ULTRAVIOLET LIGHT INDUCED DEGRADATION OF PATULIN AND ASCORBIC ACID IN APPLE ...

189 Pages·2010·1.69 MB·English

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.
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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
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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
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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 (C =1000 ppb) (top) No UV
0
(bottom) after UV dose of 5.04 J/cm2……………………….……………….…………..57
Figure 3-3: Effect of incident intensity on the degradation of patulin (C =1000 ppb) in
0
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 (C =1000 ppb) in
0
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
(C =1000 ppb). Each data point represents average of three measurements ± standard
0
deviation.…………………………………………………………….…………………...65