DEVELOPMENT OF AUTHENTICITY METHODS FOR APPLE AND PEAR JUICES A Thesis ...

193 Pages·2017·2.34 MB·English

by  Jamie Willems

DEVELOPMENT OF AUTHENTICITY
METHODS FOR APPLE AND PEAR JUICES

A Thesis Submitted to the College of
Graduate and Postdoctoral Studies
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy in the
Department of Food and Bioproduct Sciences
University of Saskatchewan
Saskatoon, Saskatchewan, Canada

By
Jamie Willems
2017

© Copyright Jamie Willems, August 2017. All rights reserved
PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from
the University of Saskatchewan, I agree that the Libraries of this University may make it freely available
for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part,
for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in
their absence, by the Head of the Department or the Dean of the College in which my thesis work was done.
It is understood that any copying, publication, or use of this thesis or parts thereof for financial gain shall
not be allowed without my written permission. It is also understood that due recognition shall be given to
me and to the University of Saskatchewan in any scholarly use which may be made of any material in my
thesis.
Requests for permission to copy or to make other use of material in this thesis in whole or part
should be addressed to:

Head Department of Food and Bioproduct Sciences
University of Saskatchewan
Saskatoon, Saskatchewan, S7N 5A8
Canada

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ABSTRACT

Due to its widespread global consumption and economic value, fruit juice is a common
target for adulteration through debasing by unscrupulous producers. Two common methods of fruit
juice adulteration are debasing with commercial sweeteners and juice-to-juice adulteration. The
overarching goal of this research was to develop methods to detect the undeclared addition of less
expensive commercial sweeteners to pear juice and the undeclared addition of apple to pear and
pear to apple juice.
Methods to detect the undeclared addition of high fructose corn syrup (HFCS), hydrolyzed
inulin syrup (HIS) and total invert sugar (TIS) to commercial pear juice were developed through
oligosaccharide profiling employing high performance anion exchange chromatography with
pulsed amperometric detection (HPAE-PAD) and capillary gas chromatography with flame
ionization detection (CGC-FID). Based on the application of these methods to intentional pear
juice debasing, these three commercial sweeteners could be detected at levels of 0.5-3.0% (v/v).
Coupled with the developed authenticity analysis for pear juice, the developed profiling
methods were used to examine the carbohydrate/oligosaccharide profile of pear juice as a function
of commercial processing. Chromatographic results showed that the majority of
carbohydrate/oligosaccharide formation occurred during the mashing stage of juice production
where enzymes (i.e. pectinases, hemicellulases and amylases) are employed. The remaining
processing stages were found to have a minimal impact on the carbohydrate/oligosaccharide
profile of commercial pear juice.
Methods for the detection of juice-to-juice debasing between apple and pear juices were
developed using phenolic profiling. High performance liquid chromatography with photodiode
array detection (HPLC-PDA) was used to determine the phenolic profiles of commercial apple and
pear juice concentrates from major world production regions. The phenolic profiles were used to
identify fingerprint compounds for use in juice-to-juice adulteration detection. One phenolic
marker was identified in apple juice and two in pear juice (excluding arbutin). These compounds
were analyzed by UV-vis and NMR spectroscopic methods, and high resolution MS and LC-
MS/MS spectrometry. Results from these analyses identified the fingerprint compounds as 4-O-p-
coumarylquinic acid in commercial apple juice, and isorhamnetin-3-O-rutinoside and abscisic acid
in commercial pear juice.
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The total phenolic content and antioxidant activities of the 27 apple and 32 pear juices used
throughout this research were determined by the Folin-Ciocalteu (total phenolic content), HPLC-
PDA (total phenolic chromatographic index), Trolox equivalent antioxidant activity (TEAC) and
DPPH methods. The total phenolic content of apple and pear juices were found to be 294.7 ± 128.2
and 246.4 ± 45.1 ppm GAE and the total phenolic chromatographic indices were 128.8 ± 44.9 and
211.7 ± 57.2 ppm, respectively. The TEAC of apple and pear juices were found to be 130.8 ± 60.8
and 150.8 ± 63.9 mM Trolox/100 mL, while the DPPH radical scavenging abilities were 21.5 ±
12.1 and 13.6 ± 5.5 mL of DPPH/mL of juice, respectively.

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ACKNOWLEDGMENTS

I would like to sincerely thank my supervisor, Dr. Nicholas Low for his invaluable
guidance, time and support throughout my studies. Additionally, I would like to thank the members
of my Advisory Committee, Drs. Takuji Tanaka, Chris Eskiw and Anas El-Aneed and my graduate
chairs Drs. Bob Tyler and Michael Nickerson for their time and advice. I would also like to thank
the staff of the Department of Food and Bioproduct Sciences for all of their assistance and support.
I am grateful to all my friends and co-workers in the Department, especially those in Dr. Low’s
lab for all their support and assistance.
Finally, I would like to thank all my family and friends for their support throughout my
degree. Especially, to my parents, Brad and Janice Willems, for their guidance and continuous
support.
Finical support (scholarship) was provided from the Natural Sciences and Engineering
Research Council of Canada (NSERC).

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TABLE OF CONTENTS
PERMISSION TO USE ................................................................................................................. i
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGMENTS ........................................................................................................... iv
TABLE OF CONTENTS ............................................................................................................. v
LIST OF TABLES ...................................................................................................................... xii
LIST OF FIGURES ................................................................................................................... xiv
LIST OF SYMBOLS AND ABBREVIATIONS ................................................................... xviii

1. INTRODUCTION..................................................................................................................... 1
1.1 Summary ............................................................................................................................... 1
1.2 Hypothesis............................................................................................................................. 2
1.3 Objectives ............................................................................................................................. 2

2. LITERATURE REVIEW ........................................................................................................ 4
2.1 Food Authenticity ................................................................................................................. 4
2.2 Methods for Detecting Food Adulteration ............................................................................ 5
2.2.1 Stable Isotope Ratio Analysis ........................................................................................ 5
2.2.2 Infrared Spectroscopy .................................................................................................... 8
2.2.3 Genomic Based Techniques ........................................................................................... 9
2.2.4 Chromatographic Profiling/Fingerprinting .................................................................. 11
2.3 Oligosaccharide Formation in Pear Juice ........................................................................... 15
2.3.1 Introduction .................................................................................................................. 15
2.3.2 Pear Juice Production ................................................................................................... 15
2.3.3 Enzymes Employed in Pear Juice Production ............................................................. 16
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2.3.4 Pear Polysaccharides .................................................................................................... 18
2.4 Phenolics ............................................................................................................................. 19
2.4.1 Classification of Phenolics ........................................................................................... 19
2.4.1.1 Phenolic Acids ...................................................................................................... 19
2.4.1.2 Flavonoids ............................................................................................................. 20
2.4.2 Antioxidant Potential of Phenolics .............................................................................. 22
2.4.3 Phenolic Compounds in Apple and Pear Fruit ............................................................. 24
2.4.4 Phenolic Analysis......................................................................................................... 25
2.5 Mass Spectrometry.............................................................................................................. 26
2.5.1 General Background .................................................................................................... 26
2.5.2 Ion Mobility Mass Spectrometry ................................................................................. 28
2.5.3 Phenolic Analysis by Mass Spectrometry.................................................................... 28

3. AUTHENTICITY ANALYSIS OF PEAR JUICE EMPLOYING
CHROMATOGRAPHIC FINGERPRINTING .................................................................. 30
3.1 Abstract ............................................................................................................................... 30
3.2 Introduction ......................................................................................................................... 30
3.3 Materials and Methods ........................................................................................................ 33
3.3.1 Samples ........................................................................................................................ 33
3.3.2 Chemicals ..................................................................................................................... 33
3.3.3 Major Carbohydrate and Polyol Analysis by High-Performance Liquid
Chromatography with Refractive Index Detection (HPLC-RI) .................................. 33
3.3.4 Arbutin Analysis by High-Performance Liquid Chromatography with Photodiode
Array Detection (HPLC-PDA) ................................................................................... 34
3.3.5 Pure and Intentionally Adulterated Pear Juice Oligosaccharide and Arbutin Analysis
by Capillary Gas Chromatography with Flame Ionization Detection (CGC-FID) ..... 34
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3.3.6 Pure and Intentionally Adulterated Pear Juice Oligosaccharide and Arbutin Analysis
by High-Performance Anion Exchange Chromatography with Pulsed Amperometric
Detection (HPAE-PAD).............................................................................................. 35
3.3.7 Arbutin Identification by Capillary Gas Chromatography-Mass Spectrometry (GC-
MS) ............................................................................................................................. 36
3.3.8 Arbutin Hydrolysis with β-Glucosidase ...................................................................... 36
3.3.9 Preparation of a Multiply Adulterated Pear Juice Sample with Cellobiose................. 37
3.4 Results and Discussion ....................................................................................................... 37
3.4.1 Major Carbohydrate and Sorbitol Analysis ................................................................. 37
3.4.2 Pear Juice Adulteration with High Fructose Corn Syrup............................................. 41
3.4.3 Pear Juice Adulteration with Total Invert Sugar.......................................................... 44
3.4.4 Pear Juice Adulteration with Hydrolyzed Inulin Syrup ............................................... 47
3.4.5 Detection of Juice-to-Juice Adulteration by Arbutin Analysis .................................... 50
3.4.6 Detection of Total Liquefaction by Cellobiose Detection ........................................... 53
3.5 Linkage Between Authenticity Analysis of Pear Juice and Oligosaccharide Formation
during Pear Juice Processing .............................................................................................. 55

4. OLIGOSACCHARIDE FORMATION DURING COMMERCIAL PEAR JUICE
PROCESSING ........................................................................................................................ 55
4.1 Abstract ............................................................................................................................... 56
4.2 Introduction ......................................................................................................................... 56
4.3 Materials and Methods ........................................................................................................ 58
4.3.1 Samples ........................................................................................................................ 58
4.3.2 Chemicals ..................................................................................................................... 59
4.3.3 Laboratory Scale Polysaccharide Sample Preparation ................................................ 59
4.3.4 Enzymatic Hydrolysis of Laboratory Scale Polysaccharide Samples ......................... 60
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4.3.5 Laboratory Scale Pear Juice Production ...................................................................... 60
4.3.6 Commercial Samples ................................................................................................... 61
4.3.7 Commercial Pear Juice Processing Sample Preparation for Chromatographic Analysis
..................................................................................................................................... 61
4.3.8 Galacturonic Acid and Oligosaccharide Analysis by High Performance Anion
Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) ..... 62
4.3.9 Monosaccharide Analysis by HPAE-PAD .................................................................. 62
4.3.10 Oligosaccharide Analysis by Capillary Gas Chromatography with Flame Ionization
Detection (CGC-FID) ................................................................................................. 63
4.3.11 Galacturonic Acid Analysis by CGC-FID ................................................................. 63
4.3.12 Trisaccharide Analysis by CGC-FID ......................................................................... 64
4.4 Results and Discussion ....................................................................................................... 64
4.4.1 HPAE-PAD Analysis ................................................................................................... 64
4.4.1.1 Carbohydrate Profile Changes in Commercially Produced Pear Juice as a
Function of Processing .......................................................................................... 64
4.4.1.2 Peak Identification from Laboratory Scale Pectin, Starch and Xyloglucan
Hydrolysis ............................................................................................................. 67
4.4.2 CGC-FID Analysis....................................................................................................... 74
4.4.2.1 Carbohydrate Profile Changes in Commercially Produced Pear Juice as a
Function of Processing .......................................................................................... 74
4.4.2.2 Peak Identification from Laboratory Scale Pectin, Starch and Xyloglucan
Hydrolysis ............................................................................................................. 76
4.5 Linkage Between Oligosaccharide Formation in Pear Juice and Phenolic Content of Apple
and Pear Juice ..................................................................................................................... 78

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5. PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITIES OF COMMERCIAL
APPLE AND PEAR JUICES REPRESENTING MAJOR WORLD PRODUCING
REGIONS ................................................................................................................................ 79
5.1 Abstract ............................................................................................................................... 79
5.2 Introduction ......................................................................................................................... 79
5.3 Materials and Methods ........................................................................................................ 81
5.3.1 Samples ........................................................................................................................ 81
5.3.2 Chemicals ..................................................................................................................... 81
5.3.3 Total Phenolic Chromatographic Index (TPCI) ........................................................... 81
5.3.4 Total Phenolic Content (TPC) ..................................................................................... 82
5.3.5 Juice Phenolic Fractionation ........................................................................................ 83
5.3.6 Trolox Equivalence Antioxidant Capacity (TEAC) Assay for ABTS Radical
Scavenging Activity .................................................................................................... 84
5.3.7 DPPH Radical Scavenging Assay ................................................................................ 84
5.3.8 Statistics ....................................................................................................................... 85
5.4 Results and Discussion ....................................................................................................... 85
5.4.1 Total Phenolic Content (TPC) and Total Phenolic Chromatographic Index (TPCI) ... 86
5.4.2 DPPH and ABTS Radical Scavenging Activity of Commercial Apple and Pear Juice
..................................................................................................................................... 96
5.5 Linkage Between Phenolic Content of Apple and Pear Juice and Analysis of Chlorogenic
Acid Isomers using Ion Mobility and Tandem Mass Spectrometry ................................. 100

6. ANALYSIS OF A SERIES OF CHLOROGENIC ACID ISOMERS USING
DIFFERENTIAL ION MOBILITY AND TANDEM MASS SPECTROMETRY ........ 101
6.1 Abstract ............................................................................................................................. 101
6.2 Introduction ....................................................................................................................... 102
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