange juice. The main factors affecting the quality grade include color,
defects, and flavor. Other factors are specific to the type of juice and include appearance,
reconstitution and coagulation. The color is scored as compared to USDA Orange Juice Color
Standards with a max score of 40 points, with Grade A having a minimum of 36 score points.
Defects include juice cells, pulp, seeds or portion of seeds, specks, particles of membrane, core,
peel, or any other distinctive features that adversely affect the appearance or drinking quality of
the orange juice. Defects are scored on a scale with max points of 20. Grade A orange juice is
considered practically free of defects with a minimum score of 18. Flavor is evaluated and
scored on a scale with a maximum of 40 points and separated into three categories: very good
flavor, good flavor and poor flavor. Grade A orange juice has very good flavor with a minimum
of 36 points and defined as fine, distinct, and substantially typical of orange juice extracted from
fresh mature sweet oranges and is free from off flavors of any kind. Grade B orange juice meets
the good flavor standards, ranging from 32 – 35 points, and is similar to the flavor of juice
extracted from fresh mature sweet oranges but may be slightly affected by processing,
packaging, or storage conditions. Poor flavor orange juice would score less than 32 points and is
defined to fail to meet the requirements set for good flavor. As defined, poor flavor juice would
be categorized as substandard orange juice.
The main difference between NFC and FCOJ is the concentration step in FCOJ. FCOJ
takes orange juice through a series of concentration steps taking the juice from approximately
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11.0 °Brix to 65 °Brix. There are advantages of FCOJ over NFC. The FCOJ process will strip
off-flavors and excess oil in the evaporator. The evaporator cannot be used for NFC production;
therefore a “softer” extraction is used to prevent excess oil addition. The softer squeeze might
result in lower juice yields as compared to FCOJ. One way to remove excess peel oil is to
employ centrifuges, thereby allowing maximum yield. Grade A orange juice has a maximum
limit of 0.035% by volume of recoverable oil (USDA, 1983). By being below this level,
essential oil flavor systems can be added.
Not-from concentrate orange juice undergoes a pasteurization step to reduce
microorganisms and to inactivate enzymes. The main enzyme in orange juice is pectinesterase,
PE. PE activity is a major concern in the citrus industry. PE is naturally present in the peel, rag
and pulp and is released during extraction and finishing. PE leads to cloud loss in single-strength
juice and gelation in concentrate. The thermal process needed to inactivate PE is higher than that
needed for microbial purposes.
A recent trend in the United States has seen the consumption of NFC increase from 183.1
million SSE gallons in 1990 to 629.9 million SSE gallons in 2000. This has in turn increased the
amount of Florida’s orange crop going to NFC to approximately 50% in the 1998-1999 season
(Spreen and Muraro, 2000).
Flavor Production
Off-flavor production in orange juice can be caused by many different pathways.
Sources can include enzymatic off-flavors, microbial off-flavors, packaging, processing, and
storage off-flavors. Storage off-flavors will be discussed in detail, examining the following
possible pathways: precursor development, Shickimic acid pathway, Maillard reaction and
Strecker degradation. Flavor precursors are flavorless compounds that produce flavor
compounds in consequence of enzymatic or chemical reactions that occur during maturation
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(usually enzymatic driven) or processing (usually chemically driven). Process flavors can
positive or negative depending on the food matrix and desired goal, such as in the formation of
garlic odor from flavorless precursor allin to the garlic odor alliein. In grapefruit juice one
reaction includes the formation of a characteristic grapefruit aroma of 1-p-menthene-8-thiol from
limonene by the acid catalyzed addition of hydrogen sulfide across the external double bond.
Lin et al. (2002) found 1-p-menthene-8-thiol present in concentrated grapefruit juice but not
fresh juice and suggesting that this character impact compound might be a reaction product of
thermally treated juice. The (R)-(+)-enantiomer of the 1-p-menthene-8-thiol is one of the most
potent naturally occurring volatiles with a detection threshold of 0.02 µg/L (Leffingwell, 2002).
Another citrus flavor precursor example is the breakdown of carotenoids, large C40,
tetraterpenoid compounds such as β-carotene into the smaller (C13) β-ionone (dried, fruit woody
aroma). Kanasawud and Crouzet studied the thermal degradation of β-carotene in an aqueous
medium and identified β-ionone as a volatile degradation product, showing an increase in
concentration of β-ionone with an increase in temperature (Kanasawud and Crouzet, 1990).
Terpene glycosides
Another important type of fruit flavor precursors includes terpene glycosides. In this
process, volatile terpene and norisoprenoid compounds are cleaved from nonvolatile terpene
glycosides via enzymatic or acidic hydrolysis. Terpene glycoside reactions have been studied in
many fruits including the peach, yellow plum and apricot (Krammer et al., 1991) and grapes
(Maicas and Mateo, 2005). Phosphate ester reactions are an in vivo source of terpenoid
compounds. One example is the formation of geranyl pyrophosphate (PP), neryl-PP and
dimethyl-allyl-PP from enzymatic breakdown of mevalonic acid-PP (Lindsay, 1985).
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Terpene alcohols can also be formed through acid catalyzed hydrations. A reported off-flavor
compound in orange juice is α-terpineol (Rymal et al., 1968; Tatum et al., 1975). α-Terpineol
has a floral, lilac-like aroma, but when added to orange juice a stale, musty or piney aroma has
been reported (Tatum et al., 1975). Haleva-Toledo et al. (1999) demonstrate the pathways of the
precursors, linalool and (+)-limonene, present in citrus juice, that can undergo acid catalyzed
hydration to form α-terpineol (Figure 2-1). The conversion of linalool to α-terpineol is much
faster than the reaction with (+)-limonene. However, it was noted that with the high
concentration of (+)-limonene in citrus juice, α-terpineol production is due to both linalool and
(+)-limonene equally. Perez-Lopez et al. (2006), show production of α-terpineol increases after
pasteurization of mandarin juice with a simultaneous decomposition of linalool and (+)-
limonene. Measurement of linalool, (+)-limonene, α-terpineol and terpinen-4-ol were suggested
as a tool to monitor the quality of the mandarin juice.
Shikimic acid pathway
The shikimic acid pathway starts a series of reactions that can lead to several different
classes of flavor compounds. Shikimic acid can produce other precursors such as cinnamic acid
and ferulic acid which can lead to potent aroma compounds such as eugenol, 4-vinylguaiacol and
vanillin (Lindsay, 1985). 4-Vinylguaiacol is described as possessing a peppery/spicy aroma and
is considered a major off-flavor. In orange juice it imparts an old/rotten fruit aroma (Tatum et
al., 1975; Peleg et al., 1992; Naim et al., 1988). Vanillin has also been noted in orange,
tangerine, lemon, lime and grapefruit juices (Goodner et al., 2000). The shikimic acid pathway
also plays an important role in flavor production of wines. Lopez et al. (2004), studied the aroma
compounds from mild acid hydrolysates in Spanish wine grapes. The author found the shikimic
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acid pathway produced important flavor components in the flavor of red wine such as phenolic
compounds guaiacol, 4-vinylphenol and isoeugenol as well as vanillin.
Maillard reaction
The Maillard reaction, also known as non-enzymatic browning, is a very significant
source of flavors in cooked foods. Depending on the food, Maillard reaction flavors can be
deemed positive or negative. Maillard reaction flavors in food systems such as meat (Mottram
and Leseigneur, 1990), coffee (Montavon et al., 2003), cocoa (Countet et al., 2002) and bread
(Kimpe and Keppens, 1996) are highly important and beneficial. On the other hand, the Maillard
reaction is responsible for off-flavors in food systems like fruit juices and also produce pigments
which darkened juice color (Tatum et al., 1975; Haleva-Toledo et al., 1997).
The Maillard reaction takes place between free amino groups from amino acids and
reducing sugars. Reaction products are dependent on not only the starting reducing sugars and
amino acids but are also dependent on time, temperature, water activity and pH of the system.
As with most chemical reactions, the Maillard reaction rate increases with increasing
temperature. Color formation is much greater in the Maillard reaction when the pH is above 7.
However, at lower pH compounds such as furfural and some sulfur compounds are preferentially
formed (Mottram, 1994; Mottram and Whitfield, 1994; Mottram and Leseigneur, 1990).
Compounds created from the Maillard reaction are classified into three groups: 1) Sugar
dehydration/fragmentation products including furans, pyrones, cyclopentenes, carbonyl
compounds and acids 2) Amino acid degradation products including aldehydes, sufur compounds
(e.g. hydrogen sulfide and methanethiol) and nitrogen compounds (e.g. ammonia and amines) 3)
Volatiles produced by further interactions: pyrroles, pyridines, pyrazines, imidazoles, oxoles,
thiazoles, thiophenes, di- and trithiolanes, di- and trithianes, furanthiols and compounds from
aldol condensations (Mottram, 1994).
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As previously mentioned, Maillard reaction products can be considered negative in fruit
juices. One of the main off-flavor compounds in orange juice is 2,5-dimethyl-4-hydroxy-3(2H)-
furanone sometimes called Furaneol or DMHF, which has been well documented to increase
with increasing storage time and temperature in orange juice (Tatum et al., 1975). Haleva-
Toledo et al. (1997) determined the production of Furaneol in orange juice is via the Maillard
reaction between rhamnose and arginine in the presence of the acidic matrices of ascorbic acid in
orange juice.
Strecker degradation
A closely related reaction to the Maillard reaction is Strecker degradation. In Strecker
degradation, the reaction is the oxidative deamination and decarboxylation of α-amino acids with
a dicarbonyl compound (Mottram, 1994). One main difference between Strecker degradation
and the Maillard reaction is the lack of browning products produced in Strecker degradation.
Strecker degradations produce amino acid aldehydes with one less carbon including pyrazines,
oxazoles and thiazoles as well as producing α-amino carbonyls. Strecker degradation produces
the potent methional with a potato-like aroma from the odorless amino acid, methionine.
Methional has been noted in diverse matrices including coffee (Czerny and Grosch, 2000),
cooked mussels (Le Guen et al., 2000), cheese (Milo and Reineccius, 1997), aged beer (da Costa
et al., 2004) and cashew apple nectar (Valim et al., 2003). Methional is an off-flavor in citrus
juice as has been found in grapefruit juice (Buettner and Schieberle, 1999; Lin et al., 2002) and
orange juice (Buettner and Schieberle, 2001a; Bezman et al., 2001).
Microbial
Another possible source of off-flavor compounds in orange juice is from microbial
contamination. Alicyclobacillus strains were studied as a source of medicinal off notes in orange
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juice (Gocmen et al., 2005). Three medicinal aromas were identified and attributed to guaiacol,
2,6-dibromophenol and 2,6-dichlorophenol in orange juice inoculated and incubated with
different Alicyclobacillus strains.
Packaging
An important variable in maintaining the initial orange juice flavor is packaging. A
variety of packages are available, including cans, glass, corrugate, plastics and laminates. An
ideal package would contain the juice and provide an inert system allowing no interaction
between the package, the juice and the outside environment. Glass containers are considered as
close to a totally inert package as possible; however the weight of glass containers is a
disadvantage in terms of transportation costs.
Packaging materials must be evaluated on the basis of cost, weight and ability to protect
the product. Scalping of flavors into the packaging and migration of flavors from the package
into the product are two variables that must be considered. Tetra Brik (Duerr et al., 1981; Marin
et al., 1992) as well as low density polyethylene (LDPE) (Kutty et al., 1994) have been shown to
readily scalp (+)-limonene in orange juice.
Van Lune et al., examined the adsorption of organic compounds in polyethylene
terephthalate (PET) and polyethylene naphthalate (PEN) material (Lune et al., 1997). The
premise of the study examined the importance of absorption of chemicals into plastic bottles and
how the chemicals would effect recycling and reuse by the consumer. If a consumer reuses a
plastic container, absorbed compounds may be present before refilling, causing the possibility of
migration into the product. The migration can add non-typical volatiles to the product thus
producing off-flavors. Absorption of methanol and toluene was reported to increase with an
increase in temperature and is also affected by the composition of the plastic container.
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Fauconnier et al. (2001) studied migration from high density polyethylene (HDPE) into
various liquids including hexane, ethanol, lemon terpenes and their emulsions. A phenolic
compound was shown to migrate from the HDPE into each test liquid and was most likely
attributed to an antioxidant additive. The organoleptic effect of the migration, however, was not
examined.
Orange juice aroma compounds were compared over time by Berlinet et al. (2005) using
glass and various PET containers. Of note, the study determined no statistical difference in
aroma composition between the packaging types. Aroma composition was determined to be
affected by storage over time by reactions within the juice matrix. The researchers suggest the
inherent acidic matrix of the orange juice produced acid-catalyzed reactions which lead to a loss
of aldehydes, ketones, esters, aliphatic alcohols and terpene alcohols; while increasing levels of
4-vinylguaiacol and furfural.
Van Willige et al. (2003) compared the absorption of orange juice flavor compounds in
LDPE, polycarbonate (PC) and PET containers. Polyethylene terephthalate and PC containers
showed only small decreases in limonene, myrcene and decanal through absorption; while LDPE
had a more significant loss of limonene and a smaller decrease in myrcene, valencene, pinene
and decanal. Organoleptic evaluation through duplicate triangle testing did not show a
significant difference between packages at up to 29 days of dark storage at 20°C.
Glass, monolayer PET and multilayer PET package effects on orange juice quality and
shelf life was recently studied by Ros-Chumillas et al. (2007). Ascorbic acid, vitamin C, was
evaluated as a measure of shelf life with a minimum amount of 200µg/mL. The monolayer PET
had a significantly lower shelf life at 4°C, with ascorbic acid dropping below 200µg/mL at 180
days where the multilayer PET and glass were approximately 300µg/mL levels at 300 days.
24
They concluded that the shelf life of the monolayer PET orange juice can be extended through
use of oxygen scavengers, nitrogen headspace and aluminum foil seals in the closure.
Gas Chromatography-Olfactometry
The use of gas chromatography-olfactometry (GC-O) is a technique where the gas
chromatograph separates aroma mixtures into individual components and the human nose is used
as a detector. Modern GC-O instruments use both human and instrumental detectors by splitting
the GC effluent between the sniffing port and an instrumental detector such as flame ionization
detection (FID), mass spectrometer (MS), or pulsed flame photometric detection (PFPD). GC-O
is used to determine which of the volatile compounds in a food matrix have aroma activity and
thus contribute towards the overall aroma of the sample.
The primary advantage for using a human assessor as a detector is the sensitivity and
selectivity of the human nose. The human nose can detect some volatiles at extremely low
concentrations such as bis(2-methyl-3-furyl) disulfide at a threshold level of 8.9 x 10-11 nM
(Buttery et al., 1984). This is significant as the nose is often more sensitive to some aroma-
active compounds than the best instrumental detector. The concept of aroma value has been
developed to determine if a volatile has aroma activity when direct aroma measurement is not
possible or to determine relative aroma strength. Aroma value (sometimes called odor activity
value, OAV) is defined by the ratio of the concentration of an aroma active compound divided
by its detection threshold. Aroma values assigned to a compound in a given matrix will
therefore determine if and by how much the concentration exceeds it threshold value (Mistry et
al., 1997).
How the threshold for a given aroma active compound is calculated can cause a large
variance in the reported threshold. The interaction between a compound and its matrices has an
effect on the threshold. For example, an aroma active compound will have a different threshold
25
if measured in air, water or oil. Generally, a volatile’s threshold will be higher in a food matrix
compared to water because the matrix interacts with the volatile to a greater degree than water.
Plotto et al., (2004) determined the aroma and flavor thresholds for key components in orange
juice using orange pump out (concentrated orange juice whose volatiles have not been restored).
They have reported odor thresholds up to 200 times higher in an orange juice matrix as compared
to published thresholds in water.
GC-O has been used to characterize the odorants in a variety of matrices from coffee
(Holscher and Steinhart, 1995; Akiyama et al., 2002) to wine (Chisholm et al., 1995; Cullere et
al., 2004) to orange juice (Marin et al., 1992; Rouseff et al., 2001a; Schieberle and Buettner,
2001) to orange essence oil (Hognadottir and Rouseff, 2003). Determining which compounds in
a matrix have aroma activity can impact current industrial practices. For example, traditionally
the sesquiterpene valencene is used as an indicator of quality in orange peel oils. However,
Valencene has been recently shown to not have aroma activity at concentrations typically found
in orange oil (Elston et al., 2005).
Early GC-O devices had two main limitations: nasal discomfort caused by hot dry carrier
gas and the lack of sensitivity of the chemical detector as compared to the human nose (Acree
and Barnard, 1994). Dravnieks (1971) enhanced the GC-O technique by using humidified air in
combination with the effluent. Another limitation of GC-O is evaluating individual components
outside of the original matrix (Mistry et al., 1997). GC-O does not take in effect the contribution
of the solubility of the aroma active compounds within the matrix or the interaction of the aroma
active compounds with nonvolatile components within the matrix.
GC-O methods can be categorized into three groups: dilution analysis techniques
including combined hedonic and response measurements (Charm) and aroma extract dilution
26
analysis (AEDA), time-intensity techniques such as OSME, and frequency of detection
techniques including global analysis. Each technique has advantages and disadvantages that will
be discussed.
Dilution techniques operate by sniffing the effluent of an extract in a series of dilutions,
usually in a series of 1:2 or 1:3 dilutions (Acree and Barnard, 1994). Charm analysis (Acree et
al., 1984) constructs a combined response from several experiments where the concentration of
the aroma active compound is directly proportional to the sniffed peak area. Thus a compound
that is detected after more dilutions is considered to be more potent than those compounds which
can be no longer detected after a few dilutions. The relationship between intensity response and
concentration is spelled out in Stevens’ Law: I = k(C-T)n, where I is intensity, k and n are
constants based on the type of compound, C is concentration, and T is threshold (Stevens, 1960).
For aroma, Stevens applies different values to the exponent from 0.55 for coffee odor to 0.6 for
heptane (Stevens, 1961). Charm has been used to study anosmia. Charm values are reportedly
proportional to the amount of stimulus while inversely proportional to the individual subject’s
threshold limit (Marin et al., 1988). AEDA is a dilution technique similar to Charm, where the
flavor dilution, FD, values are comparable to Charm values. However, the main difference being
that AEDA only determines dilution intensity used when calculating FD factor whereas Charm
also takes a compound’s elution duration into effect (Mistry et al., 1997). Another advantage of
AEDA is that it does not require specialized software as in the case of Charm. The main
disadvantage to both dilution techniques is the number of chromatographic runs needed to find
the largest dilution for all compounds in the sample.
Time-intensity techniques are similar to Charm as a compound’s intensity and elution
duration are determined without dilution. The original time-intensity technique is called Osme,
27
developed by da Silva et al. (1994). In Osme the assessor continuously rates the intensity of
aromas using a sliding scale from 0 being no detection to 7 being moderate to 15 being extreme.
The assessor is simultaneously rating the intensity and characterizing the aroma. Panelists need
to be trained to use the equipment as well as develop a common sensory language for
descriptors. Aroma active peaks have to be detected at least 50% of the time by panelists in
order to be considered aroma active. A combined panelist Osmegram is then constructed. An
advantage of Osme over Charm or AEDA is that no dilutions are made and therefore the number
of chromatographic runs is reduced. The main disadvantage of Osme is the aforementioned
training for panelists.
Frequency of detection methods are similar to time-intensity techniques however the
number of panelists is increased while the training per panelist is decreased or in many cases,
eliminated. One main difference between frequency of detection methods and other GC-O
methods is the aroma peak intensity is based on the frequency of detection and not related to the
perceived intensity of the compound. One main disadvantage of this method is the number of
panelists needed, ideally 8 -10 (Pollien et al., 1997). Frequency of detection has been used to
characterize odorants in cooked mussels (Le Guen et al., 2000), red wine vinegar (Charles et al.,
2000), Iberian ham (Carrapiso et al., 2002), French fries (van Loon et al., 2005), leeks (Nielsen
and Poll, 2004), and fresh and smoked salmon (Varlet et al., 2006).
Frequency of detection has also been used in comparing odorants in orange juice of
different cultivars, including blond and blood types (Arena et al., 2006). The study found
difference between blood types (Moro and Tarocco) and blond types (Washington navel and
Valencia late). One of the most intense aroma active compounds found in the blood types,
28
methyl butanoate, was not found in the blond cultivars. Conversely, linalool, was only reported
in blond cultivars
Extraction Methods
Most sample matrices are not able to be directly injected onto a gas chromatograph. The
object then lies to extract the volatile components from the sample and be able to represent the
original matrix. The two main types of extractions are solvent extraction such as liquid-liquid
and direct headspace adsorption of the volatiles onto a solid phase such as Solid Phase Micro
Extraction (SPME).
The solvent used for extraction is dependent on the nature of the food matrix. Organic
solvents are usually used in a matrix that is lipid free and includes matrices such as fruit, berries,
and alcoholic beverages. A separate preparatory procedure is needed to separate lipids from an
organic solvent extraction. When extracting lipids, there is no one standard procedure and the
method and solvent is again dependent on the food matrix (Marinetti, 1962). Often a
combination of different solvents will give the best results. One such matrix that often uses a
combination of solvents is citrus juices, where a common extraction method is with a mixture of
pentane and diethyl ether (Tonder et al., 1998; Lin et al., 2002; Bazemore et al., 2003).
Liquid-liquid extractions can give different results compared to SPME. SPME fibers
have been shown to selectively absorb volatile compounds through competition (Roberts et al.,
2000). For example, Ebeler found in brandy the polydimethylsiloxane SPME extraction was
more selective for esters and acids than liquid-liquid extractions (Ebeler et al., 2000). In citrus,
SPME is more selective for terpenoid compounds as compared to liquid-liquid extractions
(Rouseff et al., 2001a). A SPME fiber (carboxin-polydimethylsiloxane) headspace analysis of
heated orange juice resulted in 86% of the total FID peak area from 3 terpene compounds
(limonene, myrcene, and α-pinene) as compared to 24% in a liquid-liquid extraction of pentane-
29
ether. Rega, et al. (2003) worked to optimize a SPME method for use in orange juice, examining
fiber coatings, exposure time and sample equilibration time. However, the optimized SPME
conditions were skewed to minimize extraction of unpleasant odors and are therefore not fully
representative of the juice.
A recent study (Jordan et al., 2005) compared polydimethylsiloxane (PDMS) and
polyacrylate (PA) SPME fibers in orange juice at different stages in processing (fresh juice,
deaeration and pasteurization. The deaerated process, as compared to fresh juice showed the
greatest processing difference. Both fibers had similar results for alcohols and terpenes.
However, a statistically significant change in aldehydes and esters was noted only with the PA
fiber. The researchers concluded that the PA fiber is more suitable for use in studying
processing affects on orange juice.
Thiamin as a Source of Potent Sulfur Aroma Compounds
Thiamin (vitamin B1) is the second most abundant water-soluble vitamin in orange juice,
and is a more concentrated source than many foods that are better known sources of vitamin B1,
such as whole wheat bread (Nagy and Attaway, 1980; Ting and Rouseff, 1981). Thiamin is
readily degraded by thermal treatment, producing potent sulfur compounds with meaty and
roasted notes. This reaction is important in many food systems, producing flavor impact
compounds typical in meat and breads.
2-methyl-3-furanthiol
2-Methyl-3-furanthiol, MFT, is a significant thermal degradation product of thiamin. This
potent sulfur compound gives an intense savory, meaty aroma. This compound is well known in
meat flavor systems (Mottram, 1991; Grosch and Zeiler-Hilgart, 1992; Kerscher and Grosch,
1998) and has a low aroma threshold of 6.14 x 10-8 mM/L water (Munch and Schieberle, 1998).
MFT has been found in a number of different flavor systems, including coffee (Hofmann and
30
Schieberle, 2002; Tressl and Silwar, 1981), cooked brown rice (Jezussek et al., 2002), beer
(Lermusieau et al., 2001), reconstituted grapefruit juice (Lin et al., 2002) and as an off-flavor in
orange juice (Bezman et al., 2001).
Bis(2-methyl-3-furyl) disulfide
Thiols are known to readily oxidize into their corresponding disulfide. Hofmann et al.,
1996 (1996) studied the oxidative stability of odor active thiols. Results show that after 10 days
of storage at 6°C, 53% of a dilute ethereal MFT solution was oxidized to its dimer, bis(2-methyl-
3-furyl) disulfide, MFT-MFT. Bis(2-methyl-3-furyl) disulfide has also been reported in meat
flavor systems (Evers et al., 1976; Farmer and Mottram, 1990). Bis(2-methyl-3-furyl) disulfide,
portraying a savory, meaty aroma is responsible for the most potent food aroma to date, having
an odor threshold of 8.9 x 10-11 mM water (Buttery et al., 1984). The same study also
determined MFT-MFT to be responsible for the characteristic odor of vitamin B1.
Thiamin degradation pathway
The thermal degradation pathway, determined by van der Linde and coworkers (1979),
involves the rupturing of the C-N bond between the pyrmidine and thiazoles moieties of thiamin
by a hydroxyl ion attack (Figure 2-2). The thiazole moiety (III) then degrades to form other
potent aroma-active thiazoles such as 4,5-dimethylthiazole (roasted meat) and 4-methylthiazole
(green hazelnut).
However, from an aroma perspective, the hydrolysis of the thiazole ring in the thiamin
hydrochloride (Figure 2-2) leads to a key aroma intermediate, 5-hydroxy-3-mercapto-2-
pentanone (VI). This intermediate produces many aroma active thiophenes and furans, including
MFT (van der Linde et al., 1979; Guntert et al., 1990; Guntert et al., 1992).
31
Alternate pathways for the production of 2-methyl-3-furanthiol
Another pathway for the production of MFT is through the Maillard reaction. Meynier et
al. (1995) observed the formation of MFT in a cysteine/ribose model system where the MFT
formation was greatly increased at a lower pH of 4.5 with almost a 2.5 fold increase from pH 5.0
and a 10 fold increase from pH 6.0.
Whitfield et al. (1999), studied the reaction between 4-hydroxy-5-methyl-3(2H)-furanone
(norfuraneol) and cysteine or hydrogen sulfide. MFT was found in both the norfuraneol/cysteine
and norfuraneol/hydrogen sulfide systems at similar concentrations. The author suggests that
this points to only hydrogen sulfide being necessary and not needing other cysteine degradation
compounds. Cerny et al. (2003), further investigated the possible source MFT from norfuraneol
a model system of cysteine, ribose and norfuraneol. A 13C5-labeled ribose and norfuraneol were
reacted with cysteine. The resulting MFT contained some of the 13C-label 93% of the time,
suggesting that the more probable source being the cysteine/ribose reaction.
A study by Bolton et al. (1994) combined thiamin and cysteine in model systems. Four
model systems were examined for MFT formation using combinations of thiamin, cysteine,
labeled cysteine and D-xylose at a pH range of 5.5 to 5.8. Of interest, the only model system
that MFT was not detected in was the only system without thiamin addition, suggesting the
primary mechanism for the formation of MFT, under the conditions of the model system,
involves thiamin degradation. In the two model systems using labeled cysteine, only a net 8% of
the MFT contained the labeled sulfur, 34S, from cysteine as compared to the unlabeled cysteine
model solution.
Of note, much of the thiamin degradation studies have been carried out at elevated
temperatures on meat systems rather than exploring thiamin degradation in other matrices such
as orange juice that would not receive the elevated temperatures as compared to the cooking of
32
meat. Ramaswamy et al. (1990) determined the kinetics of thiamin degradation in an aqueous
solution at temperatures ranging from 110°C to 150°C to be first order reactions. Van der Linde
et al. (1979) determined that MFT is a product of 5-hydroxy-3-mercapto-2-pentanone from a
breakdown of thiamin at 130°C in an aqueous system.
Hartman and co-workers (1984b) studied the effect of water activity, aw, in a model meat
system containing thiamin, with heat treatment at 135°C for 30 minutes. Results show a higher
aw produced more boiled meat-like aroma such as MFT while the lower aw system produced
more roasted meat-like aromas including 2-methylthiophene with a roast beef aroma.
Meynier and Mottroam (1995) studied pH effect in model meat systems with thermal
reactions at 140°C. The study determined a cysteine model system at a lower pH of 4.5
produced the highest amount of MFT.
One study does look at MFT at a lower temperature of 6°C (Hofmann et al., 1996), with
the purpose of determining the oxidative stability of odor-active thiols including MFT. MFT
was shown to have the highest concentration over the 10 day storage in n-pentane and
dichloromethane where the concentration readily decreased in a diethyl ether system.
Conversely, MFT-MFT showed the highest formation rate in diethyl ether, with very little being
formed in a dichloromethane or n-pentane system.
33
34
OH
OH
d-Limonene
α-Terpineol
+HOH -H+
H+, -HOH
Linalool
+
+
H+, HOH
Figure 2-1. Pathways for α-terpineol formation from linalool and (+)-limonene (Haleva-Toledo et al., 1999).
N
N
N+
SOH
NH2
N
NNH2
OH N
SOH
OH-H3O+
N
N
NH
SOH
NH2
CHO
O
SH
OH
H3O+
HCOOH
N
N
NH2
NH2
Cl-
+
(A)
(B) (C)
(D)
++
(E) (F)
Figure 2-2. Thiamin thermal degradation pathways. A =thiamin hydrochloride, B = pyrimidine
moiety, C = thiazoles moiety, D = diaminopyrimidine, E = formic acid, and F = 5-hydroxy-3-mercapto-2-pentanone. Adapted from (van der Linde et al., 1979; Guntert et al., 1990; Mottram, 1991).
CHAPTER 3 AN AROMA COMPARISON BETWEEN ORANGE JUICES OF DIFFERING
ORGANOLEPTIC QUALITIES
Introduction
Orange juice is ranked number one in fruit juice consumption in America (Pollack et al.,
2003). One of the major attributes consumers are looking for is flavor. Considerable research
has been spent examining the volatile components that are responsible for the desired aroma and
flavor in orange juice. Much of this research has involved the use of thermally abusive storage
studies to determine changes in volatile content and formation of off-flavor compounds. The
assumption being that elevated thermal temperatures will produce a larger quantity of storage
off-flavors in a shorter period of time. Thermal abuse studies will also produce storage off-
flavors in higher concentrations making volatile identification easier. Tatum et al. (1975) stored
single-strength canned orange juice at 35°C for up to 12 weeks and identified ten degradation
compounds. Of the degradation compounds, three exhibited negative aroma impact in the
orange juice: α-terpineol, 2,5-dimethyl-3(2H)-furanone (Furaneol or DMHF) and 4-
vinylguaiacol. These three compounds were determined to be above their taste thresholds; and
when added to a control orange juice imparted a characteristic aroma of heat-abused juice.
Moshonas and Shaw (1989) noticed an increase of α-terpineol during storage. Tonder et
al. (1998) studied stored reconstituted orange juice for up to 12 months at 20°C. Earlier studies,
(Walsh et al., 1997; Peleg et al., 1992; Naim et al., 1997) show minimal formation of both 4-
vinyl guaiacol and Furaneol at temperatures under 30°C.
Chemical reaction rates are known to increase with a rise in temperature. This is
explained through the Arrhenius equation and the relationship between temperature and the rate
at which a reaction takes place. The relationship is explained in the following equation:
35
k = Ae-Ea/RT
where k is the rate constant, A is the frequency factor (specific to a particular reaction), e is the
math quantity or exponent, Ea is the activation energy or minimum energy required for the
reaction, R is the gas constant and T is temperature in °K. Through this equation, either a
temperature increase or a decrease in Ea results in an increase in reaction rate. In orange juice,
an increased reaction rate would derive from temperature as a decrease in Ea being would need a
catalyst which would not normally be present in juice. A general rule of thumb for reactions
around ambient temperature states that for every 10°C increase in temperature a reaction rate
doubles. However, in a complex matrix such as orange juice, the reaction rates of competing
reactions can differ considerably. The dominant reaction at a temperature of 40 to 50°C may not
be the dominant reaction at a lower temperature range of 4 to 20°C. The dominant reactions that
produce specific off-flavors at higher storage temperatures may not be the same reactions that
produce off-flavors that develop at lower storage temperatures. Therefore, the reactions that
produce flavor changes under typical industrial storage conditions may not be the same as those
which occur under an accelerated storage study. The purpose of my study was to evaluate flavor
differences in products obtained from supermarkets without subjecting the samples to additional
thermal abuse and determine which aroma active compounds differentiate between poor quality
and good quality flavor.
Materials and Methods
Survey of commercial orange juice
Juices for this survey were collected from local supermarkets and consisted of orange
juice reconstituted from concentrate produced in Florida. All juices were within the product
expiration dates and contained the Florida Seal of Approval on the container. The juices were
formed a market basket survey of orange juice, categorizing each juice into one of three
36
categories: above average, average, and below average flavor quality based on an informal
organoleptic panel. One above average juice and one below average juice were chosen to
compare the extremes between the categories. The above average quality RECON juice was
purchased refrigerated in a gable-top carton; while the below average flavor quality juice was a
canned RECON juice packaged purchased at ambient temperature. Both juices were chilled for
sensory evaluation.
Chemicals
The following chemicals were obtained commercially from Aldrich (Milwaukee, WI): 1-
Figure 5-1. SPME headspace samples of GC-O aromagrams comparing day 7 and 42, where peak intensities were inverted for day 42 data. Peak number corresponds to compound numbers in Table 5-1.
93
S
SH
3-Thiophenethiol
(peak 6)
SO
2-Acetylthiophene(peak 8)
O
S S
2-methyl-3-(methyldithio) furan
(peak 11)
N
S
4,5-Dimethylthiazole
(peak 5)
SO
2-Formyl-5-methylthiophene
(peak 10)
Figure 5-2. Structures of select aroma active sulfur compounds detected in the model orange juice solution. Peak numbers in parentheses correspond to peak numbers in Table 5-1.
94
2.0 3.0 4.0 5.0 6.01.0
Sul
fury
Frui
ty/g
reen
Trop
ical
frui
ty
Roa
sted
mea
t
2-Methyl-3-furanthiol
Hyd
roge
n su
lfide
Dim
ethy
lsul
fide
Time (min)
GC
-O re
spon
sePF
PD re
spon
se
Figure 5-3. Comparison between PFPD chromatogram and corresponding aromagram from a model orange juice solution stored for 7 days at 35°C. First 6 min shown, to clearly illustrate which of the early PFPD peaks were aroma active as well as to demonstrate that there was no sulfur activity associated with peaks 2 and 3. SPME injection using a DB-5 column. See methods section for additional experimental details.
95
96
0
0.0002
0.0004
0.0006
0.0008
0.001
0.0012
0.0014
0 10 20 30 40 50 60Time (days)
Con
cent
ratio
n (m
M/L
)
2-Methyl-3-furanthiol
Bis-(2-methyl-3-furly) disulfide
Figure 5-4. MFT and MFT-MFT concentrations in thiamin model orange juice solutions stored at 35°C in the absence of light as determined by PFPD.
CONCLUSIONS
The underlying objective for my study was to determine what factors can affect the quality
of orange juice that a consumer purchases and which of these factors can be manipulated to
provide the highest quality of orange juice to the consumer. Factors that can affect the quality
include determining what aroma impact compounds contribute to quality orange juice as well as
compounds that would negatively contribute towards the flavor. Other factors that can affect the
quality of orange juice include temperature, packaging and flavor precursors such as thiamin.
Aroma impact compounds were determined in commercially purchased orange juices that
were determined organoleptically to be of differing quality. Aldehydes including hexanal,
heptanal, octanal, nonanal, decanal, undecanal and geranial were determined to contribute to the
above average quality orange juice; where as known off-flavors 4-vinylguaiacol and methional
contributed to the detriment of the below average juice.
A second study determined how the aroma impact compounds from the above study
change over time, temperature and packaging. Aldehydes including (Z)-3-hexenal (green banana
aroma), octanal (lemon aroma) and decanal (woody, green aroma) diminished and/or were lost
over time and temperature. Off-flavor compounds such as carvone (licorice aroma) and m-cresol
(manure aroma) were not found at day 0 and were formed over time. Polyethylene terephthalate
samples had known off-flavor compounds that were not in glass samples, including 2-methyl-3-
The last study determined the probable source of the off-flavor compounds 2-methyl-3-
furanthiol and bis(2-methyl-3-furyl) disulfide through a model orange juice study to be the
second most abundant water soluble vitamin in orange juice, thiamin.
Orange juice manufacturers can use the information from this study to tailor add-back
flavor packages with the aroma active compounds that contribute to quality orange juice.
97
Manufacturers can also take into account the type of packaging that is used and the shelf-life of
the product at higher real world temperatures and the affect it has on orange juice quality.
Finally, with a recent trend towards fortification of orange juice and beverages with vitamins and
phytochemicals, for example calcium fortified orange juice; this study shows that the levels of
thiamin present in orange juice can cause off-flavor production in an orange juice matrix.
Additional fortification with thiamin would cause an increase in the off-flavors 2-methyl-3-
furanthiol and bis(2-methyl-3-furyl) disulfide.
98
99
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BIOGRAPHICAL SKETCH
J. Glen Dreher grew up in West Palm Beach, FL. He attended Purdue University and
graduated in 1997 with a B.S. in Food Science. In spring 1999 he entered graduate school at the
University of Florida in food science. He spent a year in Gainesville, FL taking course work and
then relocated to Winter Haven, FL for his doctoral research at the Citrus Research and
Education Center in Lake Alfred, FL. He took a job at Jim Beam Brands in Clermont, KY
February, 2003 working in new product development. He has since completed his Ph.D. in