ultraviolet light induced degradation of patulin and ascorbic acid in apple
TRANSCRIPT
The Pennsylvania State University
The Graduate School
Department of Food Science
ULTRAVIOLET LIGHT INDUCED DEGRADATION OF PATULIN AND
ASCORBIC ACID IN APPLE JUICE
A Dissertation in
Food Science
by
Rohan V. Tikekar
2010 Rohan V. Tikekar
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
May 2010
The dissertation of Rohan V. Tikekar was reviewed and approved* by the following:
Luke F. LaBorde
Associate Professor of Food Science
Thesis Co-Advisor
Co-Chair of Committee
Ramaswamy C. Anantheswaran
Professor of Food Science
Thesis Co-advisor
Co-Chair of Committee
Hassan Gourama
Associate Professor of Food Science
Ali Demirci
Associate Professor of Agricultural and Biological Engineering
John D. Floros
Professor of Food Science
Head of the Department of Food Science
*Signatures are on file in the Graduate School
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ABSTRACT
The overall goal of this research was to study the effect of UV processing on
patulin (a mycotoxin commonly found in apple cider and juice) and ascorbic acid
(vitamin C) in model apple juice system and in apple juice.
The first objective was to study the kinetics of patulin degradation during
exposure to UV light in 0.5% malic acid buffer (model apple juice system). A collimated-
beam batch UV (254 nm) apparatus was used. The effects of added ascorbic acid (AA),
tannic acid, and suspended solids on patulin degradation in 0.5% malic acid buffer were
studied using Box-Behnken design. Results showed a first order degradation kinetics for
patulin. The degradation rate constant (cm2/J) was not significantly affected by incident
intensity (0.8-1.8 mJ/cm2) (p>0.05), buffer pH (3.0-3.6) (p>0.05) and initial
concentration of patulin (0-1000 ppb) (p>0.05). Presence of tannic acid, (0-1 g/L) and
suspended particles (0-100 NTU) significantly reduced the patulin degradation rate
constant (p<0.05), while AA (0-100 mg/L) did not affect the reaction rate constant
(p>0.05).
The second objective was to study the UV induced degradation of AA in 0.5%
malic acid buffer (apple juice model system) and in apple juice. AA degradation occurred
more rapidly in juice compared to 0.5% malic acid. Further studies demonstrated that UV
degradation of AA in 0.5% malic acid was more rapid at higher UV dose levels and that
reaction deviated from zero order. AA degradation did not change significantly (p>0.05)
between pH 2.4 and 3.3, but increased as the pH of the buffer was raised from 3.3 to 5.5
(p<0.05). Increasing malic acid concentration between 0.1 and 1%, at a constant pH of
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3.3, increased AA degradation (p<0.05) although there was no difference between 0.5
and 1.0 % (p>0.05). With increasing concentration of tannic acid in buffer, AA
degradation rate decreased significantly (p<0.05), possibly due to competitive absorption
of UV light. Addition of 10% sucrose to buffer showed no significant effects (p>0.05),
but addition of 10% glucose decreased AA degradation (p<0.05). However, addition of
10% fructose increased AA degradation significantly (p<0.05), perhaps due to breakdown
products of this sugars reacting with AA. AA degradation in malic acid and in apple
juice continued during storage in the absence of light. Post UV treatment degradation was
more rapid at higher initial UV dose levels and at higher storage temperature.
The third objective was to understand the mechanism of UV induced AA
degradation. Electron paramagnetic resonance (EPR) spectroscopy studies demonstrated
that ascorbate radicals formed in AA solutions in phosphate buffer at pH 7.0 and in malic
acid buffer between pH 3.3 and 6.0. Lesser amounts of ascorbate radicals formed at lower
pH levels and only trace amounts were detected at pH 3.3. Ascorbate radicals in UV
treated AA solutions continued to form at higher rates than that for identically stored
untreated AA solution. High pressure liquid chromatography-mass spectroscopy (HPLC-
MS) analysis of UV treated samples demonstrated that as AA levels decreased,
dehydroascorbic acid (DHA) and 2, 3-diketogulonic acid (DKGA) levels increased. We
propose that UV processing of AA leads to formation of ascorbate radical that leads to
the formation of DHA, which further degrades into DKGA.
v
TABLE OF CONTENTS
LIST OF FIGURES ………………………………………………………………………x
LIST OF TABLES ....................................................................................................... ….xv
ACKNOWLEDGEMENTS ......................................................................................... …xvi
Chapter 1 Introduction ............................................................................................ …...1
Chapter 2 Literature review and statement of problem ....................................... …...3
2.1 Patulin …………….………………….……………………………………..……3
2.1.1 Role of patulin and other mycotoxins in fungi……………………...3
2.1.2 Patulin occurrence in apple products………………………………..6
2.1.3 Patulin toxicology…………………………………………………...7
2.1.4 Processing stability of patulin ………………………………………9
2.1.5 Alternative technologies for patulin reduction ……………………..9
2.2 Ascorbic acid ………………………………………………………………..11
2.2.1 Chemistry and antioxidant activity of ascorbic acid ………………11
2.2.2 Physiological role of ascorbic acid ………………………………..15
2.3 Ultraviolet light processing of foods………………………………………...16
2.3.1 Mode of action …………………………………………………… 16
2.3.2 UV dose measurement …………………………………………… 19
2.3.3 Factors influencing the efficacy of the UV treatment ……………..22
2.3.4 Processing equipment ……………………………………………..24
2.3.5 UV processing of food products…………………………………...28
2.3.5.1 Fresh fruits and vegetables ……………………………....28
2.3.5.2 Meat, poultry and dairy products………………………...30
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2.3.5.3 Fruit juices……………………………………………….30
2.3.6 Stability of ascorbic acid during UV processing of juice …………31
2.4 Statement of problem ………………………………………………………..32
2.4.1 Specific objectives. ………….…………………………………….33
2.5 references ……………………………………………………………………35
Chapter 3 Patulin degradation in a model apple juice system during ultraviolet
light processing………………………...…………………......................................48
3.1 Introduction ………………………………………………………………….49
3.2 Materials and methods ………………………………………………………51
3.2.1 UV treatment equipment ………………………………………..…51
3.2.2 UV dose measurement …………………………………………….53
3.2.3 Sample preparation…...……………………………………………54
3.2.4 Extraction and quantification of patulin …………………………..55
3.2.5 HPLC analysis……………………………………………………..56
3.2.6 Data analysis………..……………………………………………...56
3.2.7 Statistical analysis ………………………………………………....59
3.3 Results and discussion…………………………………………………….....60
3.3.1 Patulin degradation in malic acid …………….................................60
3.3.2 Effect of initial concentration……………………………………...63
3.3.3 Effect of pH………………………………………………………..63
3.3.4 Effect of ascorbic acid, tannic acid and suspended particles...….....66
3.4 Conclusions……………………………………………………………..........69
3.5 References …………………………………………………………………...73
vii
Chapter 4 Ascorbic acid degradation in a model juice system and in apple juice
during ultraviolet light processing and storage..…………………...……………77
4.1 Introduction ………………………………………………………………….78
4.2 Materials and methods ………………………………………………………81
4.2.1 UV processing equipment …………………………………………81
4.2.2 UV dose measurement …………………………………………….84
4.2.3 Apple juice ……… ………………………………………………..84
4.2.4 HPLC analysis …………………………………………………….84
4.2.5 Data analysis ………………………………………………………85
4.2.6 Statistical analysis …………………………………………………87
4.3 Results and discussion ………………………………………………………87
4.3.1 Comparison of AA degradation in apple juice and juice model
system……………………………………………………..……………..87
4.3.2 Kinetics of AA degradation in 0.5% malic acid …………………..88
4.3.3 Effect of pH……… ………………………………………………..92
4.3.4 Effect of malic acid concentration………………………..………..94
4.3.5 Effect of absorbance ………………………………………………94
4.3.6 Effect of sugars…………. ……………………………………….100
4.3.7 Interaction of tannic acid and fructose in buffer …………………103
4.3.8 Post UV-treatment effects on AA degradation ……………..……105
4.4 Conclusions…………………………………………………………………109
4.5 References ………………………………………………………………….110
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Chapter 5 Ultraviolet light induced degradation of ascorbic acid: Identification of
degradation products and a proposal for a reaction mechanism ...……...........115
5.1 Introduction ………………………………………………………………...116
5.2 Materials and methods ……………………………………………………..117
5.2.1 Reagents ………………………………………………………….118
5.2.2 UV treatment equipment ………………………………………....118
5.2.3 UV dose measurement ……………………………………….......120
5.2.4 Electron spin resonance (ESR) spectroscopy ……………………121
5.2.5 HPLC-MS ………………………………………………………..121
5.3 Results and discussion ……………………………………………………..122
5.3.1 ESR analysis …………………………………………………......122
5.3.1.1 AA degradation kinetics ……………………………….124
5.3.1.2 Effect of fructose on AA degradation rate ……………..127
5.3.1.3 Post-UV processing storage degradation of AA ……….130
5.3.1.4 Detection of ascorbate radical in malic acid buffer ……132
5.3.2 HPLC-MS analysis …………………………………………..…..135
5.4 Conclusions……………………………………………………………..…..139
5.5 References ………………………………………………………………….141
Chapter 6 Overall conclusions and suggestions for future work ………………….144
6.1 Overall conclusions…………………………………………………………144
6.2 Suggestions for future work………………………………………………...145
Appendix A Patulin degradation in model apple cider system ……………………….148
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Appendix B Validation of model apple juice system …………………………………152
Appendix C Patulin degradation in apple juice ………………………………...……..153
Appendix D Effect of furan on degradation rate of patulin…………………...……….161
Appendix E Ascorbic acid degradation rate in malic acid buffer during UV processing
using Cidersure 1500 …………………………………………………………………..162
Appendix F Degradation of patulin and ascorbic acid in apple cider and apple juice
during the UV processing using Cidersure® continuous reactor…………………….…163
12000
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LIST OF FIGURES
Figure 2-1: Structure of patulin…………………………………………………………..4
Figure 2-2: Reaction scheme for ascorbic acid degradation ……………………………13
Figure 2-3: UV induced microbial death curve…………………………………………18
Figure 2-4: Bench-top batch UV reactor ………………………………………………..25
Figure 2-5: (a) Design of CiderSure® continuous UV system (Courtesy: Phil Hartman,
FPE, Macedon NY) (b) Cross section of the process tube ……………………………...26
Figure 3-1: Schematic representation of collimated UV beam equipment……………...52
Figure 3-2: Representative HPLC chromatograms of patulin (C0=1000 ppb) (top) No UV
(bottom) after UV dose of 5.04 J/cm2……………………….……………….…………..57
Figure 3-3: Effect of incident intensity on the degradation of patulin (C0=1000 ppb) in
0.5% malic acid buffer (pH 3.3). Each data point represents average of three
measurements ± standard deviation……………………………………………………...61
Figure 3-4 Effect of incident intensity on the degradation of patulin (C0=1000 ppb) in
0.5% malic acid buffer (pH 3.3). Each data point represents average of three
measurements ± standard deviation.………………………………………….………….62
Figure 3-5: Effect of initial patulin concentration on the rate of degradation in 0.5%
malic acid buffer (pH 3.3). Each data point represents an average of three measurements
± standard deviation……………………………………………..……………………….64
Figure 3-6: Effect of malic acid buffer pH on the rate of degradation rate of patulin
(C0=1000 ppb). Each data point represents average of three measurements ± standard
deviation.…………………………………………………………….…………………...65
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Figure 3-7: Surface plots for Box-Behnken design for model apple juice system
at (a) 50 NTU (b) 100 NTU ………………..……………………………………………71
Figure 4-1: Schematic representation of the collimated beam batch UV reactor……….82
Figure 4-2: A representative HPLC chromatogram of AA (AA0= 100mg/L) (a) control
(b) after UV dose of 5.04 J/cm2 ………….………..…………………………………….86
Figure 4-3: UV degradation of AA in apple juice (AA0=170 mg/L, pH 3.5) and in 0.5 %
malic acid (AA0=190 mg/L, pH 3.3). Each data point represents an average of three
measurements + standard deviation………………………………………….….……....89
Figure 4-4: UV degradation of AA in 0.5% malic acid buffer (pH 3.3) at varying initial
AA0 concentrations. Each data point represents an average of three measurements +
standard deviation ……………………………………………………………………....91
Figure 4-5: Effect of pH on UV degradation of AA in 0.5% malic acid buffer. Each data
point represents an average of three measurements + standard deviation……………....93
Figure 4-6: Effect of malic acid concentration (pH = 3.3) on UV degradation of AA
(AA0= 100 mg/L). Each data point represents an average of three measurements +
standard deviation……………………………..…………………………………………95
Figure 4-7: UV induced AA degradation (AA0= 100 mg/L) in distilled water (pH 6.0).
Each data point represents the average of two measurements…………………………..96
Figure 4-8: Effect of added caramel (60 mg/L) on the UV induced degradation rate of
ascorbic acid AA (AA0= 150 mg/L) in malic acid (pH 3.3). Each data point represents an
average of two measurements + standard deviation…………………………………….98
xii
Figure 4-9: Effect of tannic acid concentration on degradation of AA (AA0=100 mg/L)
in 0.5% malic acid (pH 3.3). Each data point represents an average of three
measurements + standard deviation…………………………………………………….99
Figure 4-10: Effect of fructose (10%), glucose (10%) and sucrose (10%) on UV induced
degradation of AA (AA0= 100 mg/L) in 0.5% malic acid (pH 3.3). Each data point
represents an average of three measurements + standard deviation……………………101
Figure 4-11: Dependence of UV induced AA degradation (AA0= 100 mg/L) on fructose
concentration added to 0.5% malic acid (pH 3.3). Each data point represents an average
of three measurements + standard deviation……………………………………………102
Figure 4-12: AA (AA0=200 mg/L) degradation rate in malic acid buffer simultaneously
incorporated with tannic acid (200 mg/L) and fructose (5% w/v) as compared AA
degradation in malic acid buffer and apple juice. Each data point represents an average of
three measurements ± standard deviation ……………………………………………...104
Figure 4-13: Effect of initial UV dose on post-processing storage degradation of AA
(AA0=100 mg/L) in buffer (pH 3.3) at 25 °C. Each data point represents an average of
three measurements + standard deviation………………………………………………106
Figure 4-14: Effect of storage temperature (4 °C and 25
°C) on UV treated (5.76 J/cm
2)
samples (AA0=100 mg/L) in 0.5% malic acid (pH 3.3). Each data point represents an
average of three measurements + standard deviation…………………………………..107
Figure 4-15: Post processing degradation of AA (AA0=200 mg/L) in UV treated apple
juice (1.2 J/cm2) and then stored at 4 °C. Each data point represents an average of three
measurements + standard deviation…………………………………………………….108
Figure 5-1: Schematic representation of the batch UV system………………………..119
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Figure 5-2: Ascorbate radical standard generated in AAPH (250 mM) and ascorbic acid
4mM in 10 mM pH 7.0 phosphate buffer at pH 7.0 ( 1:1 v/v) . aH = hyperfine coupling
constant, C1 = crest height 1, C2 = crest height 2, T1 = trough height 1. Absolute peak
height = (C1 + C2 )/2 + T1 ……………………………………………………………...123
Figure 5-3: ESR spectrum for ascorbate radical generated in phosphate buffer (pH 7.0) before (a)
and after (b) UV exposure for 1 hour (Incident intensity = 1.4 mW/cm2). …………………..125
Figure 5-4: Comparison of AA (AA0= 450 mg/L) degradation determined by HPLC and
AA radical formation determined by EPR in phosphate buffer (pH 7.0) (Incident UV
intensity = 1.4 mW/cm2 ). ESR data points represent the average of three measurements ±
standard deviation. HPLC data points represent the average of two measurements ±
standard deviation. …………………..…………………………………………………126
Figure 5-5: Comparison of AA (AA0= 450 mg/L) degradation determined by HPLC and
AA radical formation determined by ESR in phosphate buffer (pH 7.0) containing 10%
(w/v) fructose (Incident UV intensity = 1.4 mW/cm2). ESR data points represent the
average of three measurements ± standard deviation. HPLC data points represent the
average of two measurements ± standard deviation….………………………………...128
Figure 5-6: Presence of AA radical after UV treatment (10.08 J/cm2) (AA0=450 mg/L) in
phosphate buffer (pH 7.0) held at 21 °C. Each data point represents an average of three
measurements ± standard deviation…………………………………………………….131
Figure 5-7: Effect of malic acid buffer pH on signal strength of ascorbate radical peak in
EPR after 1 hr of UV exposure at incident intensity of 1.4 mW/cm2. (a) pH 3.3 (b) pH 4.2
(c) pH 6.0……………..………………………………………………………………...133
xiv
Figure 5-8: Representative HPLC-MS chromatogram of products formed after UV
exposure of AA in 0.5% malic solution (pH 3.3) for 3 hours (Incident intensity = 1.4
mW/cm2). (a) DHA (b) AA (c) DKGA. (AA0=400 mg/L)………………...…………..136
Figure 5-9: Degradation of AA and formation of DHA in malic acid buffer (pH 3.3) after
exposure to UV light (Incident intensity = 1.4 mW/cm2) determined by HPLC-MS. Data
is an average of two measurements ± standard deviation. (AA0=400 mg/L)…………..137
Figure 5-10: Formation of DKGA in malic acid buffer (pH 3.3) after exposure to UV
light (Incident intensity = 1.4 mW/cm2) determined by HPLC-MS. Data is an average of
two measurements ± standard deviation. (AA0=400 mg/L)..…………………………..139
Figure 5-11: Proposed mechanism for UV induced degradation of AA………………140
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LIST OF TABLES
Table 2-1: UV dose required for 1-log reduction of common food borne microorganisms
(adapted from Coohil and Sagripanti, 2008) …………………………………………….20
Table 3-1: Experimental design and corresponding patulin degradation rate constant for
model apple juice system ………………………………………………………………..67
Table 3-2: ANOVA table for statistical model for patulin degradation in model apple
juice (- indicates data not shown as statistically not significant)………………………...68
Table 3-3: Fit statistic for the model. Master model represents the statistic for model that
includes all parameters and Predictive model includes only significant parameters…….70
Table 4-1: Absorbance values (254 nm) for chemical compounds used in
experiments………………………………………………………………………………90
Table 5-1: EPR peak heights representing the amount of ascorbate radical present in
0.5% malic acid as a function of pH and UV exposure time (Incident intensity 1.4
mW/cm2)………………………………………………………………………………..134
xvi
ACKNOWLEDGEMENTS
I would like to this opportunity to thank my advisors Dr. Luke LaBorde and Dr.
Anantheswaran for being the mentors throughout the course of this dissertation. Their
unwavering support and direction made this endeavor possible. They helped me to
improve my technical skills, realize my limitations and overcome them. I am grateful to
them for providing me the financial support without which I would not have achieved
this. I am greatly indebted to them.
I would like to express my gratitude towards my committee members Dr. Hassan
Gourama and Dr. Ali Demirci for their constant guidance and insights. They helped me to
hone my skills in various facets of research.
I am grateful to Dr. Ryan Elias, Dr. John Coupland and Dr. Devin Peterson for
allowing me to use the facilities in their laboratories and for the stimulating discussions
that provided the project with momentum. I would like to appreciate all the graduate
students, departmental support staff and faculty for making my stay in the department
such a pleasant experience.
Thank you Smaro, Julius, Minal, Tanuj, Lisa and Sunando for being such great
friends and lab mates. Special thanks to Smaro for being so patient with my experimental
work. Thank you Jyotsna, for your love and support.
I am greatly indebted to my parents for their unwavering support and
encouragement. Their love and support helped me see through the tough times. I am
forever grateful to them for their numerous sacrifices for me.
1
Chapter 1
Introduction
The food processing industry is witnessing an increasing interest in non-thermal
food processing technologies such as ultraviolet (UV) irradiation. Compared to thermal
disinfection treatments, UV radiation may have fewer adverse quality effects and operate
at lower costs (Koutchma, 2009). UV technology is extensively used in water purification
and wastewater treatment (Legrini et al., 1993); however it has only recently found
applications in food processing. Although UV processing has been applied to variety of
food products such as fresh fruits, vegetables, and meats, it has been found to be most
effective for fruit juices as they offer better penetration to UV light. UV processing has
been successfully applied to apple cider to ensure the FDA mandated 5-log reduction in
human pathogens such as Escherichia coli O157:H7 and Cryptosporidium parvum.
Occurrence of patulin in apple products is a chronic and recurrent phenomenon.
Patulin is a mycotoxin produced by fungi such as Aspergillus spp. and Penicillium
expansum which often infest apples. Patulin has been shown to be cytotoxic, mutagenic
and teratogenic in animals and human cell lines. As a result, US FDA has set a limit of 50
μg/L in apple products (US FDA 2001). Patulin is thermostable and therefore
conventional pasteurization does not reduce patulin loads significantly. Several
alternative technologies have been developed to address this issue, but they have been
met with a limited success. A significant proportion of apple cider is now being
2
processed with UV light. Therefore, there is an incentive to study if patulin is sensitive to
UV light.
UV processing is still in its infancy and considerable research on the effect of UV
light on several food chemicals is necessary before widespread commercialization of this
technology can occur. Ascorbic acid (AA) (vitamin C) is a suitable representative
compound to study the severity of UV processing. AA is present in fruit juices either
naturally or through fortification. AA is one of the more reactive compounds and its loss
during processing is used as an indicator of processing severity. Therefore, it was decided
to study the sensitivity of AA towards UV light.
This dissertation was focused on studying the feasibility UV processing as an
effective technology for reducing the patulin load and investigates the sensitivity of AA
towards UV light. Effect of several factors on the rate of degradation of these compounds
was investigated and the degradative pathway for AA was identified.
3
Chapter 2
Review of literature and statement of problem
2.1 PATULIN
2.1.1 Role of patulin and other mycotoxins in fungi
Mycotoxins are predominantly produced by several fungal genera including
Aspergillus, Claviceps, Penicillium and Fusarium spp. (Moss, 2002). Patulin (4-
hydroxyl-4H-furo (3, 2c)-pyran-2 (6H)-on) (figure 2-1) is produced by Byssochlamys,
Eupenicillium, Penciillium, Aspergillus and Paecilomyces spp (Moake et al.; 2005). By
definition mycotoxins are secondary metabolites produced by fungi that are toxic to
vertebrates and other animal groups in low concentrations (Bennett, 1987). There are
multiple generic reasons for synthesizing toxins. Because mycotoxins are secondary
metabolites, they have little or no role to play in the growth of the organism. These are
small molecules with molecular weight less than 1000Da and because of this do not
generate immune-response in hosts. Some mycotoxins are produced as a response to the
stress conditions, mainly the exhaustion of the limiting nutrient. As the limiting nutrient
decreases to a critical level, the cell enters a stationary or maintenance phase where the
dry weight accumulates into the cell, as carbon source is still in abundance. This leads to
biosynthesis of secondary metabolites including mycotoxins. Their concentrations vary
with time as they may be converted to other compounds (Griffin, 1981).
4
Figure 2-1: Structure of patulin
5
Toxin production in fungi is known to be correlated with the environment they inhabit.
Ecologically most of the Penicillium species are saprophytic, meaning they live on dead
or decaying matter or in soil (Pitt, 1994). These substrates are also inhabited by other
organisms such as bacteria, protozoa and other lower animals. Mycotoxins may serve to
protect fungi by forming a defense mechanism against these organisms. For example,
patulin has been shown to inhibit 75 species of gram positive and gram negative bacteria
and has been shown to have antiviral and anti-protozoal activity (Ciegler et al.; 1971). In
early 1900‟s patulin was used as an antibiotic ointment (Ciegler et al., 1971). This
suggests that these compounds may help the fungus to grow preferentially in an
environment in which competing bacteria, viruses and protozoa co-exist.
Another function of mycotoxin may be to impart pathogenesis in plants by the
fungus. It is known that fungal mutants that are incapable of producing the toxin show
much less virulence than their toxin producing counterparts. For instance, patulin has
been shown to be phytotoxic, and its role in pathogenesis by Penicillium has been
documented earlier (Desjardin and Hohn, 1997). On similar lines the mycotoxin by
Fusarium graminearum was found to be crucial in imparting pathogenecity in maize
(Harris et al., 1999). Thus, these toxins may weaken the defense mechanism of the host
cell thereby making them more susceptible to the fungal infection. These toxins may
impart phytotoxicity by changing the permeability of the cell membrane of the host that
leads to the cell death while some may cause cell death by generating excessive oxidative
stress ( as in the case with patulin) (Speijers, 2004). Thuss, research findings suggest that
the essential role of fungal toxins is to make the fungus more competitive and pathogenic
and consequently better suited for survival in the environment it inhabits.
6
2.1.2 Patulin occurrence in apple products
P. expansum is commonly found on apples and is primarily responsible for the
presence of patulin in apples (Doores, 1983). The fungus can penetrate through bruised or
mechanically damaged fruits during storage causing spoilage losses (Rosenberger, 2001).
The concentration of patulin in infected apples varies widely and can reach levels as high
as 5000 ppb to 360,000 ppb (Harwig et al., 1973; Lindroth and Niskanen, 1978). An FDA
survey conducted in 1993 reported that nearly 20% of the apple juice samples contained
more than the permitted 50 ppb limit (Kashtock, 2003). Smaller processors are especially
likely to contribute to high levels of patulin, as low quality apples are used to make cider
(Brackett and Marth, 1979a). The amount of patulin in apple juice is influenced by the
strain of the organism (Paster et al., 1995), the variety of the apple (Jackson et al., 2003),
the pH of the growth environment (Damaglou et al., 1986), and the number of apples
used that show signs of infectious lesions (Sydenham et al., 1997). Ritieni (2003) studied
the occurrence of patulin in apple juice, clarified apple juice, infant formula and apple
vinegar in Italy. The average concentration in 11 positive samples out of 33 was found to
be 26.7 µg/L (ppb). Apple vinegar samples were negative. In baby foods, 2 out 10
samples were contaminated with an average concentration of 15.4 µg/L. Studies on South
African commercial apple products conducted between 1996 and 1998 revealed that 8 out
of 31 juice samples were contaminated with patulin concentration ranging between 5 and
45 µg/L. Out of 10 infant juices, 6 samples contained patulin in the range of 5 and 20
µg/L (Leggott and Shephard 2001). In another study conducted in Iran, 33% of fruit juice
samples and 56% of apple juice concentrates contained patulin above the mandated 50
7
μg/L with maximum levels as high as 285 µg/L and 148 μg/L in juice and concentrate
respectively (Cheranghali et al. 2005). In a recent survey by Harris et al. (2009) patulin
occurrence in apple juice and cider in the state of Michigan was studied. Of the 493
samples from 108 cider mills throughout the state, 18% contained patulin at levels greater
than 4 ppb with 11 samples (or 2%) contained greater than 50 ppb patulin. In this study,
higher occurrence of patulin was found in thermally pasteurized samples as compared to
UV processed and un-processed samples. Among the grocery samples, 23% of 159
samples contained patulin with 18 samples (or 11%) containing greater than 50 ppb
patulin. Some samples contained as high as 2700 ppb patulin. With the growing
globalization of agricultural market, the patulin occurrence is not contained within a
specific geographical area and has a potential to impact apple processing industries in the
farthest corners of the world. Therefore, patulin content in apple may pose a significant
problem for the apple industry as considerable proportion of apples are imported into the
US from countries such as China, New Zealand, Argentina, Chile, Brazil (USDA, 2007).
In accordance with 21 CFR part 120 (HACCP), juice manufacturers must identify
the hazards and consideration should be given to multiple factors that can potentially
cause hazards; presence of natural toxins being one of them. From literature it is evident
that occurrence of patulin in apple products in fairly common and juice manufacturers
need to monitor the levels of patulin in apple products. Thus, patulin control in juice
products is necessary from the regulatory standpoint.
2.1.3 Patulin toxicology
8
Patulin could be considered as one of the most hazardous mycotoxins.
Mutagenicity of patulin is debatable, and studies showed variable results. Wurgler et al.
(1991) showed that patulin did not induce reverse mutations in Salmonella typhimurium
TA 102, while more recent studies by Schumacher and Metzler (2005) showed patulin to
be mutagenic at the hypoxanthine-guanine phosphoribosyltransferase gene locus in
cultured Chinese hamster V79 cells at regular, elevated and reduced levels of glutathione
(GSH), an antioxidant enzyme naturally present in cells. Patulin increased the GSH levels
in all three types of cells and was more toxic to GSH depleted cells as compared to the
other two types. It also showed concentration dependent increase in mutagenicity
(maximum of 270 mutants per 106
cells at 2.5 µM concentration) at sub-cytotoxic levels.
Patulin cytotoxicity mechanisms were studied by Barhoumi and Burghardt (1996).
Patulin at up to 1000 μM caused depletion of GSH, increased the oxidative stress by
generating reactive oxygen species (ROS), caused membrane depolarization and
simultaneously increased Ca+2
and cytoplasmic acidification in vitro. Lipid peroxiation
and loss of structural integrity of plasma membrane were also reported (Speijers 2004).
Ciegler et al. (1976) found patulin to be teratogenic in chicken embryo at 1-2 µg/egg.
Carcinogenicity of patulin remains questionable owing to very limited number of studies
and discrepancy in the data interpretation (Speijers, 2004). Studies by Osswald et al.,
(1978) showed no increase in tumor incidence in rats while Wouters and Speijers (1996)
found increased fore-stomach pappilomas and glandular stomach adenomas in the
treatment group. Speijers (2004) provided a comprehensive review of the state of the
knowledge on the toxicological attributes of patulin. The US Food and Drug
Administration (FDA, 2000) used the study by Becci et al. (1981) to establish a "No
9
Observed Adverse Effect Level" (NOAEL) for patulin of 0.3 mg/kg body weight per
week. After including a 100 fold safety factor, a maximum Provisional Tolerable Daily
Intake (PTDI) of 0.43 µg /kg body weight per day was established. The current FDA
limit for patulin occurrence in apple products based on toxicological data and juice intake
estimates is 50 ppb (μg/L) (US FDA, 2000)
2.1.4 Processing stability of patulin
Despite the variability in the results, it can be said that the thermal processing
cannot inactivate patulin completely. Patulin in apple juice was not degraded by a heat
treatment of 80 °C for 10–20 min, and there was no significant decrease during storage
for 3 weeks at 22°C (Scott and Somers, 1968). Lovett et al. (1973) reported that patulin is
more stable at lower pH and is resistant to destruction at 105-125° C. Brackett and Marth
(1979b) confirmed these results when they reported patulin half-life values of 55 and 2.6
days respectively in juice held at 25oC at pH 6.0 and 8.0. Kubacki (1986) reported a 20%
patulin reduction in apple juice heated to 120oC for 30 min although 30 min at 80
oC had
no effect. These results show that there are multiple factors that need to be taken into
account to study the toxin stability and conventional pasteurization cannot be relied upon
to be an effective way of eliminating patulin in foods.
2.1.5 Alternative technologies for patulin reduction
Apart from established techniques such as sorting damaged apples and culling,
several novel techniques have been developed for reduction of patulin in juices. Jackson
et al (2003) showed that washing of apples can reduce the patulin load by 10-100% but
10
can also serve as a source of contamination if the water is re-used. They also showed that
no patulin was detected in tree-picked culled apples stored for 4-6 weeks at 0-2 °C, while
un-culled apples contained patulin at the levels of 0.97-64 μg/L. Moake et al. (2005) have
extensively reviewed some of the patulin control strategies.
Conventional physical techniques such as clarification and ultra filtration have
been shown to significantly reduce patulin levels (Acar et al., 1998). Application of
ozone for 15 s led to a significant reduction of patulin and also a corresponding reduction
in toxicity (McKenzie, 1997). Activated charcoal at 3 g/L concentration has been shown
to reduce the patulin load by 50% (Kadakal and Nas, 2002). Complete disappearance of
patulin (at the initial concentration of 2 mg/kg) was observed when apple juice was
exposed to 2.5 kGy of gamma radiation (Zegota et al., 1988).
Stinson et al. (1976) showed that alcoholic fermentation of apple juice reduced
the patulin concentration by 99%. Similar results were reported by Burroughs (1977).
Gourama (1997) proposed the use of Lactobacillus spp. to hinder the growth of
Penicillium spp. and hence the mycotoxins production.
Patulin forms an adduct with cysteine and thus reduces its toxicity (Lindroth and
Wright, 1978). Addition of other electrophiles such as ascorbic acid (Kokkinidou et al.,
2007) has been tested to be an effective method to reduce the patulin load. Drusch et al.
(2007) showed that stability of patulin was reduced in presence of ascorbic acid. Patulin
was reduced by 70% when ascorbic acid was present as compared 30% without ascorbic
acid. The free radicals generated during the oxidation of ascorbic acid to dehydroascorbic
acid were attributed to the degradation of patulin. In another study, 500 ppm ascorbic
acid reduced the patulin levels by 50% (Aytac and Acar, 1994). 80% patulin loss was
11
observed in apple juice after 8 days when 3000 ppm ascorbic acid was added and samples
were stored at 25 °C (Brackett and Marth, 1979b). Activity of ascorbic acid against
patulin is highly temperature dependent and is negligible at refrigerated storage
temperatures (Kokkinidou et al., 2007). Sulphur dioxide at 100 ppm level caused 42%
reduction in patulin levels (Moake et al., 2005). Another study showed 90% reduction in
patulin content within 48 hours when 2000 ppm sulphur dioxide was added (Burroughs,
1977). Fliege and Metzler (2000) showed that patulin irreversibly forms an adduct with
glutathione thereby reducing its toxicity. No study involving UV induced degradation of
patulin has been reported.
2.2 ASCORBIC ACID
Ascorbic acid (2-(1, 2-dihydroxyethyl)-4, 5-dihydroxy-furan-3-one) is a water
soluble vitamin commonly known as vitamin C. In this section the chemistry, antioxidant
activity, and physiological role will be reviewed.
2.2.1 Chemistry and antioxidant activity of ascorbic acid
Ascorbic acid (AA) is a six carbon compound with a five membered lactone ring.
The lactone group has an ene-diol group adjacent to the carbonyl group. This conjugated
arrangement makes ascorbic acid a highly reactive compound and an excellent reducing
agent (Buettner and Jurkiewicz, 1996). The pKa1 of ascorbic acid is approximately 4.2
and therefore it is present in predominantly dissociated form at physiological pH. The
mono-anion form is called ascorbate. Ascorbate, after losing an electron generates an
ascorbate radical intermediate which further leads to the formation of dehydroascorbic
12
acid (DHA). DHA is the first stable oxidized form of AA. DHA can be reduced back to
AA in vivo and therefore still retains vitamin C activity. However, DHA is relatively
unstable and is irreversibly hydrolyzed to 2, 3- diketogulonic acid (DKGA), which does
not show vitamin C activity. (Gregory III, 2008). The entire AA degradation reaction
scheme is given in figure 2-2.
Ascorbate radical is a stable radical as compared to other radicals such as OH.
with a half-life of approximately 50 s at pH 7.4 (Somani, 1996). This long half-life allows
ascorbate radical to be detected by techniques such as electron spin resonance (ESR)
spectroscopy and spectrophotometry (Bielski and Richter, 1975). The stability of
ascorbate radical and one of the lowest reduction potential for A.-/AH
- couple are
contributing factors to the excellent antioxidant nature of AA (Buettner and Jurkiewicz,
1996). The ascorbate radical could be generated in two ways- (1) by AA acting as a
sacrificial antioxidant and (2) by the auto-oxidation of AA in presence of transition metal
ions.
The scheme for the antioxidant activity of AA is as follows:
X X.
AscH- + X
. Asc
. + XH
13
Figure 2-2: Reaction scheme for ascorbic acid degradation (Gregory III, 2008)
Ascorbic Acid Ascorbate radical
Dehydroascorbic Acid 2, 3-Diketogulonic acid
-e-
-H+
+H2O
Ascorbate ion
H
14
Asc.
DHA
Where X is a food chemical (such as lipids) which is prone to oxidation. Thus, the radical
X is quenched by Ascorbate that in turn generates ascorbate radical. The stability of the
ascorbate radical arrests the further propagation of the oxidation reaction. Thus, the
oxidation reaction of compound X is terminated at the initiation stage itself and a
potentially harmful radical X. is now substituted by relatively stable and non superoxide
forming Ascorbate radical (Buettner and Jurkiewicz, 1996). The ascorbate radical may
then further breakdown to form DHA. Ascorbate radical concentration is usually
considered as an indicator of the oxidative stress in the system. Hubel et al. (1998) found
that the plasma ascorbate radical concentration as measured by ESR were significantly
higher in women with preeclampsia as compared to normal pregnancy, indicating
increased oxidative stress. Sharma and Buettner (1993) observed similar increase in
ascorbate radical concentration in plasma exposed to continuous radical mediated
oxidative stress. Pietri et al. (1994) found that ascorbate radical was a reliable marker for
oxidative stress during an open heart surgery. From these studies it is evident that
ascorbate acts as a terminal antioxidant in biological systems. AA and its derivatives are
widely used in food systems as antioxidants in order to extend product shelf life. Cort
(1974) showed that at 0.01% concentration ascorbyl palmitate,a lipid soluble derivative
can successfully be used to extend the shelf life of vegetable oils. Ascorbic acid in
conjunction with tocopherols can show synergistic effects in antioxidant activity (Niki,
1991). Yi et al. (1991) showed synergistic action of ascorbic acid and σ-tocopherol in
reducing the auto-oxidation rate of fish oil. The antioxidant capacity of most of the fruit
15
juices is at least partially attributed to presence of ascorbic acid (Miller and Rice-Evans,
1997; Netzel et al., 2002; Gardner et al., 2000).
Ascorbic acid dianion can auto-oxidize in presence of oxygen generating
ascorbate radical (Buettner, 1990).
AA2-
+ O2 A.-
+ O2
.-
This explains the presence of ascorbate radical peak by ascorbic acid solution in distilled
water as determined by the Electron Paramagnetic Resonance EPR) spectroscopy. Auto-
oxidation of AA is increased in the presence of transition metal ions (Van Duijin et al.,
1998) which catalyze this reaction. Because of this, AA at low concentration acts as a
pro-oxidant while at higher concentration it acts as an antioxidant. (Buettner and
Jurkiewicz, 1996; Kanner et al., 2006).
2.2.2 Physiological role of ascorbic acid
Role of ascorbic acid in curing scurvy is well studied. Ascorbic acid at the dose of
approximately 10 mg/day can prevent and cure scurvy in adult humans (Hodges et al.,
1971). Ascorbic acid has several important functions in the body such as acting as a co-
substrate for dioxygenases and hydroxylation of proteins (Englard and Sifter, 1986). May
(2000) has described multiple mechanisms by which ascorbic acid can repair the
vasodilations and endothelial dysfunctions that helps in preventing diseases such as
atherosclerosis. Arrigoni and Tullio (2002) have extensively reviewed physiological
functions of ascorbic acid. Ascorbic acid has a significant role to play in regulation of
transcription and stabilizations of specific mRNAs in cell (Arrigoni and Tullio, 2002).
16
Fraga et al. (1991) showed that AA can prevent oxidative damage to the DNA in human
sperms. Cameron et al. (1979) reviewed the anticancer activity of ascorbic acid. Cameron
and Pauling (1979) in a clinical trial studied the anticancer potential of ascorbic acid
where 100 terminally ill cancer patients received ascorbic acid supplement while 1000
control patients received identical treatment but no ascorbic acid supplementation. They
observed that the survival period for treatment group (210 days) was 4.2 times higher
than the control group (50 days). Chen et al. (2005) found that ascorbic acid at
pharmacologic concentration (20 mM) could selectively kill cancer cells in a study using
cell lines in vitro. This was attributed to possible generation of hydrogen peroxide in the
cell system by ascorbic acid. Thus, it is evident that ascorbic acid has multitude of
functions in human physiology.
2.3 ULTRAVIOLET LIGHT (UV) PROCESSING OF FOODS
The food processing industry is witnessing an increasing interest in non-thermal
food processing technologies including the use of ultraviolet (UV) irradiation. Compared
to thermal disinfection treatments, UV radiation may have fewer adverse quality effects
and operate at lower costs (Koutchma, 2009). Although, UV technology is extensively
used in water purification and wastewater treatment (Legrini et al., 1993), it has only
recently found applications in juice processing. UV processing is still in its infancy and
considerable research is needed before more widespread commercialization can occur.
2.3.1 Mode of action
17
The ultraviolet region of the light spectrum consists of a range of wavelengths
between 200 and 400 nm. This region is divided into three types- UV-A (315-400 nm)
which causes skin tanning, UV-B (280-315 nm) which can lead to skin cancer, and UV-C
(200-280 nm) which has germicidal activity (Sastry et al., 2000). Ultraviolet light causes
microbial lethality through dimerization of adjacent pyrimidine molecules (thymine or
cytosine) on the same DNA strand. This cross linking makes DNA unsuitable for
transcription, thus halting the protein biosynthesis and eventually leading to cell death.
Because the maximum absorption of UV light for DNA is 254 nm, this wavelength is
considered to be the most effective for reducing microbial populations. Exposure to UV
light can also cause mutations that lead to cellular injuries and death (Guerrero-Beltran
and Barbosa-Canovas, 2004).
The radiant intensity of UV light incident per unit surface area is termed
irradiance and is expressed as mW/cm2. The amount of UV energy incident per unit
surface area over a period of exposure time is termed UV dose. This value is therefore
calculated by multiplying irradiance by the exposure time and may be expressed as
mJ/cm2, J/m
2, or mW-s/cm
2. Some researchers have expressed dose as a function of
treated juice volume (J/l) (Keyser et al.; 2008).
Microbial inactivation kinetics for ultraviolet light typically follows a sigmoidal
curve (figure 2-3), where an initial shoulder is followed by a linear phase and then
eventual tailing. In the shoulder region, death rates are low because most microorganisms
18
Figure 2-3: UV induced microbial death curve (Sastry et al.; 2000)
0
1
2
3
4
5
6
7
0 5 10 15
Linear
Tailing
Mic
rob
ial L
og
re
du
ctio
n
Shoulder
UV dose (mJ/cm2)
19
are capable of reversing low to moderate DNA damage to some extent. Excision repair
occurs through enzymatic replacement of dimerized thymine with un-dimerized
molecules. Photoreactivation (light mediated repair) is another repair mechanism in
which the enzyme photolyase splits dimerized thymine by absorption of UVA radiation
(Coohil and Sagripanti, 2008). At higher dose levels, repair mechanisms are less effective
and cell death increases as dose levels increase. For this reason, dose levels should be
selected so they are adequate to achieve irreversible DNA damage. The last region of the
death curve, the tailing phase, describes the behavior of microbial cells that have higher
intrinsic resistance to UV light.
In table 2-1, dose levels necessary to achieve a 1-log reduction of select
microorganisms of importance in foods are shown (Coohil and Sagripanti, 2008). These
data show that Bacillus subtilis spores are 6-7 times more UV resistant than their
corresponding vegetative cells. Molds and yeasts are in general more resistant to UV light
than bacterial cells. Differences in microbial resistance can largely be explained by the
relative opacity of the spore-coat or cell wall which protects the cell contents from UV
exposure or the ability of cells to repair UV damage.
2.3.2 UV dose measurement
It is necessary to accurately measure UV dose levels to validate the efficacy of a
UV treatment or to routinely verify that the system is working according to the
specifications. A radiometer may be used to directly measure the incident UV irradiance
20
Table 2-1: UV dose required for 1-log reduction of common food borne
microorganisms (adapted from Coohil and Sagripanti, 2008)
Microorganism Dose (J/m2) for 1-log reduction
Bacillus subtilis vegetative cells 40-60
Bacillus subtilis spores 260
Campylobacter jejuni 11
Escherichia coli 20-40
Listeria monocytogenes 50
Salmonella typhimurium 80
Shigella paradysentriae 17
Vibrio cholerae 11
Yersinia entercolitica 13
Saccharomyces cerevisiae 75
21
striking a surface. However, in continuous systems, a more practical method may be to
use indirect techniques such as chemical actinometry or biodosimetry.
Chemical actinometry utilizes a chemical compound that is sensitive to the
wavelength of interest and which degrades in proportion to the applied light energy.
Therefore, the dose can be determined by measuring the extent of UV induced
photochemical degradation. HHEVC (4, 4‟, 4‟‟ –tris-di-B-hydroxyethyl
aminotriphenylacetonitrile) dye is an example of a chemical actinometer that has been
used to measure UV dose applied to apple cider (Adhikari et al., 2005)
Biodosimetry is similar to chemical actinometry except that microbial lethality is
used to measure dose instead of degradation of a chemical compound. A calibration curve
is prepared by first testing the sensitivity of the microorganisms to UV light (number of
log reduction/dose) and then a known quantity is added to the treatment sample. The log
reduction achieved in the sample is then fitted into the calibration curve to determine the
dose level. Because this method measures the effect of UV dose on an actual human
pathogen or surrogate with similar UV sensitivity, it is considered the gold standard for
instrument calibration (Koutchma, 2009).
Another approach for measuring the UV dose is mathematical modeling and
numerical simulation of UV dose distribution. Mathematical modeling can serve multiple
purposes- 1. Predict microbial lethality in given food system based on its attributes such
as flow rate and absorbance coefficient, source intensity and other equipment
specifications and inactivation kinetics of microorganism of concern 2. Understand the
UV dose distribution within UV reactor and predict the location of least-treated liquid 3.
22
Suggest design modifications for improving the efficacy of the reactors (Koutchma,
2009). Unluturk et al. (2004) developed a numerical model to describe the flow pattern
and the residence time distribution for particles in a thin film reactor, Cidersure 1500
(FPE, Macedon, NY). Results showed a non-uniform UV distribution across the reactor.
However, the developed model slightly over-estimated the UV dose. Nevertheless, the
results matched fairly with the experimental values as obtained by biodosimetry. Thus,
mathematical modeling can be used as a first step in predicting the microbial lethality for
the given food-reactor combination and some conclusions can be drawn regarding
feasibility of the process. However, empirical studies would be required to confirm the
predictions.
2.3.3 Factors influencing the efficacy of UV treatment
The efficacy of UV process treatments is determined by the intensity of the light
that reaches the food source (irradiance) and attenuation of light as it passes through the
food.
Monochromatic UV light systems use low pressure mercury lamps capable of
producing light at a narrow wavelength range around 254 nm. Surface irradiance is
linearly proportional to the intensity of the light source but decreases with the square of
the distance between the source and the surface. Therefore, it can be expected that a high
intensity UV lamp placed close to the food surface will require lower treatment times to
achieve a given lethality. However, very high UV intensities can lead to increases in
temperature which may have deleterious effects on food quality. Moreover, if
23
temperature rise is not taken into account, it may become a confounding variable that will
cause process efficiency calculations to become misleading.
Attenuation of light is the most important factor that determines the efficacy of
UV light. According to Beer-Lambert‟s law (equation 2-1), as light enters a liquid
medium, light intensity decreases exponentially with increasing absorption coefficient (α)
and liquid film thickness (t). Equation 2-2 shows that the average intensity of exposure
(Iavg) in a liquid is calculated by integrating It over the film thickness.
It= I0 exp (-αt) Eq. (2-1)
Iavg= Eq. (2-2)
Where, It= Irradiance at thickness t (mW/cm2), I0= Incident irradiance (mW/cm
2),
α=absorption coefficient (mm-1
) and t=film thickness (mm)
The absorption coefficient describes the extent to which light intensity decreases
as it passes through the liquid (Koutchma, 2008). In a highly transparent liquid (low
absorption coefficient), light penetration and thus intensity at any depth will be greater
than that in a less transparent liquid (high absorption coefficient). Juice products typically
contain a variety of UV absorbing compounds such as polyphenols, anthocyanins, and
ascorbic acid which increase the absorption coefficient of the medium. In addition, juice
insoluble solids such as pectin, cellulose, hemicellulose, and protein may reduce average
24
intensity by scattering. Insoluble substances also decrease the amount of light that enters
the liquid by reflecting it away from the surface. The net effect from these factors is to
decrease the average intensity (Iavg) of light which must be overcome by increasing
exposure times (higher dose levels) or reducing the film depth (Sizer and
Balasubramaniam, 1999).
Fan and Geveke (2007) studied the UV absorption pattern of several compounds
found in apple cider. Sugars (glucose, sucrose and fructose) showed very little
absorbance between 240-300 nm, although fructose showed significantly higher
absorbance than glucose and sucrose. Malic acid absorbed significantly below 240 nm
while the absorbance decreased dramatically beyond that. Ascorbic acid (10 ppm)
showed strong absorbance between 220 and 300 nm even at such low concentrations.
Polyphenols, present in significant quantities in apple cider, are known to absorb in the
UV region (Ying et al., 2009). Suspended particles cause attenuation of light through
scattering, blocking and absorption of light. Apple cider contains high quantity of
suspended particles (800-1000 NTU) that can dramatically reduce the penetration of light
(Koutchma, 2009). pH (tested between 3-5) and brix (9.7-16.5°) of apple cider do not
have a significant effect on the penetration of UV light (Koutchma, et al., 2004).
2.3.4 Processing equipment
Both batch and continuous UV reactors can be used to treat juice samples
(Figures 2-4, 2-5a). Batch UV radiators are typically used to obtain empirical data in
bench-top laboratory experiments. Single or multiple UV lamps direct light downward
25
Figure 2-4: Bench-top batch UV reactor
Sample in a Petri plate with stirrer
UV lamp
Collimator
Collimated beams
26
Figure 2-5: (a) Design of CiderSure® continuous UV system (Courtesy: Phil
Hartman, FPE, Macedon NY) (b) Cross section of the process tube
Blower Box
(a)
(b)
Outer stainless steel wall
Clearance for the flow of
juice
Quartz sleeve
Pump motor
and pump
Flow sensor
Absorption coefficient sensor
assembly
Process tube
UV lamps
Clamps
Product inlet and
outlet
27
through a collimator, which is essentially a length of tube painted on the inside surface
with a non-reflecting material. This ensures that the incident UV light approaches the
surface of the sample in a perpendicular manner. The sample may be held in a petri-dish
placed at the end of the tube with ample mechanical stirring to ensure a uniform dose
throughout the sample volume. UV irradiance levels can be varied by adjusting the
collimator length. As collimator length is increased, the distribution of light energy across
the sample surface becomes more uniform. It is not possible to achieve 100% collimation
since this would require an infinitely long collimator tube. Thus, the level of collimation
actually obtained may be limited by the availability of a light source with sufficient
intensity to achieve meaningful experimental results (Kuo et al., 2003).
Continuous systems, more closely representing commercial applications, typically
consist of multiple UV lamps arranged within a quartz sleeve. The liquid flows through a
narrow clearance between the quartz sleeve and an outer stainless steel wall (figure 2-5b).
It is desirable to minimize the thickness of the clearing to maximize average intensity
levels and thus allowing higher flow rates while achieving necessary dose levels. To
ensure thorough mixing and uniform exposure of the liquid, it is desirable to achieve
turbulent flow as it passes through the system.
The current US juice HACCP regulation (21CFR Part 120) requires all juice
processors to treat their products in a manner that is capable of achieving a 5-log
reduction in pathogenic microorganisms. To date only one UV system, CiderSure®
developed by FPE Inc. (Macedon, NY, USA) meets these standard set by the FDA. This
continuous UV system achieves consistent microbial lethality over a range of turbidity
levels through the use of a UV light sensor that automatically adjusts the flow rate to
28
maintain a constant dose level. The dose level of the CiderSure® system should be
routinely verified using E.coli K12 or E. coli O157:H7. This technology offers a lower
cost alternative to the more expensive heat pasteurization systems in addition to
improvement in product quality.
Pulsed light UV technology is a recently developed disinfection method in which
food is subjected to very high intensity light pulses (1-5 J/cm2). Compared to non-pulse
systems, greater penetration depths can be achieved and thus may find application with
viscous or solid foods (Sharma and Demirci, 2003; Hillegas and Demirci, 2003) . By
varying the frequency of the light pulses, higher pulse intensities can be achieved with
fewer adverse quality effects. Nevertheless, temperature increase can be significant
causing overheating or cooking of the product and this factor must be taken into account
in lethality calculations (Oms- Oliu et al., 2009). This technology is still in the
experimental phase and to date there are no commercial juice applications.
2.3.5 UV processing of food products
2.3.5.1 Fresh fruits and vegetables
Suitability of UV processing to increase the shelf life has been studied for a
variety of fruits and vegetables. Marquenie et al. (2002) tested UV-C treatment to retard
fungal infestation by Botrytis cinerea and Monilinia fructigena on strawberries and
cherries during storage. UV doses of 0.05-1.5 J/cm2 significantly reduced fungal growth
in strawberries but not in cherries. The highest doses of 1 and 1.5 J/cm2 caused adverse
effects on strawberries such as browning and drying of leaves but also retarded the
softening rate during storage. Application of UV- C light for extending shelf life of
29
lettuce was studied by Allende and Artes (2003). Red oak leaf lettuce was exposed to UV
dose of up-to 8.1 kJ/m2. UV increased the respiration rates of the leaves as compared to
control and considerably reduced the growth of psychrotrophic organisms, coliforms and
yeasts but not the lactic acid bacteria. Fonseca and Rushing (2006) studied effect of UV-
C (254 nm) on the diced watermelon in terms of quality and microbial population. UV
dose of 4.1 kJ/m2 reduced the microbial load by at least 1 log at the end of storage time
without significantly affecting juice leakage, color or any other visual attributes.
Gonzalel-Aguilar et al. (2001) found that UV-C can prevent decay and maintain the post
harvest quality of „Tommy Atkins‟ mangoes. Mangoes were treated with UV for 10 or 20
min and stored at 5 or 20 °C. 10 min exposure was effective in maintaining firmness and
reduce the decay symptoms at both the temperatures. 20 minute exposure led to a
decrease in organic acid and an increase in sugar content of mangoes while 10 minute
exposure did not show any significant change. Lamikanra et al. (2001) described the
changes the UV light can impart on the composition of volatile compounds in
cantaloupes. UV exposure to up to 60 minute decreased the concentration of aliphatic
esters by 60% as compared to fresh-cut cantaloupes. Exposure to UV light led to
synthesis of terpenoids such as geranylacetone and terpinyl acetate, which in turn reduced
the microbial load in cantaloupes by 0.5 logs. Cantos et al. (2000) showed that treating
Napoleon table grapes with UV-B and UV-C radiation led to an increase in resveratrol ( a
health benefitting phytoalexin) derivatives content of grapes by 2-3 fold. In another study
Cantos et al. (2003) found that wine made of UV-C treated grapes contained 2 and 1.5
times more resveratrol than the control. Arakawa (1988) reported similar increase in
anthocyanins content when apples were exposed to UV-B radiation.
30
It is evident that UV processing can be used for fresh fruits and vegetables, but
the efficacy of the treatment is limited by penetration of light, non-uniform exposure to
UV light and possible adverse effects on the quality attributes due to UV induced stress.
2.3.5.2 Meat, poultry and dairy products
Wong et al. (1998) found that UV light can be effectively used to reduce the load
of Escherichia coli and Salmonella senftenberg in pork skin and pork muscle. The D-
value for E. coli at 100 μW/cm2 was found to be 1370 s for pork muscle and 1282 s for
pork skin. The D-values for S. senftenberg were found to be 1163 s for pork muscle and
595 s for pork skin. Sumner et al. (1996) reported that UV light (254 nm) can reduce the
load of Salmonella typhimurium on poultry skin by 80% at a dose level of 2 mJ/cm2. A
UV dose of 0.3 J/cm2 in boneless chicken breast filet led to at least 2-log reduction in the
load of Listeria monocytogenes without significantly affecting the meat color (Lyon et
al., 2007). Mahmoud and Ghaly (2004) reported the application of UV reactor for the
sterilization of cheese whey. Several design modifications were suggested to overcome
poor transmittance of UV light through the medium. Milk and fish products contain
significant amount of saturated and unsaturated fats (USDA food database). Since, UV
light has a tendency to oxidize these lipids leading to generation of rancid or oxidized
flavors, application of UV technology in these food products categories would be limited.
2.3.5.3 Fruit juices
Limited studies have been reported on UV processing of fruit juices. Apple juice
and cider are the most studied products for UV processing and considerable research on
the effects of UV light on flavor profile, color changes, and microbial lethality have been
conducted. Microbial studies (Quintero- Ramos et al., 2004; Koutchma et al.; 2004) have
31
determined that a dose level of 14.3 mJ/cm2 is sufficient to achieve a 5-log reduction in
E. coli O157:H7 in apple cider. These data provided the scientific basis for FDA‟s
approval of UV processing to achieve the mandated 5-log reduction in human pathogens.
Other studies comparing UV processing and heat treatments showed improved sensory
quality of apple cider, although the shelf life of UV treated cider was lower than
pasteurized cider due to greater UV resistance of yeasts and molds (Tandon et al.; 2003).
Tran and Farid (2004) found that orange juice treated at dose levels of 97.0 and
119.0 mJ/cm2 reduced populations of aerobic microorganisms and yeasts and molds
(YM) by 1 log respectively. The higher dose required for orange juice compared to apple
cider was attributed to the greater turbidity of orange juice. Additionally, it was
determined that deactivation of pectin methyl esterase, an enzyme responsible for cloud
loss and juice precipitation, was not significantly affected.
Keyser et al. (2008) studied microbial viability in various juice samples including
guava and pineapple blend, orange juice, strawberry nectar, mango nectar and tropical
juices as a function of UV exposure (0-2066 J/l) using a PureUV® (Milnerton, South
Africa) system. The highest lethality per unit dose was achieved in apple juice, the most
transparent of the juices tested (230 J/l) for 5-log reduction of E.coli K12, 3.5-log
reduction in aerobic plate count (APC) and 3-log reduction in yeast and mold count
(YM)). Lower reduction at the same dose levels in orange juice (0.25-log reduction in
APC and 0.07-log reduction in YM) and tropical juice (0.3-log reduction in both APC
and YM) were attributed to suspended particles and fibrous tissue.
2.3.6 Stability of ascorbic acid during UV processing of juice
32
Ascorbic acid loss during a processing technique is routinely used as an indicator
of severity of a processing method. Sparse data is available on UV induced ascorbic acid
degradation kinetics. In a study by Koutchma et al. (2009) it was found that ascorbic acid
degradation followed zero order with a rate constant of -0.025/s. The degradation rate
was found to be inversely proportional to the absorption coefficient of the juice. Tran and
Farid (2004) reported 17% ascorbic acid degradation when orange juice was exposed to
100 mJ/cm2. Approximately 30% reduction in AA at dose of 14.32 mJ/cm
2 in apple cider
was reported by Adzahan (2006). The dose required to achieve 5-log microbial reduction
in each juice sample would vary due to differences in turbidity and absorbance
coefficient and therefore corresponding ascorbic acid loss also would be expected to
vary. These studies were directed at specific juice systems and therefore it is difficult to
extrapolate these results to other juices. Therefore, it is necessary to acquire some basic
understanding of UV induced degradation of ascorbic acid that could be translated for
other fruit juices.
2.4 STATEMENT OF PROBLEM
From the review of literature, it is evident that patulin persistently occurs in apple
products. Removal of patulin from these products has been met with limited success,
since patulin is thermostable and the alternative techniques proposed add a processing
step that may prove to be expensive. Therefore, there is an incentive to develop a new
technique for reduction of patulin load in apple products. We hypothesize that since
33
patulin absorbs in the UV region (276 nm peak absorbance), it may degrade when
exposed to UV light.
The effects of UV processing on bioactive compounds in juice need to be studied
before wide-spread commercialization of this technology can occur. In order to
understand the severity of UV processing on food chemicals, we decided to study the
degradation of ascorbic acid when exposed to UV light. Although naturally apple juice
contains fairly low amount of ascorbic acid (10 mg/L), it is incorporated in most of the
fruit products including apple juice at 1-2 recommended dietary allowance (RDA) levels
and is a compound susceptible to degradation owing to its reactivity. Ascorbic acid is
also commonly used as an indicator of severity of a processing technique. Similar to
patulin, ascorbic acid also absorbs in UV region (peak absorbance at 245 nm) and
therefore may be prone to UV induced degradation.
The overall goal of this research was to study the effect of UV processing on
patulin and ascorbic acid in model apple juice system and in apple juice.
2.4.1 Specific objectives
1. Study the UV induced degradation kinetics of patulin in model apple
juice and identify the effects of various factors such as initial concentration of
patulin, incident intensity, pH of the model system and presence of juice
components such as tannic acid, ascorbic acid and suspended particles on the
degradation rate constant.
34
2. Study the UV induced degradation kinetics of ascorbic acid (AA) in
model apple juice and apple juice and investigate the effects of factors such as
initial concentration of ascorbic acid, pH of the model system, sugars and
presence of UV absorbing compounds on the degradation rate of AA.
Additionally, post-processing storage degradation of AA will be studied.
3. Identify the mechanism of UV induced degradation of AA.
35
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48
Chapter 3
Patulin degradation in a model apple juice system during ultraviolet
light processing
ABSTRACT
The objective of this research was to study the kinetics of patulin degradation
during exposure to UV light in a model apple juice system. A collimated beam batch UV
(254 nm) apparatus was used for the UV treatment. 0.5% malic acid buffer was used as a
model apple juice system. The effects of added ascorbic acid, tannic acid, and suspended
solids on patulin degradation were studied using Box-Behnken design. Results showed a
first order degradation kinetics for patulin in malic acid buffer. The degradation rate
constant (cm2/J) was not significantly affected by incident intensity (0.8-1.8 mJ/cm
2)
(p>0.05), buffer pH (3.0-3.6) (p>0.05) and initial concentration of patulin (0-1000 ppb)
(p>0.05). Tannic acid, (0-1 g/L) and suspended particles (0-100 NTU) significantly
reduced the patulin degradation rate constant (p<0.05), while ascorbic acid (01-00 mg/L)
did not affect the reaction rate constant (p>0.05). Tannic acid reduced the rate possibly by
competitive absorption of UV light; while suspended particles scatter light and thus may
reduce the amount of light available for degradation.
49
3.1 INTRODUCTION
Patulin (4-hydroxy-4H-furo-[3,2c]pyran-2[6H]one) is a mycotoxin produced by
several species of apple decay molds including Penicillium, Aspergillus, and
Byssochlamys. P. expansum is the most common decay organism in apples and is
responsible for the majority of patulin found in apples (Doores, 1983). Bruising or
mechanical damage to fruits can increase penetration of the mold into apples and thus
levels of patulin (Rosenberger, 2001).
The concentration of patulin in decayed apples varies widely from 5000 ppb to
360,000 ppb (Lindroth and Niskanen, 1978). A U.S. Food and Drug Administration
(FDA) survey conducted in 1993 reported that nearly one fifth of the apple juice samples
contained more than the FDA mandated 50 ppb limit (Kashtock, 2003). Smaller
processors that use low quality or dropped apples to make cider are especially at the risk
of contributing to high levels (Brackett and Marth, 1979). The amount of patulin in apple
juice is influenced by the strain of the organism (Paster et al., 1995), the variety of the
apple (Jackson et al., 2003), the pH of the growth environment (Damaglou et al., 1986),
and the number of apples processed that show signs of infectious lesions (Sydenham et
al., 1997). A survey was conducted recently by Harris et al. (2009) to measure the patulin
occurrence in apple juice and cider in the state of Michigan. About 18% of 493 samples
from 108 cider mills throughout the state contained patulin at levels greater than 4 ppb
with 11 samples or 2% of the samples containing greater than 50 ppb patulin. Some
samples contained as high as 2700 ppb patulin. Patulin has been shown to be mutagenic,
teratogenic, and carcinogenic in animals (Mayer and Legator, 1969; Ceigler et al., 1976,
50
Taniwaki et al., 1991; Alves et al., 2000). Therefore, US FDA (2001) has set the
maximum level of 50 ppb patulin in the apple products.
Previous studies show that patulin is relatively thermostable (Scott and Somers,
1968; Lovett et al., 1973). Alternative technologies studied to reduce patulin levels
include the addition of electrophiles such as cysteine, (Lindroth and Wright, 1978)
ascorbic acid (Drusch et al., 2007), and ozone (McKenzie, 1997). Physical methods
studied include the use of activated charcoal (Kadakal and Nas, 2002) and ionizing
irradiation (Zagota et al., 1998). These have met with limited success due to cost factors
and a requirement for additional processing steps.
UV light has long been used for drinking water purification, waste water
treatment and surface sterilization/disinfection in food and pharmaceutical processing
plants (Legrini et al.; 1993; Guerrero-Beltran and Barbosa- Canovas, 2004). With the
growing interest in minimally processed foods that have improved quality and nutrient
retention, UV processing is a non-thermal processing technology worthy of exploring.
Reviews by Sastry et al. (2001), Bintsis et al. (2000) and Guerrero-Beltran and Barbosa-
Canovas (2004) provide comprehensive insights into the current usage of UV technology
in the food industry. UV light with 254 nm wavelength (termed as germicidal) has
maximum efficacy against microorganisms due to the fact that DNA absorbs UV light at
254 nm and causes cross linking of thymine molecules, thus disabling DNA transcription
and leading to cell death (Donahue et al., 2004)
Considerable research has been conducted to study the effects of UV light on the
flavor profile, color changes and microbial lethality in apple juice. Studies have shown
that UV light is capable of achieving the FDA mandated 5-log or greater reduction of
51
human pathogens in apple juice (specifically Escherichia coli O157:H7 and
Cryptosporidium parvum) when exposed to a dose level of 14.32 mJ/cm2 (Donahue et al.,
2004; Quintero-Ramos et al., 2004; Koutchma et al., 2004; Murakami et al., 2005). UV
treatments have successfully been used without affecting sensory or nutritional quality
significantly (Tandon et al., 2000; Choi and Nielson, 2004; Donahue et al., 2004). Thus
many smaller processors are utilizing this technology as a lower cost alternative to heat
pasteurization for producing safe cider and juice products.
No prior studies have been reported on the effects of UV light on patulin. It was
hypothesized that patulin will undergo photo-induced degradation when exposed to UV
light since its peak absorbance is 276 nm, which is close to the germicidal UV
wavelength of 254 nm. Our objectives were to (1) study the kinetics of UV induced
degradation of patulin in model apple juice (2) study the effects of UV absorbing or
scattering juice components such as polyphenols ( as represented by tannic acid),
ascorbic acid and suspended particles on the rate of degradation in model apple juice.
3.2 MATERIALS AND METHODS
3.2.1 UV treatment equipment
All experiments were carried out using a bench-top batch collimated beam UV
reactor (figure 3-1). The reactor consisted of three UV lamps (254 nm, 10 W, Atlantic
Ultraviolet Inc., Hauppauge, NY) mounted within a shielded horizontal cylindrical holder
fitted over a vertical tube (100 mm diameter) of varying length. Collimation was
achieved by painting the inside surface of the vertical tube with UV absorbing black
paint. Based on the length of the tube, the calculated maximum incident angle was no o
52
Figure 3-1: Schematic representation of collimated UV beam equipment
Sample in a Petri plate with stirrer
UV lamp
Collimator
Collimated beams
53
greater than 20°. Incident intensity (mW/cm2) was measured by placing a radiometer
(Model: UVP-J225, UVP LLC, Upland, CA) at the bottom of the tube at a length equal
tthe distance between the light source and the surface of the sample. The incident
intensity was varied by changing the length of the collimator tube. Variation of incident
intensity over the entire sample surface area was less than 1%. This slight error was
neglected because the sample was continuously stirred with a mechanical stir bar (300
rpm).
All experiments were carried out by adding a 20-ml sample into the bottom of a
plastic petri-dish (100 mm X 10 mm) and exposing it to UV light at various time
intervals. All UV treatments were performed at 21 (±1) °C. At each time interval, 0.6 ml
was withdrawn from a treated and an untreated (control) sample for HPLC analysis.
Sample withdrawal and the resultant reduction in the depth of the liquid accounted for a
theoretical incident intensity reduction of no more than 0.03 mW/cm2.
3.2.2 UV dose measurement
The UV dose (J/cm2) was calculated by multiplying the incident intensity as
measured by the radiometer at the surface of the liquid under treatment with the exposure
time in seconds (equation 3-1).
D= I×t Eq. (3-1)
Where, D= UV dose (J/cm2), I= Incident intensity (W/cm
2), t= duration of
exposure (second)
54
The incident intensity or irradiance range at the surface of the liquid was between
0.8-3.3 mW/cm2.
3.2.3 Sample preparation
All the chemicals such as malic acid, patulin, ascorbic acid, tannic acid were
procured from Sigma Aldrich (St. Louis, MO).The model apple juice system consisted of
0.5% malic acid buffer (Sigma Chemicals, Saint Louis, MO) solution with pH adjusted to
desired level (3-3.6) by adding NaOH solution. Since malic acid determines the pH of
apple juice (Koutchma et al., 2004), it was chosen to form the model system. The pKa1
for malic acid is 3.4 (Evangelista et al., 1996) and therefore can effectively act as a buffer
for the pH range chosen in these experiments (pH=3-3.6) Appropriate amounts of stock
patulin solution were added to malic acid buffer to make the final concentrations of up to
1000 ppb.
In order to study the effect of UV absorbing or scattering components on patulin
degradation, ascorbic acid (AA), tannic acid (TA) (a complex mixture of water soluble,
UV absorbing, polyphenolic glucose esters of gallic acid) and suspended particles (NT)
(in order to generate turbidity) were incorporated to 0.5% malic acid buffer. Polyphenols
is a class of compounds that contains several different molecules. In order to avoid the
interactions that may occur between these molecules and thus confound the results, a
single type of molecule (tannic acid) was chosen. The turbidity (NT) was varied by
adding filtered apple sauce (not fortified with ascorbic acid) (Motto‟s® from Wal-Mart,
State College PA) to the malic acid buffer followed by stirring and then filtering
excessively large particles using coarse filter paper (Kimwipes®
). The mean particle size
55
as measured by laser scattering particle size distribution analyzer (Horiba, LA-920,
Horiba Inc. Irvine, CA) was 4.78 µm which was within the same range as reported by
Koutchma et al. (2004) where it was observed that 65-70% of the suspended particles in
apple cider range from 1- 26 µm. Turbidity was measured using a Hach® turbidimeter
(Model 2100P, Hach Inc., Loveland, CO). Apple juice has been reported to contain 0.35
g/L tannic acid equivalent of polyphenols, 10-15 mg/L ascorbic acid and 1-15 NTU
(Nephelometric Turbidity Units) suspended particles (Picinelli et al., 1996; USDA food
database; Koutchma et al., 2004).
To study the effects of these three juice components on the rate of patulin
degradation and their possible interactions, „Box-Behnken model‟ with 3 levels of
variables was used. The ranges chosen for the experimental design were broader than the
reported values to account for variations in product composition and processing methods.
Factors and the levels used in the experimental design were- AA concentration 0-100
mg/L, TA concentration 0-1 g/L and turbidity (NT) 0-100 NTU with the rate constant
(cm2/J) for patulin degradation used as a response variable. The rate constant was
calculated by exposing the sample for 120 minutes and measuring the patulin content
after 20 minute interval. Thus, for each condition the rate constant was obtained from 7
data points for patulin concentrations plotted over UV dose.
3.2.4 Extraction and quantification of patulin
Polyphenols and ascorbic acid interfere with patulin quantification using HPLC.
Therefore, patulin was extracted from the model system using OASIS® solid phase
extraction syringes (Waters Inc., Milford MA) as described in the AOAC protocol
56
developed by Trucksess and Tang (2001). The average extraction efficiency was
determined to be 92% and was independent of concentration of patulin in the sample
between 50 and 1000 ppb (p>0.05).
3.2.5 High Performance Liquid Chromatography (HPLC) analysis
Patulin was quantified by a Waters HPLC system (pump model: 600;
Autosampler: 71P; photodiode array (PDA) detector: 2998, Waters Inc., Milford MA)
using a hybrid C-18 reverse phase / cation exchange column (Primesep-D, 4.6 mm × 150
mm, particle size 5 μm, SIELC Inc., Prospects Heights, IL). The mobile phase consisted
of 95% water, 5% acetonitrile, and 0.1% formic acid (pH 1.8 ±0.05 adjusted with HCl).
The PDA detector wavelength range was 220-300 nm. The flow rate of the mobile phase
was set at 1 ml/min and the injection volume was 20 µL. The peak absorbance for
detection was 276 nm. Runs were performed in the isocratic mode. The identity of patulin
peak was confirmed by injecting pure compound in HPLC. Representative HPLC
chromatograms for patulin are shown in figure 3-2.
3.2.6 Data analysis
The data was fitted into two different first order equations. Equation 3-2 is first
order kinetics in terms of duration of exposure.
(3-2)
57
Figure 3-2: Representative HPLC chromatograms of patulin (C0=1000 ppb) (a) No
UV (b) after UV dose of 5.04 J/cm2
(b)
(a)
58
Where, Ct= Concentration after exposure for time t, C0= initial concentration, k= rate
constant (s-1
)
Equation 3-3 represents first order kinetics in terms of UV dose. Equation 3-3 was
derived from equation 3-2 by incorporating duration of exposure into UV dose (D= I×t,
where I= incident intensity and t= duration of exposure).
Eq. (3-3)
Where, CD= Concentration after exposure to UV dose D (J/cm2), C0= initial
concentration, k= rate constant (cm2/J), where D=I×t, I-UV intensity (mW/cm2) and t=
duration of exposure (s)
Additionally, the data was represented by a value called D50, which was defined as
UV dose required to achieve 50% patulin reduction and was obtained by the rate
constants in terms of UV dose.
Conventionally the average UV intensity is calculated by integrating the intensity
over the liquid film thickness using absorbance coefficient of the liquid medium
(equation 3-4, 3- 5) (Murakami et al., 2006).
It= I0 exp (-αt) Eq. (3-4)
Iavg= Eq. (3-5)
59
Where, It= Irradiance at thickness t (mW/cm2), I0= Incident irradiance (mW/cm
2),
α=absorbance coefficient (mm-1
) and t=film thickness (mm)
The penetration of UV light through the medium is inversely proportional to the
absorbance coefficient of the medium. The absorbance coefficient strongly depends on
the composition of the medium. In this study, because of the presence of components
such as tannic acid in the model system which have high absorbance at 254 nm (at 0.5
g/L and 1 g/L levels tannic acid showed apparent absorbance of 10.6 and 20.5
respectively), the penetration of UV light through the medium would be minimal.
Therefore, the results were expressed using „incident intensity at the surface‟ of the
medium and not the average intensity. Another reason for deviating from the
conventional way of measuring the absorbance coefficient was the fact that the juice
components, in addition to absorbing UV light may also react with each other chemically,
which may alter the rate of patulin degradation. For example, although ascorbic acid
absorbs in the UV region (peak absorbance 245 nm) it also degrades patulin (Drusch et
al.; 2007). Thus, in such a situation absorbance coefficient may not be the best indicator
of the efficacy of UV light for patulin degradation.
3.2.7 Statistical analysis
Statistical analysis was carried out using Microsoft® Excel (version 2007) SAS
®
(version 9.1 SAS Inc., Cary NC) and Minitab® 15 (Minitab Inc., State College, PA).
Student‟s t-test or ANOVA was used to check the statistical significance between the
treatments at 95% confidence level.
60
3.3 RESULTS AND DISCUSSION
3.3.1 Patulin degradation in malic acid
Figure 3-3 shows the effect of incident intensity (0.8-3.3 mW/cm2) on the rate of
patulin degradation in 0.5% malic acid buffer (pH 3.3). Data shows that the UV induced
degradation followed first order kinetics (r2>0.99). The average rate constants in terms of
duration of exposure for 0.8, 1.4, 1.8 and 3.3 mW/cm2 were 0.009 ±0.00, 0.0185 ±
0.0005, 0.01 ±0.002, 0.05 ±0.002 min-1
, respectively and were significantly different
(p<0.05) except for 1.4 and 1.8 mW/cm2 (p>0.05). The rate constants in terms of duration
of exposure decreased with the decrease of incident intensity. This was expected as with
the decrease in intensity, the number of photos reaching the surface of the medium per
unit time decreased. In figure 3-4, data from figure 3-3 was plotted in terms of UV dose.
This transformation was performed using equation 3-1 and equation 3-3. Despite the
transformation, degradation reaction followed first order. The rate constants in terms of
UV dose for 0.8, 1.4, 1.8 and 3.3 mW/cm2 were 0.19 ±0.005, 0.2 ±0.005, 0.2 ±0.02, 0.25
±0.01 cm2/J, respectively and were statistically not significant (p>0.05) except for the
rate constant at 3.3 mW/cm2 which was significantly different from those at 0.8 and 1.4
mW/cm2 (p<0.05). The D50 values for 0.8, 1.4, 1.8 and 3.3 mW/cm
2 intensity were
3.52±0.1, 3.15±0.14, 3.43±0.36, 2.77±0.11, respectively. The D50 values were not
significantly different except for 3.3 mW/cm2, which had significantly lower D50 than
those at 0.8 and 1.4 mW/cm2. Thus, although the rate constant in terms of duration of
exposure were significantly different for varying intensities, the rate constants in terms of
UV dose were not. This can be explained by the fact that when the data was plotted
61
Figure 3-3: Effect of incident intensity on the degradation of patulin (C0=1000 ppb)
in 0.5% malic acid buffer (pH 3.3). Each data point represents average of three
measurements ± standard deviation.
62
Figure 3-4: Effect of incident intensity on the degradation of patulin (C0=1000 ppb)
in 0.5% malic acid buffer (pH 3.3). Each data point represents average of three
measurements ± standard deviation.
63
against the UV dose, lower intensity was compensated by higher exposure times.
Therefore, plotting the data with respect to UV dose has its merits over plotting the data
against time, as it is more robust against slight variations in intensity. Henceforth, the
data will be plotted against the UV dose and rate constants will be shown in terms of UV
dose. Future experiments were conducted at the incident intensity range of 1.4-1.8
mW/cm2 and thus the rate of patulin degradation was not affected by slight variations in
the intensity.
3.3.2 Effect of initial concentration
Figure 3-5 shows the effect of initial concentration of patulin (C0= 100-1000 ppb)
on the rate constant of patulin degradation in 0.5% malic acid buffer (pH 3.3). The
average rate constants for 100, 500 and 1000 ppb initial concentrations were 0.25 ±0.15,
0.22 ±0.01, and 0.22 ±0.015 cm2/J, respectively. The rate constant for patulin degradation
was not significantly affected by the initial patulin concentration between 100 and 1000
ppb (p>0.05). The D50 values for 100, 500 and 1000 ppb patulin were 2.74 ±0.16, 3.15
±0.14, 3.06 ±0.15 respectively and were not significantly different from each other
(p>0.05).
3.3.3 Effect of buffer pH
Figure 3.6 shows effect of malic acid buffer pH (3.0-3.6) on the rate of patulin
(C0=1000 ppb) degradation. The average rate constants for pH 3.0, 3.3 and 3.6 were
determined to be 0.21 ±0.015, 0.21 ±0.02, 0.22 ±0.001 cm2/J. The pH of buffer had no
significant impact on the rate constant of patulin degradation (p>0.05) through the tested
64
Figure 3-5: Effect of initial patulin concentration on the rate of degradation in 0.5%
malic acid buffer (pH 3.3). Each data point represents an average of three
measurements ± standard deviation.
10
100
0 2 4 6 8
1000 ppb
500 ppb
100 ppb
% p
atu
lin
rem
ain
ing
UV dose (J/cm2)
65
Figure 3-6: Effect of malic acid buffer pH on the rate of degradation rate of patulin
(C0=1000 ppb). Each data point represents average of three measurements ±
standard deviation.
10
100
0 1 2 3 4 5 6 7 8
pH 3.0
pH 3.3
pH 3.6
UV dose (J/cm2)
% p
atu
lin
rem
ain
ing
66
range. The D50 values for pH 3.0, 3.3 and 3.6 were 3.06 ±0.023, 3.24 ±0.25, 3.33 ±0.36
J/cm2
and were not significantly different (p>0.05).
3.3.4 Effect of ascorbic acid, tannic acid and suspended particles
From the previous experiments, it was evident that pH, initial concentration of
patulin and the incident intensity did not affect the rate of patulin degradation and
therefore were not included in the subsequent Box-Behnken design. The experimental
conditions and the results for the Box-Behnken design are shown in table 3-1. ANOVA
results (table 3-2) showed that tannic acid reduced the rate constant of patulin
degradation (p<0.05), while AA did not show significant influence (p>0.05). Suspended
particles did not significantly alter the rate (p>0.05), but the interaction parameter with
tannic acid was significant (p<0.05). Therefore, only ascorbic acid was excluded from the
statistical model equation shown in equation 3-6.
Rate constant for patulin degradation (cm2/J) = 0.17034 - 0.385921*TA +
0.000517*NT + 0.225371*TA*TA - 0.000713*TA*NT Eq. (3-6)
Where, TA- Tannic acid concentration (g/L), NT- Turbidity (NTU)
Tannic acid absorbs in the UV region. The apparent absorbance values (Aapp) for
0.5 g/L and 1 g/L tannic acid were 10.6 and 20.5 respectively. Thus, tannic acid possibly
competes with patulin for the photons thereby reducing the degradation rate. Ascorbic
acid has been reported to cause patulin degradation (Drusch et al., 2007; Kokkinidou et
al.; 2008) in apple juice. However, in our experiments, ascorbic acid showed no
67
Table 3-1: Experimental design and corresponding patulin (C0=1000 ppb)
degradation rate constant for model apple juice system
Run AA ( mg/L) TA(g/L)
Suspended
particles (NTU)
Rate
constant
(cm2/J)
D50
(J/cm2)
1 0 0 50 0.2107 3.29
2 0 1 50 0.000 0.00
3 100 0 50 0.1742 3.98
4 100 1 50 0.000 0.00
5 50 0 0 0.1643 4.22
6 50 0 100 0.2356 2.94
7 50 1 0 0.000 0.00
8 50 1 100 0.000 0.00
9 0 0.5 0 0.0373 18.58
10 100 0.5 0 0.0435 15.93
11 0 0.5 100 0.0432 16.05
12 100 0.5 100 0.0306 22.65
13 50 0.5 50 0.0436 15.9
14 50 0.5 50 0.0480 14.44
15 50 0.5 50 0.0461 15.04
68
Table 3-2: ANOVA table for statistical model for patulin degradation in model
apple juice (- indicates data not shown as statistically not significant)
Source SS MS F Pr>F
AA 0.076989 0.076989 456.316 -
TA 0.000517 0.000517 3.06316 0.0001
NT - - - 0.1106
AA*AA - - - -
AA*TA - - - -
AA*NT - - - -
TA*TA 0.011852 0.011852 70.2448 0.0001
TA*NT 0.0017871 0.0017871 7.532806 0.0206
NT*NT - - - -
Lack of fit 0.000913 0.000228 1.7677 0.25369
69
interaction with patulin. Suspended particles scatter light. Thus, particles present at the
surface may reflect the light away from the buffer medium and less photons may be
available for patulin degradation.
The surface plots for the model are shown in figure 3-7. The fit statistic for the
model is provided in table 3-3. The lack of fit for the model was not significant (p>0.05)
indicating that the data fits the model reasonably.
From the D50 values, it is evident that even in the condition with the highest rate
constant (0.5% malic acid buffer), the energy required for 50% reduction of patulin (3.06
J/cm2) is much higher than that needed for 5-log reduction of E.coli O157:H7 (14.32
mJ/cm2) (Donahue et al.; 2004). Therefore, although UV processing could potentially be
used for reducing the patulin load, such high doses will likely incur significant quality
losses. Further investigation is needed to study the sensory attributes of the apple juice
treated at high UV dose. Additionally, future work is needed to identify UV induced
degradation products of patulin and to determine the toxicity of these products.
3.4 CONCLUSIONS
UV induced degradation of patulin followed a first order kinetics in model apple
juice. Degradation rate constants in terms of UV dose were independent of incident
intensity between 0.8-1.8 mW/cm2, initial concentration (100-1000 ppb) and buffer pH
(3.0-3.6) (p>0.05). Patulin degradation rate was reduced by tannic acid, and suspended
particles possibly due to competitive absorption and scattering of UV light respectively.
70
Table 3-3: Fit statistic for the model. Master model represents the statistic for model
that includes all parameters and Predictive model includes only significant
parameters.
Parameter Master Model Predictive model
Mean -0.07181 -0.07181
R-square 99.01% 98.17%
Adj. r-square 97.22% 97.44%
RMSE 0.013546 0.012989
CV -18.8646 -18.0891
71
Figure 3-7: Surface plots for the Box-Behnken design for model apple juice system
at (a) 50 NTU (b) 100 NTU
(b) Suspended particles=100 NTU
0.2
0
0
100
TA AA
Rat
e co
nst
ant
Rat
e co
nst
ant
0.2
0
0
TA AA
100
(a) Suspended particles=50 NTU
72
Ascorbic acid (0-100 mg/L) did not affect the patulin degradation rate (p>0.05). Thus,
UV processing can reduce the patulin loads in apple juice; however the high dose
requirements may cause severe damage to the organoleptic attributes of the product.
Further research is necessary to identify the degradation products of patulin and study
their toxicity.
73
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apples. Journal of Food Protection. 55, 902 – 904.
Trucksess, M.; Tang, Y. 2000. Solid phase extraction method for patulin in apple juice
and unfiltered apple juice. Methods in Molecular Biology. 157, 205-214.
US FDA, (2001). http://www.cfsan.fda.gov/~dms/patubck2.html Accessed on April 15th 2009,
7:16 pm
Zegota, H.; Zegota, A.; Bachman, S. 1988. Effect of irradiation on the patulin content
and chemical composition of apple juice concentrate. Zeitschrift für
ebensmitteluntersuchung und -Forschung. 187(3), 235-238.
USDA food composition data for unsweetened, unfortified apple juice.
http://www.nal.usda.gov/fnic/foodcomp/cgi-bin/list_nut_edit.pl
77
Chapter 4
Ascorbic acid degradation in a model apple juice system and in apple
juice during ultraviolet light processing and storage
ABSTRACT
Ultraviolet light induced degradation of ascorbic acid (AA) in an apple juice
model system and in apple juice was studied using a collimated beam batch UV reactor.
Despite a considerably higher UV254 absorbance of apple juice samples, AA degradation,
measured by HPLC, occurred more rapidly in juice compared to 0.5% malic acid system.
Model system studies demonstrated that UV degradation of AA was more rapid at higher
dose levels and that reaction accelerated with increasing exposure time. AA degradation
significantly (p<0.05) increased as the pH of the medium was raised from 2.4 to 5.5,
although not from 2.4 to 3.3. Increasing malic acid concentration between 0.1 and 1%,
while maintaining pH constant at 3.3, increased AA degradation (p<0.05) although there
was no difference between 0.5 and 1.0 %. Tannic acid, used to study the effects of UV
absorbing compounds in juice, decreased AA degradation rate with increasing
concentration due to competitive absorption of UV light. Ten percent sucrose, fructose,
and glucose had variable effects on AA degradation. Sucrose showed no significant
78
effects and glucose slightly decreased AA degradation. However, fructose dramatically
increased AA degradation, perhaps due to breakdown products of this sugar, and the
effect was proportional to the amount added. AA degradation in the model system and in
apple juice continued during storage in the dark. Post UV treatment degradation was
more rapid at higher initial UV dose levels and higher storage temperature.
4.1 INTRODUCTION
Non-thermal food processing technologies are attracting growing interest in food
industry owing to their benefits over thermal processing techniques such as better flavor
and texture retention, less severe effects on food micronutrients, relatively fresher
appearance of food products and potential energy savings (Koutchma, 2009). Ultraviolet
light (UV) processing of foods is one of the emerging non-thermal processing techniques
and is being widely studied in different areas of food processing such as extending the
shelf life of fresh fruits and vegetables (Gonzalez-Aguilar et al. 2001; Fonseca and
Rushing, 2006) increasing the phytochemical content in fresh fruits (Cantos et al, 2000),
and sanitization of food contact surfaces (Guerrero-Beltran and Barbosa-Canovas, 2004).
UV light has been effectively used to treat water (Legrini et al.; 1993). However, it is
only recently being studied for utilization as a food processing technique. There are two
distinct UV processing methods (1) pulsed UV- very high intensity light containing
significant proportion of UV range energy (200-400 nm) and (2) monochromatic UV-
where almost 90% of the energy is from a single wavelength of 254 nm (conventionally
referred to as UV-C). Advantages and disadvantages of each have been described
elsewhere (Tikekar et al., 2010). In this study, a monochromatic UV system is used.
79
The mechanism of UV induced microbial lethality is well understood. The peak
absorbance of DNA is close to 254 nm. Absorption of UV light by DNA causes
dimerization of adjacent pyrimidine molecules (thymine or cytosine) on the same DNA
strand. This cross linking makes DNA unsuitable for transcription, thus halting the
protein biosynthesis and eventually leading to cell death (Sastry et al., 2000). Exposure to
UV light can also cause mutations that lead to cellular injuries and death. UV is effective
against vegetative cells of bacteria while yeasts are more resistant. Bacterial spores are
15-20 times more resistant than corresponding vegetative cells (Coohill and Sagripanti,
2008).
The penetration and therefore the effectiveness of UV light are inversely
proportional to the absorbance of the medium as measured by the attenuation coefficient
(mm-1
) which is defined as the change in the absorbance per unit path length of the
medium. Solid foods have high attenuation coefficients and therefore the penetration of
light is negligible. Hence, UV can be used only for the surface treatment of solid foods.
However, liquid foods have a finite and relatively lower attenuation coefficient and
therefore offer better penetration for UV light. The attenuation coefficient is determined
by the chemical and physical composition of the food. Juices contain chemical
compounds that absorb in the UV region that contribute to higher attenuation
coefficients, and thus lower the amount of light available for microbial lethality (Beltran
and Canovas, 2004; Koutchma 2008). Therefore, the rate of UV induced microbial
lethality and the desired UV dose strongly depends on the medium in which the
microorganisms are present.
80
UV processing can reduce the load of pathogenic microorganisms, specifically
Escherichia coli O157:H7 and Cryptosporidium parvum by 5-log in apple cider at the
dose level of 14.32 mJ/cm2 (Koutchma et al., 2004; Quintero- Ramos et al., 2004; US
FDA, 2001). But as this technology is being implemented for other juices, it is evident
that much higher doses are needed to products such as orange juice or red grape juice
owing to their turbidity and presence of UV absorbing components. UV processing has
been successfully applied to various other juices such as orange juice, guava juice and
mango nectars with satisfactory microbial lethality, although the doses required to
achieve a specific log-reduction vary considerably (Keyser et al. 2004; Tran and Farid,
2004).
Many food chemicals absorb in the UV region, including ascorbic acid (AA)
(Buettner and Jurkiewicz, 1996).The absorption of light causes excitation of these
molecules which then take part in several photochemical reactions leading to their
degradation (Kagan, 1993). Apples do not contain high levels of AA. However, most
commercially available apple juice is fortified with the vitamin. Depending upon the type
of juice, the UV dose applied may increase and thus increasing the possibility of AA
degradation. Therefore, it is necessary to investigate the fate of AA when exposed to UV
light.
Ascorbic acid (2-(1,2-dihydroxyethyl)-4,5-dihydroxy-furan-3-one) is a water
soluble vitamin. It has been extensively studied for its chemistry, physiological activity
and its role as an antioxidant and therefore as a nutraceuticals (Buettner and Jurkiewicz,
1996; Cameron et al., 1979; Englard and Seifter 1986). The research suggests that AA
protects against UV induced oxidation reactions by quenching the radicals that are
81
generated (Fuchs and Kern, 1998; Shindo et al., 1993) as a result of exposure to the UV
light. But sparse data is available pertaining to the sensitivity of AA itself to the UV light.
A study indicated an approximately 30% reduction in AA at a dose level of 14.32 mJ/cm2
in apple cider (Adzahan, 2006). Koutchma et al. (2009) determined that AA degradation
followed zero order in apple juice with a rate constant of -0.025 s-1
. The degradation rate
was inversely proportional to the absorption coefficient of the juice. Tran and Farid
(2004) reported 17% AA degradation when orange juice was exposed to 100 mJ/cm2.The
extent of degradation would be strongly dependent upon the dose level and the type of
juice involved. Therefore, it is necessary to acquire some basic understanding of UV
induced degradation of AA that could be translated for other fruit juices as well.
Our objectives are to investigate the fate of AA when it is exposed to UV light
and how the rate of degradation is affected by chemical and physical juice characteristics.
4.2 MATERIALS AND METHODS
4.2.1 UV treatment equipment
All experiments were carried out using a bench-top batch collimated beam UV
reactor (figure 4-1). The reactor consisted of three UV lamps (254 nm, 10 W, Atlantic
Ultraviolet Inc., Hauppauge, NY) mounted within a shielded horizontal cylindrical holder
fitted over a vertical tube (100 mm diameter X 100 mm length). Collimation was
achieved by painting the inside surface of the vertical tube with UV absorbing black
paint. Based on the length of the tube, the calculated maximum incident angle was no
greater than 20°. Incident intensity (mW/cm2) was measured by placing a radiometer
(Model: UVP-J225, UVP LLC, Upland, CA) at the bottom of the tube at a length equal to
82
Figure 4-1: Schematic representation of the collimated beam batch UV reactor
Sample in a Petri plate with stirrer
UV lamp
Collimator
Collimated beams
83
the distance between the light source and the surface of the sample. Variation of incident
intensity over the entire sample surface area was less than 1%. This slight error was
neglected because the sample was continuously stirred with a mechanical stir bar (300
rpm).
Malic acid buffer (0.5% unless otherwise stated) was used as a model system.
Samples were prepared by varying the amounts of tannic acid (Sigma Aldrich, St Louis,
MO), sucrose, glucose, fructose (Sigma Aldrich, St. Louis MO) and caramel () in the
buffer. pH was adjusted by adding NaOH or HCl solution. Absorbance values at 254 nm
for each constituent at levels used in experiments were determined using a
spectrophotometer (Model: Helios Gamma, Thermo Scientific, Waltham MA). When
values were outside the range of the spectrophotometer, an appropriate dilution was
made, the absorbance measured, and the apparent absorption calculated based on the
dilution factor.
All experiments were carried out by adding a 20-ml sample into the bottom of a
plastic petri-dish (100 mm X 10 mm) and exposing it to UV light at various time
intervals. All UV treatments were performed at 21 (±1) °C. At each time interval, 0.6 ml
was withdrawn from a treated and an untreated (control) sample for HPLC analysis.
Sample withdrawal and the resultant reduction in the depth of the liquid accounted for a
theoretical incident intensity reduction of no more than 0.03 mW/cm2.
Storage study was carried out with 0.5% malic acid or apple juice treated with
appropriate doses of UV light. Samples were held in a temperature controlled water bath
(Thermo Neslab HX 300, Thermo Scientific, Waltham, MA) at 4 or 25 °C and were
84
protected from exposure to ambient light by covering them with aluminum foil. After
each time interval, the amount of AA remaining was determined by HPLC.
4.2.2 UV dose measurement
UV dose (J/cm2) was calculated by multiplying the incident intensity (W/cm
2) at
the sample surface by exposure time in seconds (equation 4-1).
D= I×t Eq. (4-1)
Where, D= UV dose (J/cm2), I= Incident intensity (W/cm
2), t= duration of
exposure (second)
Incident intensity or irradiance range at the surface of the liquid was between 1.4-
1.8 mW/cm2 due to variations in the source intensity of the bulb.
4.2.3 Apple juice
Un-pasteurized apple cider was purchased from a local apple cider producer. The
sample was clarified by centrifugation (Beckman Coulter Model Avanti J-26 XPI,
Fullerton, CA) at 15000 g for 45 minutes. The final turbidity was < 3 NTU as measured
by a digital turbidimeter (Hach® (Model 2100P, Hach Inc., Loveland, CO). The apple
juice was stored at -15 °C until further use.
4.2.4 High performance liquid chromatography (HPLC) analysis
85
AA was quantified by a Waters HPLC system (pump model: 600; Autosampler:
71P; photodiode array (PDA) detector: 2998, Waters Inc., Milford MA) using a hybrid C-
18 reverse phase / cation exchange column (Primesep-D, 4.6 mm × 150 mm, particle size
5 μm, SIELC Inc., Prospects Heights, IL). The mobile phase consisted of 95% water, 5%
acetonitrile, and 0.1% formic acid (pH 1.8 ±0.05 adjusted with HCl). The PDA detector
wavelength range was 220-300 nm. Injection volume was 20 µL. AA showed peak
absorbance at 245 nm with an elution time of 2.2 min (figure 4-2). Peak identity was
confirmed by injecting samples of pure AA in solution.
4.2.5 Data analysis
The relative rates for AA degradation were characterized by two methods. When
the data could be described by zero order reaction kinetics, Equation 4-2 was used to
calculate the rate constant (min-1
).
Ct= C0- kt Eq. (4-2)
Where, Ct is the concentration (mg/L) of AA at any UV exposure time t (min), C0 = the
initial concentration (mg/L) of AA, and k = the zero order reaction rate constant (min-1
)
When the data did not fit into a zero or higher order equation, the data were fitted
to a second order polynomial function (equation 4-3).
y= ax2 + bx + c Eq. (4-3)
Where, y= % AA remaining; x= UV dose (J/cm2); and a, b, c are derived coefficients.
The polynomial fits for the data showed r2
values > 0.98 in all the experiments, with no
86
Figure 4-2: A representative HPLC chromatogram of AA (AA0= 100mg/L) (a)
control (b) after UV dose of 5.04 J/cm2
(a)
(b)
87
systematic error observed in the plots of residuals versus fits. Therefore, the polynomial
equation derived for each curve was used to calculate the UV dose required to achieve a
50% reduction in AA, where the value of x for y = 50 was solved using the quadratic
formula. The term D50 is used in this study to designate these values.
4.2.6 Statistical Analysis
Statistical analysis for significant differences between treatments was carried out
either by single-factor Analysis of Variance (ANOVA) or student‟s t-test using
Microsoft® Excel 2007 (Redmond, WA).
4.3 RESULTS AND DISCUSSION
In this study, we measured only the amount of ascorbic acid present and not the
total vitamin C activity as measured by combined ascorbic and dehydroascorbic acid
(DHA) concentration. This approach was used by Tran and Farid (2004) to study the
effect of UV light on AA in orange juice, Adzahan (2006) to study AA loss during UV
exposure and Burdurlu et al. (2006) to study the storage loss of AA in citrus concentrate.
It was confirmed that DHA was not retained on our HPLC column by spiking samples
with the pure compound. Therefore, the values for AA do not include DHA, Since the
DHA content in apple juice is low, ranging from 5-10% of AA concentration and is
known to be unstable (Beherens and Medere, 1987), the determination of AA in samples
is a suitable predictor for vitamin C loss in UV treated juice.
4.3.1 Comparison of AA degradation in apple juice and juice model system
88
UV induced degradation of AA in apple juice (AAo = 170 mg/L, pH 3.5) and in
0.5% malic acid (AAo = 190 mg/L, pH 3.3) as a function of UV dose is compared in
figure 4-3. Degradation occurred in both samples. However, AA degraded more rapidly
in apple juice as indicated by a D50 value of 2.5 J/cm2 for juice compared to 14.2 J/cm
2
for the model system. This result was unexpected since the absorbance value at 254 nm
of the apple juice used in this experiment was considerably higher than the 0.5% malic
acid model system (table 4-1). Apple juice is a complex mixture of compounds, many of
which absorb in the UV region. It would be expected that these UV absorbing
compounds decrease the quantity of light that interacts with AA molecules and thus slow
the degradation of AA. To explore this paradox, the effects of individual chemical
constituents in apple juice on UV induced degradation of AA were studied in the malic
acid model system.
4.3.2 Kinetics of AA degradation in 0.5% malic acid
UV induced AA degradation in 0.5% malic acid (pH 3.3) at initial AA
concentrations (AAo) of 25, 50, 100, 150 and 200 mg/L is shown in figure 4-4. At 100,
150, and 200 mg/L, the graph was linear for up to 40, 60, and 75 min, respectively,
representing zero order kinetics. At greater exposure times, however, the reaction rate
deviated from linearity. At AAo concentrations of 25 and 50 mg/L, deviation from zero
order kinetics was not apparent. Zero order rate constants obtained from the linear portion
of each curve (kavg = 0. 0.55 + 0.037 min-1
) were not significantly (p>0.05) different at
each AAo level suggesting that AA degradation follows a similar degradation mechanism
during the initial part of the reaction. Koutchma (2009) reported that UV induced AA
89
Figure 4-3: UV degradation of AA in apple juice (AA0=170 mg/L, pH 3.5) and in 0.5
% malic acid (AA0=190 mg/L, pH 3.3). Each data point represents an average of
three measurements + standard deviation.
0
20
40
60
80
100
120
140
160
180
200
0 5 10 15 20
Buffer
Juice
Co
nce
ntr
ati
on
of
AA
(m
g/L
)
UV dose (J/cm2)
90
Table 4-1: Absorbance values (254 nm) for chemical compounds used in
experiments
Chemical
Constituent Concentration
Absorbance (254
nm)
(10 mm path)
Malic acid 0.10 % 0.07
0.50 % 0.30
1.0 % 0.50
Tannic acid 25 mg/L 0.55
100 mg/L 2.24
200 mg/L 4.58
Fructose 10.0 % 0.16
Glucose 10.0 % 0.007
Sucrose 10.0 % 0.065
Apple juice Single strength
17.5
91
Figure 4-4: UV degradation of AA in 0.5% malic acid buffer (pH 3.3) at varying
initial AA0 concentrations. Each data point represents an average of three
measurements + standard deviation.
Co
nce
ntr
ati
on
of
AA
(m
g/L
)
UV exposure (minutes)
92
degradation followed zero order kinetics at AAo concentrations between 341-660 mg/L.
However, the experiments in that study were conducted with apple juice and at exposure
times that reached only up to 11.7 min which corresponds to 20-40% decreases in AA. In
this study, 0.5% malic acid was the reaction medium and exposure times were up to 200
min which corresponds to up to 90% reduction in AA concentration. The observed
acceleration of AA disappearance greater than that predicted by the zero order kinetics
equation suggests that secondary reactions are occurring at higher UV incident intensities
and longer exposure times.
Because the data for the entire course of the AA degradation does not fit a zero
order kinetic model, each reaction will henceforth be graphically presented as a function
of dose levels and D50 values will be used to numerically compare reaction rates. An
added advantage of using this approach is that day to day variations in the intensity of the
UV lamps are corrected by measuring incident intensity before each experiment and then
calculating the dose value that corresponds to each exposure time. Although the quadratic
function used to calculate D50 values does not shed any light on possible reaction
mechanisms, it serves to quantitatively compare the effect of individual chemical
constituents on UV induced AA degradation.
4.3.3 Effect of pH
The effect of pH on UV induced degradation of AA (AA0 = 50 mg/L) in 0.5%
malic acid is shown in figure 4-5. D50 values were significantly affected by pH (p<0.05)
between pH 2.4 (4.77 J/cm2)
and pH 5.5 (2.72 J/cm2). However, D50 values between pH
2.4 (4.77 J/cm2)
and 3.3 (4.23 J/cm2) did not significantly differ (p>0.05). The lower dose
93
Figure 4-5: Effect of pH on UV degradation of AA in 0.5% malic acid buffer. Each
data point represents an average of three measurements + standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8
pH 2.4
pH 3.3
pH 5.5
UV dose (J/cm2)
% A
A r
ema
inin
g
94
necessary to achieve a 50% reduction in AA at pH 5.5 indicates that AA is more
susceptible to UV degradation at higher pH values. Differences in AA degradation cannot
be explained by UV254 absorbance (254 nm) at each pH value. A254 values at pH 2.4, 3.3,
and 5.5 were 0.17, 0.26, and 0.29. Since the effect was more pronounced at a pH value
above the pKa1 for AA (pKa1= 4.2), this suggests that the dissociated form of AA is more
prone to chemical reaction than the un-dissociated molecule. Since fruit juices vary in
pH, this characteristic must be taken into consideration when predicting AA losses during
UV processing.
4.3.4 Effect of malic acid concentration
Figure 4-6 shows UV degradation of AA (AA0= 100 mg/L) at malic acid
concentrations between 0.1 and 1.0% and at a constant pH of 3.3. The D50 value for the
reaction in 0.1% malic acid (10.13 J/cm2) was significantly greater than that obtained at
0.5% (7.15 J/cm2) and 1% (7.89 J/cm
2) malic acid. Thus malic acid appears to have a
positive effect on the AA degradation reaction in the model system. This is an interesting
result given that malic acid absorbs light in the UV region (table 4-1) and would be
expected to reduce the amount of light reaching AA molecules. It is possible that malic
acid is susceptible to UV degradation which may form compounds triggering side
reactions that accelerate the destruction of AA. Figure 4-7 demonstrates that malic acid is
not essential for UV induced AA degradation since the reaction also occurs in distilled
water.
4.3.5 Effect of absorbance
95
Figure 4-6: Effect of malic acid concentration (pH = 3.3) on UV degradation of AA
(AA0= 100 mg/L). Each data point represents an average of three measurements +
standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
0.10%
0.50%
1%
% A
A r
ema
inin
g
UV dose (J/cm2)
96
Figure 4-7: UV induced AA degradation (AA0= 100 mg/L) in distilled water (pH
6.0). Each data point represents the average of two measurements.
0
20
40
60
80
100
120
0 2 4 6 8 10 12
UV dose (J/cm2)
Co
nce
ntr
ati
on
of
AA
(m
g/L
)
97
In previous studies (Koutchma et. al, 2004; Murakami, et al., 2006), caramel
solutions were used to study the effect of changes in the absorbance of the medium on the
rate of UV destruction of microorganisms. In those studies, microbial destruction
decreased with increasing amounts of caramel. However, preliminary experiments
showed that UV induced AA degradation was accelerated by the addition of caramel
(figure 4-8). Caramel is a dark-brown liquid containing a complex mixture of polymeric
compounds formed from unsaturated (5- and 6- membered ring), cyclic compounds
(Schwarz, et al., 2008). It is hypothesized that unknown compounds formed during the
preparation of caramel have a positive effect on AA degradation that more than
compensates for increases in absorbance. To avoid this confounding effect, tannic acid, a
compound not naturally present in apple juice, was used to study the effect of absorbance
on AA degradation. Tannic acid is a complex mixture of water soluble, UV absorbing,
polyphenolic glucose esters of gallic acid.
The effect of tannic acid concentration on AA degradation (AA0=100 mg/L) in
malic acid buffer (pH 3.3) is shown in figure 4-9. Tannic acid did raise the absorbance of
the reaction solution (table 4-1) and AA degradation significantly (p<0.05) decreased
when the concentration was increased from 0 to 200 mg/L. This was evidenced by higher
D50 values with increasing tannic acid. D50 values at 0, 25, 100, and 200 mg/L tannic acid
were 7.06, 9.25, 11.04, and 14.97 J/cm2, respectively. The values were significantly
different (p<0.05) except for 0 and 25 mg/L (p>0.05). In this experiment, tannic acid
served primarily as a surrogate for polyphenols which are naturally present in apple juice
and which can also be expected to reduce absorbance due to their UV light absorbing
properties. Thus, it can be expected that, in juice products containing higher polyphenol
98
Figure 4-8: Effect of added caramel (60 mg/L) on the UV induced degradation rate
of ascorbic acid AA (AA0= 150 mg/L) in malic acid (pH 3.3). Each data point
represents a single measurement.
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
60 mg/l caramel
No caramel
% A
A r
ema
inin
g
UV dose (J/cm2)
99
Figure 4-9: Effect of tannic acid concentration on degradation of AA (AA0=100
mg/L) in 0.5% malic acid (pH 3.3). Each data point represents an average of three
measurements + standard deviation.
0
20
40
60
80
100
0 2 4 6 8 10 12
200 mg/l
100 mg/l
25 mg/l
0 mg/l
% A
A r
ema
inin
g
UV dose (J/cm2)
100
levels, lower reductions in AA would occur at a given dose level. It should be noted that
tannic acid, as is the case with other polyphenols, has antioxidant properties (Scalbert et
al., 2005) and it is possible that it may also have some level of protective effect on AA
degradation by quenching oxidative free radicals.
4.3.6 Effect of sugars
The effect of 10% (w/v) glucose, fructose, or sucrose on UV degradation of AA
(AA0= 100 mg/L) in 0.5% malic at pH 3.3 is shown in figure 4-10. D50 values for the
control (no added sugars), sucrose, glucose, and fructose, were 7.16, 6.97, 8.86, and 1.46
J/cm2 respectively. Thus sucrose has no significant effect, glucose has a slight, but
significant (p<0.05) protective effect, and fructose results in a dramatic and significant
acceleration of AA degradation. A significant concentration effect was observed for
fructose (figure 4-11). D50 values at 0, 2, 5 and 10% fructose were 7.14, 4.77, 2.91 and
1.46 J/cm2, respectively.
Differences in the effects of sugars on AA degradation cannot be explained by
absorbance values for each solution. In fact, the absorbance of 10% fructose at 254 nm
was considerably greater than that for glucose or sucrose (table 4-1). Triantaphylides et
al. (1984) reported that fructose is susceptible to photolytic reactions when exposed to
UV light and that the open chain configuration is prone to UV light while the ring form is
comparatively inert. The study reported that fructose has a relatively less stable ring
structure compared to glucose with approximately 0.8% and 0.024% of each respective
sugar existing in the open chain form. Binkley and Binkley (1998) reported that, when
exposed to UV light, the carbonyl group in the open chain form of fructose can undergo
101
Figure 4-10: Effect of fructose (10%), glucose (10%) and sucrose (10%) on UV
induced degradation of AA (AA0= 100 mg/L) in 0.5% malic acid (pH 3.3). Each data
point represents an average of three measurements + standard deviation.
0
20
40
60
80
100
0 5 10 15
No sugar
10% fructose
10% glucose
10% sucrose
% A
A r
ema
inin
g
UV dose (J/cm2)
102
Figure 4-11: Dependence of UV induced AA degradation (AA0= 100 mg/L) on
fructose concentration added to 0.5% malic acid (pH 3.3). Each data point
represents an average of three measurements + standard deviation.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12
0 % fructose
2% fructose
5% fructose
10% fructose% A
A r
ema
inin
g
UV dose (J/cm2)
103
Norrish type-1 reactions leading to the formation of hydroxyalkyl and acyl radicals.
Sucrose, which cannot exist in an open chain form, would thus be expected to be
comparatively unaffected by UV light which is consistent with the results in figure 4-9.
It can be hypothesized that radicals formed by UV photolysis of fructose may in
turn react with AA, itself a potent radical scavenger and sacrificial antioxidant. Given the
known chemistry of AA degradation, ascorbate radicals may then be generated which
continue to degrade into dehydroascorbic acid as well as further end products (Gregory
III, 2008).
4.3.7 Interaction of tannic acid and fructose in buffer
An experiment was carried out to mimic apple juice conditions and identify if
presence of fructose in juice fully explains the higher rate of AA degradation in apple
juice. Figure 4-12 shows the effect of simultaneous addition of tannic acid (200 mg/L)
and fructose (5% w/v) in malic acid buffer on AA (AA0=200 mg/L) degradation rate. The
average D50 value for the system with tannic acid and fructose was 12.41±0.63 J/cm2 and
was not significantly different from the average D50 value of 13.47±0.93 J/cm2 for AA in
0.5% malic acid buffer alone. Thus, it can be observed that the rate lowering effect of
tannic acid was offset by fructose. Nevertheless, the AA degradation rate in this system
was still much lower than that in apple juice, suggesting that fructose is not the only
component that accelerates the rate of reaction and there may be several other chemical
components that may exert similar effect.
104
Figure 4-12: AA (AA0=200 mg/L) degradation rate in malic acid buffer
simultaneously incorporated with tannic acid (200 mg/L) and fructose (5% w/v) as
compared AA degradation in malic acid buffer and apple juice. Each data point
represents an average of three measurements ± standard deviation
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15
200 mg/l TA + 5% fructose
0.5% malic acid buffer
Apple juice%
AA
rem
ain
ing
UV dose (J/cm2)
105
4.3.8 Post UV treatment effects on AA degradation
Figure 4-13 shows that AA degradation in 0.5% malic acid occurs during storage
and that the rate of degradation increases as a function of initial UV dose. After UV
treatments of 0, 0.96, 1.92, and 5.76 J/cm2, AA levels were reduced to 100, 85, 72, and
52 mg/L, respectively. Among UV treated samples held for 17 hours at 25 °C, AA
retention was 76, 65, 43, and 27%, respectively.
Storage temperature had a significant (p<0.05) effect on the extent to which AA
degraded in treated and untreated samples (figure 4-14). At 4 °C, there was no significant
(p>0.05) change in AA for up to 150 hours. However, in UV treated samples, only 30%
of the initial AA remained in samples held under the same conditions. In untreated
samples stored at 25 °C, AA degraded rapidly with only 30% remaining after 43 hours.
This is compared to a 27% residual AA content in UV treated samples held for only 17
hours.
Post UV treatment degradation was also observed in apple juice samples stored at
4°C for up to 48 hours (figure 4-15). No significant change in AA concentration occurred
in untreated juice samples. However, in UV treated (1.2 J/cm2) juice, only 32% remained
after the same time interval. Similarly, Kabasakalis et al. (2000) reported that thermally
processed orange juice lost ascorbic acid at a faster rate than fresh orange juice during
storage. We hypothesize that extended degradation of AA after UV is due to initiation of
cascade of radicals that continue to degrade AA during storage. Further investigation is
necessary to detect these radicals and identify the exact mechanism.
106
Figure 4-13: Effect of initial UV dose on post-processing storage degradation of AA
(AA0=100 mg/L) in buffer (pH 3.3) at 25 °C. Each data point represents an average
of three measurements + standard deviation.
107
Figure 4-14: Effect of storage temperature (4 °C and 25
°C) on UV treated (5.76
J/cm2) samples (AA0=100 mg/L) in 0.5% malic acid (pH 3.3). Each data point
represents an average of three measurements + standard deviation.
108
Figure 4-15: Post processing degradation of AA (AA0=200 mg/L) in UV treated
apple juice (1.2 J/cm2) and then stored at 4 °C. Each data point represents an
average of three measurements + standard deviation.
109
4.4 CONCLUSIONS
AA degraded when exposed to UV light. The AA degradation rate in buffer
deviated from previously reported zero order reaction and increased with increase in UV
dose. This could be attributed to side reactions that increase the AA degradation rate.
Tannic acid decreased the AA degradation rate, most likely by increasing the attenuation
coefficient of the buffer. AA degradation was pH dependant and increased with higher
pH. Fructose accelerated the degradation reaction possibly through formation of radicals
that may trigger AA degradation. AA continued to degrade during the post-processing
storage, the rate of which was directly proportional to the initial UV exposure and
temperature of storage. Further research on identifying the degradative pathways of AA
and the end products of degradation is necessary. Although loss of AA may remain
minimal during UV processing of clear juices such as apple juice, more turbid juices such
as orange juice would require significantly higher UV dose in order to achieve the 5-log
reduction of microbial load, thus increasing the possibility of a significant AA loss.
Therefore, it is recommended to carry out AA fortification of juices after UV
processing. Commercially processed juice products may be stored in warehouses and on
the grocery store shelf for several weeks before purchasing. Since AA degradation
continues after UV processing, juice treated with this technology may contain
considerably lower levels of AA upon consumption than expected.
110
4.5 REFERENCES
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aqueous model solutions and apple juice. A PhD dissertation submitted to graduate
school, Cornell University, Ithaca, NY. 2006, 34.
Behrens, W.; Medere, R. 1987. A highly sensitive high performance liquid
chromatography method for the estimation of ascorbic and dehydroascorbic in tissues,
biological fluids and foods. Analytical Biochemistry. 165, 102-107.
Binkley, E.; Binkley, R. 1998. Unprotected carbohydrates. In Carbohydrate
Photochemistry. Edited by Binkley, E.; and Binkley, R. ACS Publications, Washington
DC. 226-227.
Buettner, G.; Jurkiewicz, B. A. 1996. Chemistry and biochemistry of Ascorbic acid. In
Cadenas, E; Packer L. Eds. Handbook of Antioxidants: Antioxidants in health and
disease. Marcel Dekker publications, New York, NY.91-115.
Burdurlu, H.; Koca, N.; Karadeniz, F. 2006. Degradation of vitamin C in citrus juice
concentrate during storage. Journal of Food Engineering. 74(2), 211-216.
Cameron, E.; Pauling, L.; Leibowitz B. 1979. Ascorbic acid and cancer: a review.
Cancer Research. 39, 663-681.
Cantos, E.; Garcia-Viguera, C.; Pascual-Teresa, S.; Tomas-Barberan, S. 2000. Effect
of postharvest ultraviolet radiation on resveratrol and other phenolics of Cv. Napoleon
table grapes. Journal of Agricultural and Food Chemistry. 48, 4606-4612.
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Coohil, T.; Sagripanti, J. 2008. Overview of the inactivation by 254 nm ultraviolet
radiation of bacteria with particular relevance to biodefense. Photochemistry and
Photobiology. 84, 1084-1090.
Englard, S.; Seifter S. 1986. The biochemical functions of ascorbic acid. Annual
Reviews in Nutrition. 6, 365-406.
Fonseca, J.; Rushing, J. 2006. Effect of ultraviolet-C on quality and microbial
population of fresh-cut watermelon. Postharvest Biology and Technology. 40, 256-261.
Fuchs, J.; Kern H. 1998. Modulation of UV-induced skin inflammation by D-alpha-
tocopherol and L-ascorbic acid: A clinical study using solar simulated radiation. Free
Radical Biology and Medicine. 25(9), 1006-1012.
Gonzalez,-Aguilar, G.; Wang, C.; Buta, J.; Krizek, D. 2001. Use of UV-C irradiation
to prevent decay and maintain post-harvest quality of ripe „Tommy Atkins‟ mangoes.
International journal of Food Science and technology. 36(7), 767-773.
Gregory III, J. 2008. Vitamins. In Food Chemistry (edited by Damodaran, S.; Parkin,
K.; Fennema, O.) 4th
edition. CRC press, Boca Raton, FL 471-473.
Guerrero-Beltran, J.; Barbosa-Canovas, G. 2004. Advantages and limitations of
processing foods by UV light. Food Science and Technology International. 10(3), 137-
147.
Kagan, J. 1993. The fundamentals. In Organic Photochemistry, Principles and
Applications. Edited by Kagan J. Academic Press, San Diego, CA. 1-26.
Kabasakalis, V.; Siopidou, D.; Moshtou, E. 2000. Ascorbic acid content of commercial
fruit juices and its rate of loss upon storage. Food Chemistry. 70, 325-328.
112
Keyser, M.; Muller, I.; Cilliers, F.; Nel, W.; Gouws, P. 2008. Ultraviolet radiation as a
non-thermal treatment for inactivation of microorganisms in fruit juice. Innovative Food
Science and Emerging Technologies. 9, 348-354.
Kokkinidou, S.; Tikekar, R.; Floros, J.; LaBorde, L. 2007. Modeling ascorbic acid
induced degradation of patulin in model juice system. Research poster at IFT-AMFE,
Chicago, USA
US FDA. Kinetics of microbial inactivation for alternative food processing technologies:
ultraviolet light.
http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesse
s/ucm100158.htm. Accessed on 10/22/2009.
Koutchma, T. 2008. UV light for processing foods. Ozone: Science and Engineering. 30,
93-98.
Koutchma, T. 2009. Advances in ultraviolet technology for non-thermal processing of
liquid foods. Food and Bioprocess Technology. 2, 138-155.
Koutchma, T.: Forney, L.; Moraru, C. 2009. UV processing effects on quality of
foods. In Ultraviolet Light in Food Technology Principles and Applications, CRC press,
Boca Raton, Fl. 107-110.
Koutchma, T.; Keller, S.; Chirtel, S.; Parisi, B. 2004. Ultraviolet disinfection of juice
products in laminar and turbulent flow reactors. Innovative Food Science and Emerging
Technologies. 5, 179-189.
Lee, H.; Wrolstad, R. 1988. Apple juice composition: sugar, nonvolatile acid, and
phenolics profiles. Journal of Association of Official Analytical chemists. 71(4), 789-794.
113
Murakami, EG, Jackson L, Madsen K, Schickedanz B. 2006. Factors affecting the
ultraviolet inactivation of Escherichia coli K12 in apple juice and a model system. J.
Food Process Eng. 29, 53-71.
Legrini, O.; Oliveros, E.; Braun, A. 1993. Photochemical processes for water
treatment. Chemical Reviews. 93, 671-698.
Picinelli, A.; Suarez, B.; Mangas, J. 1997. Analysis of polyphenols in apple products. Z
Lebensm Unters Forsch A. 204, 48-51.
Quintero-Ramos, A.; Churey, J.; Hartman, P.; Bernard, J.; Worobo, R. 2004.
Modeling of Escherichia coli inactivation by UV irradiation at different pH values in
apple cider. Journal of Food Protection. 67(6), 1153-1156.
Sastry, S.; Datta, S.; Worobo, R. 2000. Ultraviolet light. Journal of Food Safety. 65(8),
90-92.
Scalbert, A., Johnson, I.; Saltmarsh, M. 2005. Polyphenols: Antioxidant and beyond.
American Journal of Clinical Nutrition. 81, 215S-217S.
Shwartz, S.; Von Elbe, J.; Giusti, M. 2008. Colorants. Edited by Damodaran, S.;
Parkin, K.; Fennema, O. Food Chemistry. 4th
Ed.. CRC Press, Boca Raton, FL. p. 628.
Shindo, Y.; Witt, E.; Packer, L. 1993. Antioxidant defense mechanisms in murine
epidermis and dermis and their response to ultraviolet light. Journal of Investigative
Dermatology. 100, 260-265.
Tikekar, R.; LaBorde, L.; Anantheswaran, R. 2010. Ultraviolet light processing of
fruit juices. Encyclopedia of Food Agricultural and Environmental Engineering.
Accepted May 20, 2009.
114
Tran, M.; Farid, M. 2004. Ultraviolet treatment of orange juice. Innovative Food
Science and Emerging Technologies. 5, 495-502.
Triantaphylides, C.; Schuchmann, H-P.; Sonntag C. 1981. Photolysis of D-fructose in
aqueous solution. Carbohydrates Research. 100, 131-141.
115
Chapter 5
Ultraviolet light induced degradation of ascorbic acid: Identification of
degradation products and a proposal for a reaction mechanism
ABSTRACT
Ultraviolet light (254 nm) induced degradation of ascorbic acid (AA) was
reported earlier in this thesis in chapter 4. In the present chapter, end products of UV
induced AA degradation are identified and a reaction mechanism is proposed. Electron
spin resonance (ESR) spectroscopy studies demonstrated that ascorbate radicals formed
in AA solutions in phosphate buffer at pH 7.0 and in malic acid buffer between pH 3.3
and 6.0, however, lesser amounts formed at lower pH levels with only trace amounts
detected at pH 3.3. Ascorbate radicals in UV treated AA solutions continued to form for
up to 200 minutes in the dark at higher rates than that for identically stored untreated AA
solution. High pressure liquid chromatography-mass spectroscopy (HPLC-MS) analysis
of UV treated samples demonstrated that as AA levels decreased, dehydroascorbic acid
(DHA) and 2, 3-diketogulonic acid (DKGA) levels increased. From this data, it can be
suggested that UV processing of AA leads to formation of ascorbate radical that leads to
the formation of DHA which further degrades into DKGA, a non-vitamin C compound.
116
5.1 INTRODUCTION
Ultraviolet light (UV) processing is an emerging non-thermal food processing
technology with many advantages such as low operational costs, being relatively non-
destructive on food flavors and micronutrients and continuous nature of operation
(Guerrero-Beltran and Barbosa-Canovas, 2004; Koutchma, 2008, Tikekar et al., 2009 ).
UV processing has found its niche in juice processing owing to relatively better
penetration of the light. It has been implemented to impart the FDA mandated 5-log
reduction of human pathogens such as Escherichia coli O157:H7 and Cryptosporidium
parvum in apple cider (Donahue et al., 2004; Quintero-Ramos et al., 2004; Koutchma et
al., 2004; Murakami et al., 2005). UV processing has been successfully tested on other
juice products such as orange juice, tropical punch and grape juice (Keyser et al., 2008).
It is necessary to understand the impact UV processing may have on food chemicals
before it can be commercialized. Ascorbic acid (AA) was thought to be a suitable
candidate for such a study because of -1) its sensitivity to UV light (Jurkiewicz and
Buettner, 1994) and 2) it is commonly used as an indicator of the severity of a processing
technique on vitamins. Therefore, we studied the UV induced degradation of AA. It was
found that UV light degraded AA, the rate of which was strongly dependant on the
presence of a variety of juice components. Polyphenols, which are ubiquitous in the
juices, reduced the rate of degradation while fructose showed a concentration dependant
increase in the rate of degradation (Chapter 4).
AA is known to act as an antioxidant against the photo-oxidation of various
compounds, in particular unsaturated lipids (Yi et al., 1991; Tebbe et al., 1997). One
electron aerobic oxidation of AA yields the ascorbate radical, a stable species with a
117
relatively long half-life (50 s) (Somani, 1996). The primary oxidation product of the AA
radical is dehydroascorbic acid (DHA), which in vivo, can be reduced back to AA.
Therefore, both AA and DHA are considered to have vitamin C activity. DHA, however
is a relatively unstable compound and is rapidly hydrolyzed into 2, 3-diketogulonic acid
(DKGA), a compound with no vitamin C activity (Gregory III, 2008). In vivo studies by
Jurkiewicz and Buettner (1994) demonstrated that exposure of hairless mouse skin to
polychromatic UV light promotes oxidation of AA to the ascorbate radical. However, the
fate of AA exposed to UV light in vitro has not been demonstrated. Based on the known
chemistry of AA, we hypothesize that a similar degradation mechanism is responsible for
observed decreases of UV treated AA reported in chapter 4. In this chapter we seek to
identify the degradation products of UV treated AA and to propose a degradation
mechanism for this photochemical reaction.
5.2 MATERIALS AND METHODS
Electron spin resonance (ESR) spectroscopy and the high performance liquid
chromatography- mass spectroscopy (HPLC-MS) were used to study the photo-induced
mechanism of AA degradation. ESR spectroscopy exploits the paramagnetic properties of
the unpaired electron in a radical to observe its presence. The molecule is subjected to a
constant frequency of microwave energy and the magnetic field is altered until a
resonance condition is achieved. Each radical has a characteristic hyperfine coupling
constant (aH) which is used as a reference and the spectral peaks generated can be
characterized to identify the type and amount of the radical present (Andersen and
Skibsted, 2002).
118
The method described by Buettner and Jurkiewicz (1993) was used to generate an
EPR ascorbate radical spectrum. Ascorbate radical was generated by mixing 250 mM
AAPH in ultrapure water, a known radical generator (Sigma-Aldrich, St Louis, MO) with
4 mM of AA (704 mg/L) in 1: 1 proportion. No radical spin trap was used during the
ESR experiment as ascorbate radical has a relatively large half-life of 50 s (Somani,
1996).
5.2.1 Reagents
Dehydroascorbic acid, ethylenediaminetetraacetic acid (EDTA), formic acid,
malic acid, sodium phosphate monobasic and dibasic were procured from Sigma Aldrich
(St Louis, MO). Ascorbic acid powder, and acetonitrile (HPLC grade), were procured
from Fisher Chemicals (Pittsburgh, PA). 2, 2‟-azobis-2-methyl-propanimidamide,
hydrochloride was obtained from Wako Chemicals (Richmond, VA)
5.2.2 UV treatment equipment
All experiments were carried out using a bench-top batch collimated beam UV
reactor (figure 5-1). The reactor consisted of three UV lamps (254 nm, 10 W, Atlantic
Ultraviolet Inc., Hauppauge, NY) mounted within a shielded horizontal cylindrical holder
fitted over a vertical tube (100 mm diameter X 100 mm length). Collimation was
achieved by painting the inside surface of the vertical tube with UV absorbing black
paint. Based on the length of the tube, the calculated maximum incident angle was no
greater than 20°. Incident intensity (mW/cm2) was measured by placing a radiometer
(Model: UVP-J225, UVP LLC, Upland, CA) at the bottom of the tube at a length equal to
119
Figure 5-1: Schematic representation of the batch UV system
Sample in a Petri plate with stirrer
UV lamp
Collimator
Collimated beams
120
the distance between the light source and the surface of the sample. Variation of incident
intensity over the entire sample surface area was less than 1%. This slight error was
neglected because the sample was continuously stirred with a mechanical stir bar (300
rpm). All the experiments were performed at the temperature of 21 (±1) °C.
For kinetic experiments in ESR or HPLC-MS, a known amount of AA was
dissolved in either phosphate buffer (pH 7.0) or malic acid buffer (pH 3.3 unless
otherwise stated). Thirty ml of this solution was placed in a Petri dish and exposed to the
UV light. Samples were taken periodically from the treatment and the control (No UV
exposure) and immediately analyzed by ESR. For HPLC-MS studies, samples were
immediately frozen and held at -15 °C for analysis the following day.
5.2.3 UV dose measurement
UV dose (J/cm2) was calculated by multiplying the incident intensity as measured
by the radiometer with the exposure time in seconds (equation 1).
D = I × t Eq. (5-1)
Where D - UV dose (J/cm2), I = incident intensity (W/cm
2), and t = duration of exposure
(s)
The incident intensity or irradiance range at the surface of the liquid was between
1.4-1.8 mW/cm2.
121
5.2.4 Electron spin resonance (ESR) spectroscopy
A Bruker-Biospin e-scan™ X-band ESR system was used (Bruker Biospin Inc.,
Billerica, MA). Samples were loaded into a 19-bore quartz cell and placed within the
ESR cavity. The settings for the detection of ascorbate radical were as follows: center
field, 3488.225 G; sweep width, 20 G; static field, 3468.236 G; frequency, 9.77 GHz;
attenuator, 2.0; power, 37.86 mW; modulator frequency, 86 kHz; modulation amplitude,
0.69 G; modulation phase, 1.08 degree; offset, 1%; time constant, 327.68 ms; conversion
time, 20.48 ms; number of scans, 16. .
The ascorbate radical were more readily formed at neutral pH. Therefore, the ESR
experiments were performed in 10 mM phosphate buffer made in ultrapure water with the
pH adjusted to 7.0. The experiments in chapter 4 on UV induced AA degradation were
performed in 0.5% malic acid solution at pH 3.3. The presence of ascorbate radical in
0.5% malic acid buffer at pH 3.3 was detected (figure 5-7) suggesting that these radicals
are generated even at acidic pH, but the concentration of the radical was too low to be
quantified effectively by the ESR system used. Therefore, phosphate buffer adjusted to
neutral pH was used. Traces of transition metal ions caused oxidation of AA that led the
control samples to show a large ascorbate radical peak. EDTA was (5mM) was added in
phosphate buffer to sequester the traces of transition metal ions thus reducing peak height
for the control.
5.2.5 HPLC-MS
The HPLC-MS studies were conducted in 0.5% malic acid buffer with pH 3.3.
For HPLC separation a Primesep-D column 4.6 × 150 mm was used (SIELC Inc.,
122
Prospect Heights, IL). The HPLC assembly consisted of a controller (Model: SCL-10A),
a pump (Model: LC-10AD), a UV- visible range detector set at 254 nm (Model: SPD-
10A) and an autosampler (Model: SIL-10AD) (Shimadzu Scientific Instruments,
Columbia MD). The mobile phase consisted of pure acetonitrile and 5% acetonitrile in
water with 0.1% formic acid (pH 1.7). Runs were made in a linear gradient mode with
final acetonitrile concentration reaching 20%. The flow rate was set at 1 mL for HPLC,
but for the MS, a 1:4 T-splitter was used and the flow rate for MS was 250 μl/min. The
MS system (Model: MicromassTM
Quattro Micro, Waters Inc. Milford MA) was run in
electronegative single ion monitoring mode to identify and quantify the products with
molecular weight 173 (DHA in electronegative mode), 175 (AA in electronegative mode)
and 191 (DKGA in electronegative mode). The MS parameters were: capillary voltage,
3.2 kV; Cone voltage, 25 V; source temperature, 100 °C; desolvation temperature, 250
°C; gas flow, 500 l/hr; cone gas flow, 50 l/hr.
5.3 RESULTS AND DISCUSSION
5.3.1 ESR analysis
Figure 5-2 shows a representative ESR spectrum for the AAPH + AA system in
phosphate buffer. A consistent ascorbate radical duplet was observed in the ESR
spectrum at a magnetic field strength range between 3484-3487 G with a hyperfine
coupling constant (aH) of 1.8 which is in agreement with the literature value of 1.88
(Pietri et al., 1994). There are several ways of quantifying the strength of the signal and
thus the corresponding radical concentration, mainly by integrating the area under the
curve or by measuring the peak height. In this case, we used the average peak height as a
123
Figure 5-2: Ascorbate radical standard generated in AAPH (250 mM) and ascorbic
acid 4mM in 10 mM pH 7.0 phosphate buffer at pH 7.0 ( 1:1 v/v) . aH = hyperfine
coupling constant, C1 = crest height 1, C2 = crest height 2, T1 = trough height 1.
Absolute peak height = (C1 + C2)/2 + T1
C1
T1
aH
Arb
itra
ry u
nit
s
Gauss
C2
124
measure of concentration of radical as no significant peak widening (average bandwidth
of 3.51 G (±0.19)) was detected. The average height was measured by averaging the peak
heights of the two crests C1 and C2 and adding the absolute value of T1 to the average
(figure 5-2). The baseline ascorbate radical spectrum in phosphate buffer solution (pH
7.0) without any UV treatment (control) is shown in figure 5.3(a), the spectrum obtained
after the same solution was UV treated (1.4 mW/cm2) for 1 hour is shown in figure
5.3(b). This demonstrates that UV exposure of AA solutions increases the amount of AA
radicals formed compared to untreated solutions. To measure the peak height attributable
only to UV light, the treatment peak height was divided by the corresponding control
peak height to obtain a relative peak height.
5.3.1.1 AA degradation kinetics
Figure 5-4 compares AA (AA0= 450 mg/L) degradation determined by HPLC and
AA radical formation determined by ESR in phosphate buffer (pH 7.0) (Incident UV
intensity = 1.4 mW/cm2). Relative peak height increased when AA was exposed to UV
light indicating that more ascorbate radicals were generated as a result of UV treatment.
As the exposure continued, the relative peak height stabilized (60 through 240 minute),
suggesting a steady state of AA degradation. As the exposure continued, the AA
concentration progressively decreased. Hence, towards the end of the treatment AA
concentration became the rate limiting step and the relative peak height began to
decrease, eventually reaching a value of 0 suggesting that most of the AA was exhausted
and the residual concentration was too low to provide a sufficiently strong signal. The
kinetics of ascorbate radical generation (as obtained by ESR) was then compared to
125
(a)
(b)
Figure 5-3: ESR spectrum for ascorbate radical generated in phosphate buffer (pH 7.0)
before (a) and after (b) UV exposure for 1 hour (Incident intensity = 1.4 mW/cm2).
-150000
-100000
-50000
0
50000
100000
150000
3475 3480 3485 3490 3495 3500
-150000
-100000
-50000
0
50000
100000
150000
3475 3480 3485 3490 3495 3500
Arb
itra
ry U
nit
s
Gauss
Gauss
Arb
itra
ry U
nit
s
126
Figure 5-4: Comparison of AA (AA0= 450 mg/L) degradation determined by HPLC
and AA radical formation determined by ESR in phosphate buffer (pH 7.0)
(Incident UV intensity = 1.4 mW/cm2). ESR data points represent the average of
three measurements ± standard deviation. HPLC data points represent the average
of two measurements ± standard deviation.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250 300 350 400
AA radical determined by EPR
AA determined by HPLC
Duration of UV exposure (minutes)
Rel
ati
ve
EP
R p
eak
hei
gh
t fo
r A
A r
ad
ical/
Rel
ati
ve
AU
C f
or
HP
LC
127
kinetics of AA degradation (as obtained by HPLC). HPLC data showed two separate
trends. The degradation of AA by UV followed zero order kinetics within the first 240
minutes; however after this time there was a break in the linear curve and the reaction
progressed at much faster rate while maintaining the same apparent rate order. This trend
fairly matched with the onset of decrease in the relative peak height of ESR result; the
relative peak height began to decrease after 240 minutes of exposure. When ESR showed
relative peak height of 0, indicating exhaustion of AA, the HPLC data showed that there
was still about 20% AA (approximately 80 mg/L) remaining in the solution. This could
be attributed to the lower sensitivity of the ESR equipment and the transient nature of the
ascorbate radical, where it could not be detected at such a low concentration.
5.3.1.2 Effect of fructose on AA degradation rate
Figure 5-5 compares AA (AA0= 450 mg/L) degradation determined by HPLC and
AA radical formation determined by ESR in phosphate buffer (pH 7.0) containing 10%
(w/v) fructose (Incident UV intensity = 1.4 mW/cm2). In chapter 4, it was shown that
fructose increased the rate of ascorbic acid degradation and that the effect was
concentration dependant. Therefore, the goal of this study is to observe this effect by ESR
and elucidate the possible mechanism by which fructose may impart such an effect. The
ESR results corresponded with the HPLC results showing that 10% fructose indeed
increased the rate of AA degradation as compared to no fructose as demonstrated in
figure 5-4. The relative peak height reached 0 in 240 minutes in presence of fructose
(figure 5-5) compared to 330 minutes when no fructose was added (figure 5-4).
128
Figure 5-5: Comparison of AA (AA0= 450 mg/L) degradation determined by HPLC
and AA radical formation determined by ESR in phosphate buffer (pH 7.0)
containing 10% (w/v) fructose (Incident UV intensity = 1.4 mW/cm2). ESR data
points represent the average of three measurements ± standard deviation. HPLC
data points represent the average of two measurements ± standard deviation.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250 300 350 400
AA radical determined by EPR
AA determined by HPLC
Duration of UV exposure (minutes)
Rel
ati
ve
EP
R p
eak
hei
gh
t fo
r A
A r
ad
ical/
Rel
ati
ve
AU
C f
or
HP
LC
129
It is known that fructose is unstable to UV light and readily undergoes photolysis
and that the open chain configuration is sensitive to UV light while the ring form is
comparatively inert (Binkley and Binkley, 1998). Fructose has a relatively less stable ring
structure compared to glucose. It is known that only 0.024% of glucose molecules in
aqueous solution exist in the ring form, while 0.8% of fructose molecules are in the ring
form. The carbonyl group in open chain form of fructose undergoes bond cleavage at the
α-carbon atom leading to formation of hydroxyalkyl and acyl radicals when exposed to
UV light (Triantaphylides et al., 1981). It can be hypothesized that radicals formed from
fructose may in turn react with AA to yield AA radicals which further degrade.
Therefore, we expected that the presence of fructose would increase the relative peak
height in the ESR spectrum owing to higher quantities of ascorbate radicals generated at
any given time compared to when no fructose is present. However, we did not find
significant differences (p>0.05) between the relative peak heights of ascorbate radical in
solutions with or without fructose after exposure with UV light. We hypothesize that,
although the higher quantities of ascorbate radicals may be generated by fructose, they
degrade more rapidly due to the presence of fructose derived radicals. Triantaphylides et
al. (1981) reported that photolysis of oxygenated solution of fructose can form oxidative
intermediates such as hydroperoxyl (HO2
.) and superoxide (O2
.-) radicals, and hydrogen
peroxide. These oxidants may in turn cause rapid oxidation of AA and ascorbate radical
to DHA. Thus, in presence of fructose, ascorbate radicals are generated and degraded in
potentially multiple ways. Therefore, similar amounts of ascorbate radicals are observed
when either fructose is absent or present in solution. Further research is needed to detect
130
the presence of the hydroxyalkyl, acyl, or other radicals generated by fructose and to
study the reactions that occur between these radicals and ascorbic acid. It would also be
useful to identify the degradation products of UV induced degradation of fructose and to
study their reactions with AA.
5.3.1.3 Post-UV processing storage degradation of AA
In chapter 4, it was observed that UV exposed AA continued to degrade during
dark storage and that the rate increased with increasing initial UV dose and storage
temperature. In figure 5-6, relative ascorbate radical peak height of AA (AA0=600 mg/L)
after UV (10.08 J/cm2) processing and storage for up to 200 minutes at 21 °C is shown. It
is apparent that the UV treatment initiated a chain reaction mechanism in the solution that
caused further degradation to occur in the dark. The data show that ascorbate radicals
were present at level above untreated controls for as long as 90 minute after which the
relative peak height was nearly 1.0. It is important to note that, in chapter 4, AA
degradation, measured by HPLC, continued for several hours after UV processing. In this
experiment ascorbate radical is detected for comparatively less time (up to 90 min).
Nevertheless, in light of the fact that the half life of ascorbate radical is only 50 s, such
prolonged presence of ascorbate radical hinted at an occurrence of chain-reaction like
mechanism by more stable oxidative intermediates that continue to form ascorbate
radicals from AA. Also, the presence of radical might have continued to be marginally
higher than the control, but such difference could not be picked up by the ESR, thus the
relative peak height reached to 1.0 in such low time. We hypothesize that continual
degradation of AA during storage is due to possible formation of hydrogen peroxide.
131
Figure 5-6: Presence of AA radical after UV treatment (10.08 J/cm2) (AA0=600
mg/L) in phosphate buffer (pH 7.0) held at 21 °C. Each data point represents an
average of three measurements ± standard deviation.
1
1.2
1.4
1.6
1.8
2
2.2
0 50 100 150 200
Storage time (minutes)
Rel
ati
ve
pea
k h
eig
ht
132
Radiolysis of oxygenated water can form hydroxyl radical (OH.) and perhydroxyl radical
(HO2) (Seib and Tolbert, 1982). It can be hypothesized that similar products are formed
during the UV exposure. Perhydroxyl radical in turn can react with AA to generate
hydrogen peroxide (Seib and Tolbert, 1982). Hydrogen peroxide is more stable oxidant
than radicals and may remain in the solution for much longer duration than radicals.
Hydrogen peroxide may continue to react with AA to generate more ascorbate radical
and DHA during storage. This potentially explains the continued formation of ascorbate
radical and degradation of AA during storage. It is apparent from this data that these
reactions may also occur in UV treated juice products.
5.3.1.4 Detection of ascorbate radical in malic acid buffer
ESR experiments were carried out in phosphate buffer at pH 7.0, although in
chapter 4, experiments were conducted in malic acid at pH 3.3. The reasons for choosing
phosphate buffer were two-fold: 1) ascorbate radical peak signals are stronger at near
neutral pH (Buettner and Jurkiewicz, 1996) and 2) to eliminate any possible side effects
that malic acid may have on AA degradation. From the studies conducted in this chapter
using phosphate buffer, we hypothesize that the same reaction occurs in malic acid at
lower pH values. To examine this hypothesis, the pH of malic acid solution was varied
between 3.3 and 6.0 and the relative peak sizes were compared after up to 1 hr of UV
exposure (1.4 mW/cm2) (AA0=450 mg/L) (figure 5-7). Table 5-1 shows that peak size
increased significantly as the pH was increased. At pH 3.3 the ascorbate radical peak was
too weak to be distinguished from signal noise until 60 minutes of exposure. At pH 4.2
and 6.0, AA radical peak heights were more detectable at all exposure times and
133
Figure 5-7: Effect of malic acid buffer pH on signal strength of ascorbate radical
peak in ESR after 1 hr of UV exposure at incident intensity of 1.4 mW/cm2. (a) pH
3.3 (b) pH 4.2 (c) pH 6.0
3475 Gauss 3500 Gauss
pH 3.3
pH 4.2
pH 6.0
120000
0
-120000
120000
0
-120000
120000
0
-120000
(a)
(b)
(c)
134
Table 5-1: EPR peak heights representing the amount of ascorbate radical present
in 0.5% malic acid as a function of pH and UV exposure time (Incident intensity=
1.4 mW/cm2).
pH 0 min 30 min 60 min
3.3 Not detected Not detected 36847
4.2 91553 100943 87900
6.0 195200 167641 219486
135
increased with time and pH. The pKa1 for AA is 4.17. Therefore, at pH above 4.17, the
AA would be predominantly in dissociated ascorbate form from which the ascorbate
radical is formed (Buettner and Jurkiewicz, 1996). These data strongly suggest that
ascorbate radical formation, though favored at higher pH values, also occurs at pH levels
found in juice products.
5.3.2 HPLC-MS analysis
HPLC-MS experiments were conducted to identify compounds formed in UV
treated AA solution. Figure 5-8 shows that DHA, AA, and DKGA were present when AA
(AA0=400 mg/L) was treated with UV light. Simultaneous decreases in AA and increases
in DHA as a function of UV exposure time in malic acids solution (pH 3.3) are shown in
figure 5-9. At the beginning of exposure (time=0 hrs), average AA concentration was 355
mg/L and average DHA concentration was 29 mg/L. As AA concentration decreased,
DHA concentration increased. DHA is an unstable compound and is rapidly hydrolyzed
into DKGA (Gregory III, 2008). Therefore generation of DKGA (m/z 191) was followed
by MS. Because a DKGA standard was not available, the increase in the DKGA
concentration as a function of UV exposure time was plotted in terms of increase in the
relative area under the curve (AUC) compared to control (figure 5-10). It is possible that
a compound other than DKGA with the same mass to charge ratio as DKGA was
generated by the UV treatment. However, considering the ample experimental evidence
from the ESR and MS data indicating formation of DHA from AA exposed to the UV
light, formation of DKGA from DHA was likely. Additionally, the linear increase in
136
Figure 5-8: Representative HPLC-MS chromatogram of products formed after UV
exposure of AA in 0.5% malic solution (pH 3.3) for 3 hours (Incident intensity = 1.4
mW/cm2). (a) DHA (b) AA (c) DKGA. (AA0=400 mg/L)
a
b
c
137
Figure 5-9: Degradation of AA and formation of DHA in malic acid buffer (pH 3.3)
after exposure to UV light (Incident intensity = 1.4 mW/cm2) determined by HPLC-
MS. Data is an average of two measurements ± standard deviation. (AA0=400 mg/L)
0
50
100
150
200
250
300
350
400
0 1 2 3 4 5
Ascorbic acid concentration
Dehydroascorbic acid concentration
Duration of UV exposure (hours)
Co
nce
ntr
ati
on
(m
g/L
)
138
Figure 5-10: Formation of DKGA in malic acid buffer (pH 3.3) after exposure to UV
light (Incident intensity = 1.4 mW/cm2) determined by HPLC-MS. Data is an
average of two measurements ± standard deviation. (AA0=400 mg/L)
0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5
Duration of UV exposure (hours)
Rel
ati
ve
pea
k h
eig
ht
(w.r
.t.
con
trol)
139
AUC as a function of UV exposure time indicates that the product is in fact induced by
UV suggesting that it could result from the degradation of DHA.
The results obtained by ESR and HPLC-MS strongly suggest that the mechanism
for UV induced AA degradation is similar to that known to occur without UV treatment.
AA, in the dissociated ascorbate form generates ascorbate radicals and this reaction is
accelerated by UV light. AA radicals are then oxidized to DHA and ultimately to DKGA.
The proposed reaction mechanisms for UV induced AA degradation are shown in figure
5-11.
5.4 CONCLUSIONS
The ESR and HPLC-MS studies presented in this chapter demonstrate that UV
induced degradation of AA progresses via formation of ascorbate radicals, eventually
leading to the formation of DHA. DHA is unstable and quickly degrades into DKGA.
ESR studies showed that fructose increased the AA degradation rate, although the
mechanism for this effect remains unclear. We hypothesize that fructose derived radicals
formed during UV exposure account for the more rapid rate of AA degradation when
fructose is present. Post UV treatment decrease in AA concentration and persistent
presence of AA radicals during dark storage suggest occurrence of a chain-reaction like
mechanism involving more stable oxidative intermediates such as hydrogen peroxide
where ascorbate radicals continue to form from AA. Further research is needed to refine
ESR techniques such that the occurrence of these radicals as well as other degradation
products of fructose may be demonstrated.
140
(light/dark)
(light/dark)
Where, ; Ascorbate radical. Light reactions represent reactions
during processing while dark reactions represent reactions during storage
Figure 5-11: Proposed mechanism for UV induced degradation of AA
Ascorbic Acid Ascorbate radical
Dehydroascorbic Acid 2, 3-Diketogulonic acid
UV
-e-
-H+
+H2O
Ascorbate ion
H
Secondary reactions (light and dark reactions)
UV
AA degradation reaction (light reaction)
H
141
5.5 REFERENCES
Andersen, M.; Skibsted, L. 2002. Detection of early events in lipid oxidation by
electron spin resonance spectroscopy. Europian Journal of Lipid Science and
Technology. 104, 65-68.
Bielski, B. 1982. Chemistry of ascorbic acid radicals. In, Ascorbic acid: Chemistry,
metabolism and uses. Edited by Seib, P.; Tolbert, B. ACS publications, Washington DC.
93.
Binkley, E.; Binkley, R. 1998. Unprotected carbohydrates. In Carbohydrate
Photochemistry. Edited Binkley, E. and Binkley, R. 1st edition. ACS publications,
Washington DC.
Buettner, G.; Jurkiewicz, B. A. 1996. Chemistry and biochemistry of Ascorbic acid. In
Cadenas, E; Packer L. Eds. Handbook of Antioxidants: Antioxidants in health and
disease. Marcel Dekker publications, New York, NY.91-115
Donahue, D.; Canitez, N.; Bushway, A. (2004). UV inactivation of E. coli O157:H7 in
apple cider: quality, sensory and shelf-life analysis. Journal of Food Processing and
Preservation. 28, 368-387.
Gregory III, J. 2008. Chapter 7: Vitamins in Food Chemistry.4th
edition. Edited by
Damodaran S., Parkin, K., Fennema, O. CRC press, Boca Raton, FL. 468-469.
Guerrero-Beltran, J.; Barbosa-Canovas, G. 2004. Review: Advantages and limitations
on processing foods by UV light. Food Science and Technology International. 10, 137-
147.
Jurkiewicz, B. and Buettner, G. 1994. Ultraviolet light-induced free radical formation
in skin: An electron paramagnetic resonance study. Photochemistry and Photobiology.
59(1), 1-4.
142
Keyser, M.; Muller, I.; Cilliers, F.; Nel, W.; Gouws, P. 2008. Ultraviolet radiation as a
non-thermal treatment for inactivation of microorganisms in fruit juice. Innovative Food
Science and Emerging Technologies. 9, 348-354.
Koutchma, T. 2008. UV light for processing foods. Ozone: Science and Engineering.
30(1), 90-98.
Koutchma, T.; Keller, S.; Chirtel, S.; Parisi, B. 2004. Ultraviolet disinfection of juice
products in laminar and turbulent flow reactors. Innovations in Food Science and
Engineering Technology. 5, 179-189.
Murakami, E.; Jackson, L.; Medsen, K. 2006. Factors affecting the ultraviolet
inactivation of Escherichia coli K12 in apple juice and a model system. Journal Food
Processing Engineering. 29. 53-71.
Pietri, S.; Seguin, J.; Arbigny, P.; Gulcasi, M. 1994. Ascorbyl free radical: A
noninvasive marker of oxidative stress in human open-heart surgery. Free Radical
Biology and Medicine. 16(4), 523-528.
Quintero-Ramos, A.; Churey, J.; Hartman, P.; Bernard, J.; Woboro, R. 2004.
Modeling of Escherichia coli inactivation by UV irradiation at different pH values in
apple cider. Journal of Food Protection. 67(6), 1153-1156.
Somani, S. 1996. Exercise, drugs and tissue specific antioxidant system. In
Pharmacology in Exercise and Sports. Ed Somani, S.1st edition. CRC press, Boca Raton,
FL. 74.
Tebbe, B.; Wu, S.; Geilen, C.; Eberle, G.; Kodelja, V.; Orfanos, C. 1997. L-ascorbic
acid inhibits UV-A induced lipid oxidation and secretion of IL-1α and Il-6 in cultured
human keratinocytes in vitro. The Journal of Investigative Dermatology. 108(3), 302-306.
143
Tikekar, R.; LaBorde, L.; Anantheswaran, R. 2010. UV processing of fruit Juices.
Encyclopedia of Food Agricultural and Biological Engineering. Accepted May 20, 2009.
Triantaphylides, C.; Schuchmann, H-P.; Sonntag, C. 1981. Photolysis of D-fructose
in aqueous solution. Carbohydrate Research, 100, 131-141.
Yi, O-S.; Han, D.; Shin, H-K. 1991 Synergistic antioxidative effects of tocopherols and
ascorbic acid in fish oil/lecithin/water system. Journal of American Oil Chemists‟
Society. 1991. 68(11), 881-883.
144
Chapter 6
Overall conclusions and suggestions for future work
6.1 OVERALL CONCLUSIONS
UV induced degradation of patulin followed a first order kinetics in model apple
juice. Degradation rate constants in terms of UV dose were independent of incident
intensity, initial concentration of patulin and buffer pH. Patulin degradation rate was
reduced by tannic acid, and suspended particles possibly due to competitive absorption
and scattering of UV light respectively. Ascorbic acid (AA) did not affect the patulin
degradation rate constant. Thus, UV processing can potentially reduce the patulin loads in
apple juice; however the high dose requirements may cause damage to the organoleptic
attributes of the product.
AA degraded when exposed to UV light. The AA degradation rate in buffer
deviated from previously reported zero order reaction and increased with increase in UV
dose. This could be attributed to side reactions that increase the AA degradation rate.
Tannic acid decreased the AA degradation rate, most likely by increasing the attenuation
coefficient of the buffer. AA degradation was pH dependant and increased with higher
pH. Fructose accelerated the degradation reaction possibly through formation of radicals
that may trigger AA degradation. AA continued to degrade during the post-processing
storage, the rate of which was directly proportional to the initial UV exposure and
temperature of storage. Although loss of AA may remain minimal during UV processing
145
of clear juices such as apple juice, more turbid juices such as orange juice would require
significantly higher UV dose in order to achieve the 5-log reduction of microbial load,
thus increasing the possibility of a significant AA loss. Therefore, it is recommended to
carry out AA fortification of juices after UV processing. Commercially processed juice
products may be stored in warehouses and on the grocery store shelf for several weeks
before purchasing. Since AA degradation continues after UV processing, juice treated
with this technology may contain considerably lower levels of AA than expected.
The ESR and HPLC-MS studies demonstrated that UV induced degradation of
AA progresses via formation of ascorbate radicals, eventually leading to the formation of
DHA. DHA is unstable and quickly degrades into DKGA. ESR studies showed that
fructose increased the AA degradation rate, although the mechanism for this effect
remains unclear. We hypothesize that fructose derived radicals formed during UV
exposure account for the more rapid rate of AA degradation when fructose is present.
Post UV treatment decrease in AA concentration and persistent presence of AA radicals
during dark storage suggest occurrence of a chain-reaction like mechanism involving
more stable oxidative intermediates such as hydrogen peroxide where ascorbate radicals
continue to form from AA.
6.2 SUGGESTIONS FOR FUTURE WORK
The dose required to achieve 1-log reduction in patulin concentration in apple
juice was approximately 1000 times higher than that needed for 5-log reduction of
microbial load. Effect of UV light at such high dose levels on sensory attributes of apple
146
juice such as flavor, taste remains unknown. Further research is desired to identify the
impact of high levels of UV doses on apple juice quality.
The mechanism by which fructose increases the degradation rate of both patulin
and ascorbic acid is not clear. Future investigation should be directed to observe the
occurrence of hydroxyalkyl and acyl radicals generated as a result of exposure of fructose
to UV. These radicals potentially cause the increase in the degradation reaction.
Techniques such as low temperature ESR with spin-trapping can be utilized for this
purpose.
The mechanism of continued storage degradation of ascorbic acid remains
unknown. Further research should be directed at identifying this pathway, so that some
preventive steps could be taken to avoid the loss during storage.
UV induced degradation products of patulin remain unknown. Further
investigation is necessary to determine the nature and toxicity of these products.
UV processing can be effectively implemented for variety of clear and turbid
juices. The process for each juice product needs to be validated to ensure the 5-log
reduction in human pathogens. Owing to differences in chemical composition and
turbidity, each juice requires different levels of UV doses and thus the effect of these
varying doses on the sensory attributes of juices needs to be investigated.
Ascorbic acid degradation due to UV light in variety of juice products should be
studied to understand the severity of UV processing on juice bioactives.
147
Similar to ascorbic acid, various other food bioactive compounds absorb UV light
and can decompose when exposed for prolonged duration. The effect of UV light on
other bioactive compounds such as carotenoids, polyphenols and flavonoids needs to be
studied in order to fathom the nutritional losses imparted by UV light.
148
Appendix A: Patulin degradation in model apple cider system
Materials and Methods
0.5% malic acid buffer was incorporated with ascorbic acid, tannic acid (a
surrogate for polyphenols) and suspended particles and the pH was adjusted to desired
values. The range of parameters (three levels for each) selected for model apple cider
were pH 3-5, ascorbic acid (AA) concentration 0-440 mg/L, tannic acid (TA)
concentration 0-2 g/L, and turbidity (NT) 0-800 NTU for UV treatments up to 50 min
with % patulin loss selected as a response variable. These parameters were chosen to
mimic ascorbic acid fortified apple cider. AA is naturally present at very low levels in
apple cider and shelf stable apple juice and are usually fortified with AA. The levels
chosen for ascorbic acid were equivalent to 0-2 RDA.
Result and discussion
The data for the experimental design for model apple cider is shown in table 1.
Ascorbic acid, polyphenols, time, and turbidity were found to be significant factors
(p<0.05). However, pH did not significantly affect the rate of patulin degradation
(p>0.05) (table 2). The predictive model had a very weak fit with R2~36%. Ascorbic acid,
polyphenols and suspended particles reduced patulin degradation either due to
preferential absorption of UV light (ascorbic acid and polyphenols) or scattering of UV
light (suspended particles), both of which would lead to fewer quanta of light available
for the degradation reaction. Patulin degradation rates at levels used in the model apple
cider experiments were very low suggesting that patulin degradation in apple cider using
UV light is not viable.
149
Table 1: Experimental design and corresponding % patulin degradation for model
apple cider system
pH Time
(min.)
Ascorbic
acid (g/L)
Tannic
acid (g/L)
Suspended
particles (NTU)
% reduction
3 10 0.22 1 400 7.62
3 50 0.22 1 400 18.67
3 30 0.22 0 400 14.73
3 30 0 1 400 21.50
3 30 0.44 1 400 13.00
3 30 0.22 2 400 6.69
3 30 0.22 1 0 26.68
3 30 0.22 1 800 0.00
4.5 10 0.22 1 400 2.03
4.5 30 0.22 1 0 38.30
4.5 50 0.22 1 400 29.07
4.5 30 0.22 2 400 6.53
4.5 30 0.22 0 400 21.19
4.5 30 0 1 400 14.11
4.5 30 0.44 1 400 5.06
4.5 30 0.22 1 800 7.43
3.75 30 0.22 1 400 16.73
3.75 30 0.22 1 400 13.73
3.75 30 0.22 1 400 12.09
3.75 30 0.22 1 400 9.02
3.75 30 0.22 1 400 11.55
3.75 30 0.22 1 400 11.75
3.75 50 0.22 1 0 26.25
3.75 10 0.22 0 0 26.06
3.75 30 0.44 1 0 0.00
3.75 30 0 1 0 13.10
3.75 30 0.22 2 0 2.44
3.75 10 0.22 1 0 5.91
3.75 50 0.22 0 400 19.24
3.75 50 0.22 2 400 10.59
3.75 50 0 1 400 8.36
3.75 50 0.44 1 400 0.29
3.75 30 0 0 400 62.35
3.75 10 0.22 0 400 7.05
3.75 30 0 2 400 13.98
150
3.75 30 0.44 0 400 15.73
3.75 30 0.44 2 400 9.42
3.75 10 0 1 400 4.09
3.75 10 0.22 2 400 6.48
3.75 10 0.44 1 400 0.38
3.75 50 0.22 1 800 20.06
3.75 30 0.22 2 800 3.07
3.75 30 0.44 1 800 3.83
3.75 30 0.22 0 800 19.68
3.75 30 0 1 800 7.62
3.75 10 0.22 1 800 0.00
151
Table 2: Regression analysis for statistical significance of predictor variables in
model apple cider system experimental design
Predictor Coef SE Coef T P
Constant 19.04 12.75 1.49 0.143
pH 1.26 3.1 0.41 0.687
Time 0.2937 0.1133 2.59 0.013
Ascorbic acid -28.42 10.57 -2.69 0.010
Tannic acid -8.292 2.329 -3.56 0.001
Suspended particles -0.012686 0.005824 -2.18 0.035
152
Appendix B: Validation of model apple juice system
The model was verified by selecting conditions from the tested ranges of
parameters and the observed rate constants were compared with that predicted from the
model (table 3-4). The experimental values showed significant variation from the
predicted values (p<0.05). Thus although the data fitted the model well, the predictive
power of the model is low. This is possibly due to the broad range of tannic acid
concentration chosen for the experiments. The mechanism of reaction may change
depending on the concentration of tannic acid, which may interfere with predictive power
of the model. Another consequence of such broad range for tannic acid was that at 1 g/L
concentration, essentially no patulin degradation was observed and the rate constants
were 0.00 cm2/J. This may have caused discrepancy in generating the statistical model.
Nevertheless, the RSM design helped to understand the interactions between different
juice components.
Table 1: Validation of the statistical model for patulin (C0=1000 ppb) degradation in
model apple juice
Conditions
Experimental
(SD) From the model
0 mg/L AA, 0 g/L TA, 0 NTU 0.22 (0.01) 0.17
45 mg/L AA, 0.1 g/L TA, 40
NTU
0.056(0.011) 0.11
0 mg/L AA, 0.5 g/L TA, 0 NTU 0.063 (0.005) 0.031
153
Appendix C: Patulin degradation in apple juice
1 Materials and methods
Apple cider, unprocessed, unfortified with ascorbic acid was procured from a
local apple cider producer. The sample was centrifuged at 15000 g force for 30min in
order to reduce the turbidity to < 3 NTU as measured by turbidimeter. This was
considered as apple juice sample and was held under frozen condition at -15 °C until
further use. Patulin was quantified using method described in section 3.2.5, 3.2.6, the
patulin degradation rate constants were calculated using equation 3-3.
2 Results and Discussion
Degradation kinetics of patulin in apple juice is plotted in Figure 1. The
degradation rate constant obtained for the apple juice was 0.15 cm2/J. For the malic acid
buffer system, that has similar composition (0.5 g/L tannic acid buffer, 0 NTU, 0 mg/L
ascorbic acid, and pH 3.3), the rate constant was 0.037 cm2/J. Therefore, patulin degraded
in apple juice at a much greater rate than expected. It was hypothesized that there were
certain juice components in apple juice that were not included in the model which
expedite patulin degradation. Therefore, the investigation was directed to identify these
components.
Work by Fang and Geveke (2007) showed that exposure of apple juice to UV
light for extended duration (6-8 J/cm2) can lead to formations of furan at levels of 40-60
ppb in apple juice. It was hypothesized that furan may have some role to play in patulin
degradation owing to its high reactivity. Therefore, we incorporated up to 4000 ppb of
furan in malic acid to study its impact on the rate of patulin degradation. It was found that
154
Figure 1: Degradation kinetics of patulin in apple juice (starting patulin
concentration 1000 ppb). Data is an average of triplicates with standard deviation.
y = 92.15e-0.15x
R² = 0.991
0
20
40
60
80
100
0 2 4 6 8 10 12 14
Dose (J/cm2)
% p
atu
lin
155
even at such high concentrations, presence of furan did not significantly alter the rate of
patulin degradation in malic acid buffer (p>0.05) suggesting that furan had no role to play
in this observation.
Apple juice contains approximately 10% sugars with fructose (5.5%), glucose
(2.6%) and sucrose (1.26%) being the constituents (USDA food composition database).
Sugars do not absorb significantly in the UV region (figure 2) and therefore were not
included in the earlier experimental designs. Fructose, sucrose and glucose were
incorporated in malic acid buffer to find their impact on the rate of patulin degradation. It
was found that sucrose at 10% (w/v) level did not change the rate constant for patulin
degradation in malic acid (p>0.05) while glucose increased the rate only marginally
(p<0.05) (table 1). Fructose, on the other hand showed a high concentration dependant
increase in the degradation rate of patulin (figure 3). Figure 4 shows the rate constants of
patulin degradation as a function of varying concentration of fructose in malic acid
buffer. This partially explains the higher rates of degradation in apple juice as compared
to model system. In the case of apple juice the net degradation rate is due to a
combination of shielding effects from ascorbic acid, polyphenols and other UV absorbing
components and expediting effects from fructose.
It was interesting to observe fructose undergoing chemical reactions when
exposed to UV light, especially in the light of the fact that the absorbance in the UV
region is very low. Literature suggests that sugars, especially fructose are not inert to UV
light. It is shown that fructose specifically shows high sensitivity to UV light and can
undergo photolysis. The open chain configuration of sugars is prone to UV light while
the ring form is inert. Fructose has relatively less stable ring structure than glucose and
156
Figure 2: Absorbance spectra of 10% solution (in water) of glucose, fructose and
sucrose
0
0.2
0.4
0.6
0.8
1
210 230 250 270 290
fructose
glucose
sucrose
Wavelength (nm)
Ab
sorb
an
ce
157
Table 1: Effect of glucose, sucrose and fructose on the degradation rate constant of
patulin in malic acid buffer (pH 3.3) (different letters represent statistically
significant difference)
Treatment Average rate constant (cm2/J) for patulin
degradation in malic acid buffer (SD)
No sugar 0.22 (0.007)a
10% glucose 0.25 (0.009)b
10% sucrose 0.23(0.01)a
10% fructose 0.76(0.01)c
158
Figure 3: Effect of varying concentration of fructose on patulin degradation rate in
malic acid buffer (pH 3.3)
0
20
40
60
80
100
0 2 4 6 8
10% fructose
5% fructose
2% fructose
0.5% fructose
0% fructose
% p
atu
lin
Dose (J/cm2)
159
Figure 4: Patulin degradation rate constant as a function of fructose concentration
(w/v) in malic acid buffer (pH 3.3)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 2 4 6 8 10
% fructose (w/v)
Ra
te c
on
sta
nt
( cm
2/J
)
160
sucrose and about 0.8% is present in open chain form as compared to 0.024% for glucose.
The carbonyl group in open chain form can undergo bond cleavage at the α-carbon atom
(Norrish type-I reaction) leading to formation of hydroxyalkyl and acyl radicals. These
radicals further react with atmospheric oxygen to form hydroxyalkylacylperoxyl radicals
that can depending on pH form superoxide (O2∙-
) or hydroxide radicals (OH∙)
(Triantaphylides et al., 1981; Binkley and Binkley, 1998). We hypothesize that these
radicals possibly then react with patulin and eventually degrade it. As only 0.8% of
fructose absorbs UV light and undergoes these reactions, it is possible that the rate of
generation of radicals remains the rate limiting step. This justifies the concentration
dependence of patulin degradation on fructose indicating that although it may seem that
fructose is present in excess (even 0.5% fructose or 5000 mg/L is comparatively a large
quantity of fructose for 1000 ppb or 1 mg/L patulin), it is in fact not. Thus, it is likely that
it is 0.8-1% of total fructose that actually undergoes the reactions that lead to patulin
degradation. Fan and Geveke (2007) further pointed out that 5% fructose in 0.25% malic
acid buffer formed approximately 3000 ppb furan when exposed to 6-8 J/cm2 UV dose.
This further supports our postulation that the intermediate compound of fructose
degradation and not the final product which is furan is responsible for increased rate of
patulin degradation. These intermediate compounds could be radicals.
Nevertheless, it was found that 90% reduction in patulin in apple juice would
require much higher dose levels than that needed for a 5-log reduction of
microorganisms. Thus, the focus of processing conditions needs to be altered towards
patulin reduction. Because such high dose levels may significantly alter the nutritional
and sensory attributes of juice, this area needs to be studied further.
161
Appendix D: Effect of furan on degradation rate of patulin
Fan and Geveke (2007) showed that fructose degraded to furan when exposed to
UV light. We have earlier reported that fructose in presence of UV increased patulin
degradation rate. We hypothesized that the degradation product of fructose (furan) may
have caused this effect. Therefore two levels of furan (1000 and 4000 ppb) were added
into 0.5% malic acid buffer and patulin degradation rate was quantified. Results showed
that the degradation rate constants (cm2/J) did not change at two levels of furan and the
values were not significantly different from that obtained with no furan added (p>0.05).
This demonstrates that furan have no effect on patulin degradation rate.
Figure 1: Effect of varying furan concentration (1000 and 4000 ppb) on patulin
degradation rate in malic acid buffer (pH 3.3). Data is an average of duplicate
measurements. Results show that furan has no effect on patulin degradation rate.
1000 ppb
y = 98.87e-0.22x
R² = 0.998
4000 ppb
y = 98.66e-0.21x
R² = 0.999
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7
1000 ppb furan
4000 ppb furan
UV dose (J/cm2)
% p
atu
lin
162
Appendix E: Ascorbic acid degradation rate in malic acid buffer during
UV processing using Cidersure 1500
Initial concentration of ascorbic acid
(mg/L)
Rate constant ( pass-1
) (standard
deviation)
100 5.33 (0.68)a
200 6.08 (0.38)a
350 6.42 (0.69)a
Table 1: Effect of initial concentration of ascorbic acid on the degradation rate
constant in malic acid buffer (pH 3.3). The flow rate was 40 US Gallon/hour (152
litre/hour). The data is an average of triplicate measurements. Same letter
represents no significant difference (p>0.05).
163
Appendix F: Degradation of patulin and ascorbic acid in apple cider
and apple juice during the UV processing using Cidersure® continuous
reactor
ABSTRACT
Our objective was to study patulin and AA degradation kinetics in apple juice and
cider during UV processing using commercial scale Cidersure® equipment. 1000 μg/L
patulin or 200 mg/L ascorbic acid was incorporated in either apple juice or apple cider
and passed through the Cidersure® multiple times and the kinetics of degradation was
studied. The flow rate was set at 152 liter/hour (40 US gallon/hour). Both patulin and
ascorbic acid degraded faster in apple juice than in apple cider. In 20 passes, patulin
concentration was reduced by 87% in apple juice as compared to 30% in apple cider.
92% of ascorbic acid was lost in 6 passes in apple juice while the loss in apple cider was
limited to 30%. Based on L, a, b measurements, the juice color was lighter with
increasing UV dose.
1 INTRODUCTION
Cidersure 1500 is currently the only equipment commercially available that can
impart the FDA mandated 5-log reduction in human pathogens such as E.coli O157: H7
in apple cider. Therefore, we decided to study the degradation kinetics of patulin in this
120000
0
-120000
120000
0
-120000
120000
0
-120000
120000
0
-120000
120000
0
-120000
164
commercial scale UV processor. Similarly we tested the sensitivity of ascorbic acid to
UV light in this commercial setup.
2 MATERIALS AND METHODS
Apple juice concentrate was procured from Multiflow Inc. (Huntingdon valley,
PA). The concentrate was diluted to 12° brix, pH 3.5 using distilled water. The
concentrate was not fortified with ascorbic acid (AA). Apple cider (not fortified with AA)
was procured from local supermarket.
Apple juice or apple cider (3 liter batch) was incorporated with 1000 ppb patulin.
The samples were placed in a stainless steel vessel and passed through the Cidersure
multiple times. The sample was re-circulated to minimize the sample requirement. The
flow rate was set at 152 liter/hour. The samples were withdrawn at the interval of 5
passes (300 s). Patulin was extracted from the juice or cider sample as described in
section 3.2.5 and quantified using HPLC as described in section 3.2.6. The experiments
were performed in duplicate.
Apple juice or cider (5 liter batch) was incorporated with 200 mg/L AA and
processed in a similar way as patulin except samples were withdrawn after every pass
(118 s). The juice samples were directly injected into the HPLC (the method is described
in section 4.2.4) in order to quantify the AA content. Apple cider samples were filtered
through 0.8 μm filter to remove the suspended particles and then injected into the HPLC.
The experiments were performed in duplicate.
165
The L, a, b values were measured using a colorimeter (BYK-Gardner LCS II,
Columbia MD) for apple juice samples after the interval of 5 passes for patulin
experiments and every single pass for AA experiments. The net color change was
calculated by using formula given in equation 6-1 (MacDougall, 2002).
ΔE Eq. (6-1)
Where, ΔE= color change, ΔL, Δa, Δb= changes in the L, a, b values with respect to
control
Color analysis was performed only on juice samples and not the cider samples
because the instrument was capable of measuring the L, a, b values for optically clear
liquids only.
3 RESULTS AND DISCUSSION
3.1 Patulin degradation in apple juice and apple cider
Degradation kinetics of patulin (C0=1000 ppb) in apple juice and cider are shown
in figure 1. Patulin degraded faster in apple juice than in cider. 87% patulin degraded in
20 passes as compared to 30% in apple cider.
166
Figure 1: Patulin degradation (C0= 1000 ppb) kinetics in apple juice and cider. Each
data point for apple juice represents an average of two measurements ± standard
deviation.
0
20
40
60
80
100
120
0 5 10 15 20 25
Apple juice
Apple cider
167
Changes in the L, a, b values of apple juice as a function of number of passes are
shown in table 1. The Juice appeared lighter in color after UV processing, which was
confirmed by increase in L value.
3.2 Ascorbic acid degradation in apple juice and apple cider
AA (AA0=200 mg/L) degradation kinetics in apple juice and cider are plotted in
figure 2. AA degraded faster in apple juice than in apple cider. 92% AA was lost in 6
passes as compared to 30% in apple cider. In a single pass, 22% AA was lost suggesting
that considerable AA loss can occur when exposed to UV light. It was demonstrated
earlier (chapter 4) that UV exposed AA continued to degrade during storage which may
further aggravate the AA losses. Therefore, it is recommended to fortify the juices with
AA post UV processing.
Changes in the L, a, b values of apple juice as a function of number of passes are
shown in table 2. As expected, the Juice appeared lighter in color after UV processing,
which was confirmed by the increase in L value.
4 CONCLUSIONS
Patulin degradation rate was faster in apple juice than in apple cider. Patulin loss
in apple juice was 87% after 20 passes as compared to 30% in apple cider. Nevertheless,
patulin degradation rate in apple juice was slow and patulin inactivation using UV light
can incur significant color losses. AA degraded faster in apple juice than apple cider. AA
168
Figure 2: AA degradation kinetics (AA0=200 mg/L) in apple juice and cider. Each
data point represents an average of two measurements ± standard deviation.
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7
Apple cider
Apple juice
Number of passes
Number of passes
% A
A r
ema
inin
g
% p
atu
lin
rem
ain
ing
169
Table 1: Changes in L, a, b values in apple juice as a function of number of passes
during patulin inactivation. Values are average of two measurements.
Number of passes L a b ΔE
0 94.55 -4.85 24.3 0
5 97.25 -5.1 17.8 7.04
10 98.1 -5 15.1 9.86
15 98.4 -4.8 14 10.99
20 98.55 -4.7 13.2 11.8
170
Table 2: Changes in L, a, b values in apple juice as a function of number of passes
during AA degradation. Values are average of duplicates.
Number of passes L A b ΔE
0 86.9 -1.70 38.30 0.00
1 88.3 -2.70 35.70 3.32
2 90.2 -3.63 31.63 7.85
3 91.3 -4.13 29.03 10.73
4 92.2 -4.53 27.03 12.95
5 92.9 -4.73 25.70 14.43
6 93.5 -4.87 24.83 15.37
171
loss in apple juice was 92% after 6 passes as compared to 30% in apple cider. The AA %
loss/pass was as much as 22% and therefore it is recommended that fortification of AA in
juice products should be carried out after processing. Considerable color loss was
observed in apple juice during extended exposure to UV light suggesting that high doses
of UV can cause adverse effects in product quality.
172
6.5 REFERENCES
MacDougall, D. 2002. Color measurement of food: principles and practice. In Colour In
Food: Improving Quality (Edited by MacDougall, D.). CRC Press, Boca Raton, FL.
Education
Doctor of Philosophy (2010)
Department of Food Science, Pennsylvania State University, University Park, PA USA
Master of Science (2006)
Department of Food Science, Rutgers, the State University of New Jersey, NJ USA
Bachelor of Technology (2004)
Department of Food Technology and Engineering, MUICT, Mumbai, India
Professional Experience
Campbell Soup Company, (May 2005-Aug 2005) Corporate Process Safety Intern
Cadbury India Ltd., (May 2003-July 2003) R&D Intern
Parle Products Ltd., (May 2002-July 2002) Summer Intern
Publications and presentations
Peer reviewed articles:
Tikekar, R.; LaBorde, L. Anantheswaran, R.; (2010). UV processing of fruit juice.
Encyclopedia of Food Agricultural and Biological Engineering. (accepted)
Tikekar, R.; Anantheswaran, R.; LaBorde, L. (2009). Effect of UV light on patulin in model
apple juice system and in apple juice. (in preparation)
Tikekar, R.; Anantheswaran, R.; LaBorde, F. (2009). Ultraviolet light induced degradation
of ascorbic acid in a model juice system. (in preparation)
Tikekar, R.; Elias, R.; Kokkinidou, S.; Anantheswaran, R.; LaBorde, L. (2009). Ultraviolet
light induced degradation of Ascorbic acid: Mechanism of degradation and identification of
degradation products. (in preparation)
Presentations: Tikekar R., Anantheswaran R., LaBorde F. (2009). Ultraviolet light induced degradation of
ascorbic acid in a model juice system. Research poster at IFT AMFE, Anaheim, USA
Tikekar R., Anantheswaran R., LaBorde F. (2008). Modeling inactivation of patulin by UV
irradiation in model apple juice system. Research poster at FIESTA 2008, Brisbane,
Australia.
Tikekar R., Anantheswaran R., LaBorde F. (2007). Modeling inactivation of patulin by UV
irradiation in model apple juice system. Research poster at IFT-AMFE, New Orleans, USA.
Kokkinidou S., Tikekar R., Floros J., LaBorde L. (2006). Modeling ascorbic acid induced
degradation of patulin in model juice system. Research poster at IFT-AMFE, Chicago, USA.
Selected awards
Departmental teaching assistance excellence award, Penn State University ( 2009)
Frank Dudek graduate scholarship, Penn State University (2008-2009)
Albert Kleinman scholarship, Rutgers University (2006-2007)
CURRICULUM VITAÉ