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EFFECT OF THERMAL PROCESSING ON THE PHENOLIC ANTIOXIDANTS OF
COLORED POTATOES
By
BALUNKESWAR NAYAK
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
Department of Biological Systems Engineering
MAY 2011
ii
To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of
BALUNKESWAR NAYAK find it satisfactory and recommend that it be accepted.
___________________________________
Juming Tang, Ph.D., Chair
___________________________________
Shyam S Sablani, Ph.D.
___________________________________
Jose De J Berrios, Ph.D.
___________________________________
Joseph R. Powers, Ph.D.
___________________________________
Jinwen Zhang, Ph.D.
iii
ACKNOWLEDGMENTS
I express my greatest gratitude to Dr. Juming Tang for his advice, guidance and
encouragement throughout my study and research at Washington State University. I am at a loss
of words to express the importance and value of his helpful insights, understanding, and moral
support that enabled me to stay focused on my research. I also express hearty thanks to my
doctoral committee members: Drs. Shyam S Sablani, Jose De D Berrios, Joseph R. Powers, and
Jinwen Zhang for their valuable suggestions and feedback whenever I approached them with
problems.
I thank Dr. Rui Hai Liu, Associate Professor in the Department of Food Science, Cornell
University, Ithaca, NY for his guidance on experimental designs in microbiology during my stay
and research at Cornell University. I acknowledge the contributions of Dr. Sohan Birla for his
guidance in the data analysis and technical writing. I am indebted to Mr. Christopher Derito,
Cornell University for teaching me cellular antioxidant analysis.
I thank Galina Mikhaylenko for her valuable suggestions and support in conducting my
experiments smoothly in the lab. I gratefully acknowledge the assistance of James Pan and
Matthew Tom, USDA-ARS, Albany, CA for their assistance and feedback during the extrusion
experiment. I thank Frank Younce, Wayne Dewitt, and Vince Himsl for their technical support
and assistance during my instrumentation. I thank my friend Gopal and senior colleague Ram,
who were highly supportive and stood by me through the thick and thin of my doctoral study.
Last but not least, I want to express my heartfelt thanks to my parents, wife Pinky, brothers
Uma and Titu, sister Gayatri for their understanding, encouragement and patience during my
research.
iv
EFFECT OF THERMAL PROCESSING ON THE PHENOLIC ANTIOXIDANTS OF
COLORED POTATOES
ABSTRACT
by Balunkeswar Nayak, Ph.D.
Washington State University
May 2011
Chair: Juming Tang
Foods with antioxidant capacity contribute health benefits and provide protection against
certain cancers, Alzheimer‘s dementia, and cardio-vascular diseases caused by oxidative
damage. Colored potatoes are a significant source of antioxidants from polyphenols, carotenoids,
and ascorbic acid. In this research, retention of total phenolics and antioxidant activity were
studied in fresh colored potatoes, and processed flakes were prepared as potential ingredients for
snack foods using freeze-drying, drum-drying and refractance window-drying. Extruded
products prepared from purple colored potatoes (‗Purple Majesty‘ cv) and yellow peas using a
twin-screw extruder were analyzed for the effect of extrusion cooking on their antioxidant
activity and anthocyanin level. Bioavailability of the phenolic antioxidants of extruded products
was analyzed using HepG2 liver cancer cells in a cellular antioxidant assay and compared with
unprocessed samples. Antioxidant potential of degradation products from purple potato
anthocyanins during high temperature processing and contribution to the total antioxidant
activity were measured using chemical assays (DPPH and ABTS). Thermal degradation kinetics
of anthocyanins prepared from ‗Purple Majesty‘ potatoes was conducted over a temperature
range of 100 – 150 °C. Dehydrated potato flakes showed no significant losses (P > 0.05) in total
antioxidant capacity (TAC) and total phenolics (TP) content, whereas 23 – 45% losses in total
v
anthocyanins (TA) were observed during dehydration of potatoes. The quantity of TAC was
unchanged and the TP was largely retained (73 – 83%) in the extruded products prepared from
colored potatoes and yellow pea flours. Severe losses in TA (60 – 70%) of extruded products
were observed due to high temperature during extrusion cooking. The large bioavailability of the
extruded products was well supported by the ORAC antioxidant activity, total phenolics
(including free and bound fractions) and total flavonoids content irrespective of the degradation
of anthocyanins. The bioavailability in the extrudates could be attributed to the breakdown of the
conjugated phenolic antioxidants to their free forms, and formation of Maillard reaction products
with potential antioxidant activity. Thermal degradation in prepared anthocyanins followed a
first order reaction, but the degradation compounds had antioxidant potency contributing to the
TAC in the processed foods.
vi
TABLE OF CONTENT
ACKNOWLEDGMENTS ............................................................................................................. iii
ABSTRACT ................................................................................................................................... iv
LIST OF TABLES ....................................................................................................................... xiii
LIST OF FIGURES ..................................................................................................................... xvi
CHAPTER ONE
INTRODUCTION .......................................................................................................................... 1
1. SUMMARY OF RELATED STUDIES AND PROBLEM STATEMENTS............................. 1
2. OBJECTIVES ............................................................................................................................. 4
3. DISSERTATION OUTLINES ................................................................................................... 5
REFERENCES ............................................................................................................................... 9
CHAPTER TWO
EFFECT OF PROCESSING ON THE PHENOLIC ANTIOXIDANTS OF FRUITS,
VEGETABLES AND GRAINS – A REVIEW ............................................................................ 14
1. INTRODUCTION .................................................................................................................... 14
1.1. Antioxidants and Mechanism ............................................................................................ 17
1.2. Measurement of antioxidant activity ................................................................................. 19
2. PHENOLIC ANTIOXIDANTS OF FRUITS, VEGETABLE AND GRAIN .......................... 22
2.1. Effect of processing on phytochemicals ............................................................................ 27
2.1.1. Extraction .................................................................................................................... 29
2.1.2. Blanching .................................................................................................................... 31
vii
2.1.3. Cooking ....................................................................................................................... 33
2.1.4. Drying/Dehydration .................................................................................................... 37
2.1.5. Irradiation .................................................................................................................... 38
2.1.6. Extrusion ..................................................................................................................... 40
2.1.7. Non-thermal processing .............................................................................................. 41
2.1.8. Storage ........................................................................................................................ 43
2.1.9. Enzymatic/Chemical Oxidation .................................................................................. 47
3. ANTHOCYANINS ................................................................................................................... 49
3.1. Effect of processing on Anthocyanins ............................................................................... 56
3.1.1. Extraction .................................................................................................................... 56
3.1.2. Blanching .................................................................................................................... 57
3.1.3. Heating ........................................................................................................................ 59
3.1.4. Drying/Dehydration .................................................................................................... 61
3.1.5. CO2 treatment.............................................................................................................. 63
3.1.6. Addition of external ingredients ................................................................................. 63
3.1.7. Non-thermal processing .............................................................................................. 65
3.1.8. Storage ........................................................................................................................ 66
3.2. Degradation mechanism and products ............................................................................... 69
3.3. Degradation kinetics of anthocyanins ................................................................................ 72
4. SUMMARY .............................................................................................................................. 80
REFERENCES ............................................................................................................................. 82
viii
CHAPTER THREE
COLORED POTATOES (SOLANUM TUBEROSUM L.) DRIED FOR ANTIOXIDANT-RICH
VALUE-ADDED FOODS.......................................................................................................... 116
1. INTRODUCTION .................................................................................................................. 116
2. MATERIALS AND METHODS ............................................................................................ 119
2.1. Raw Materials .................................................................................................................. 119
2.2. Production of potato flakes .............................................................................................. 119
2.2.1. Freeze-drying ............................................................................................................ 120
2.2.2. Drum-drying ............................................................................................................. 120
2.2.3. Refractance window-drying ..................................................................................... 120
2.3. Chemical Analysis ........................................................................................................... 121
2.3.1. Total antioxidant capacity ........................................................................................ 121
2.3.2. Total phenolics ......................................................................................................... 123
2.3.3. Total anthocyanins .................................................................................................... 123
2.4. Color determination ......................................................................................................... 125
2.5. Statistical analysis ............................................................................................................ 125
3. RESULTS AND DISCUSSION ............................................................................................. 126
3.1. Moisture content, total antioxidant capacity (TAC), total phenolics (TP) and total
anthocyanins (TA) in raw potatoes ......................................................................................... 126
3.2. Effects of blanching on TAC, TP and TA ....................................................................... 127
3.3 Total antioxidant capacity, total phenolics and total anthocyanins in dehydrated potato
flakes ....................................................................................................................................... 130
3.4. Correlation between TP and TAC.................................................................................... 132
ix
3.5. Color in raw cultivars and dehydrated flakes .................................................................. 133
4. CONCLUSION ....................................................................................................................... 135
ACKNOWLEDGEMENTS ........................................................................................................ 136
REFERENCES ........................................................................................................................... 137
CHAPTER FOUR
EFFECT OF EXTRUSION ON THE ANTIOXIDANT CAPACITY AND COLOR
ATTRIBUTES OF EXPANDED EXTRUDATES PREPARED FROM PURPLE POTATO
AND YELLOW PEA FLOUR MIXES ...................................................................................... 142
1. INTRODUCTION .................................................................................................................. 143
2. MATERIALS AND METHODS ............................................................................................ 144
2.1. Materials .......................................................................................................................... 144
2.1.1. Production of potato flours ....................................................................................... 145
2.1.2. Sample preparation ................................................................................................... 145
2.1.3. Extrusion conditions ................................................................................................. 146
2.1.4. Experimental design.................................................................................................. 147
2.1.5. Determination of Expansion ratio ............................................................................. 147
2.1.6. Moisture content determination ................................................................................ 148
2.1.7. Pasting profile of potato flours ................................................................................. 148
2.1.8. Color evaluation ........................................................................................................ 148
2.2. Chemical Analyses........................................................................................................... 150
2.2.1. Total antioxidant capacity ......................................................................................... 150
2.2.2. Total phenolics ......................................................................................................... 151
x
2.2.3. Total anthocyanins ................................................................................................... 152
2.2.4. Browning Index ....................................................................................................... 153
2.3. Statistical analysis ............................................................................................................ 153
3. RESULTS AND DISCUSSION ............................................................................................. 154
3.1. Expansion ratio ............................................................................................................... 154
3.2. Pasting behavior of potato flours ..................................................................................... 156
3.3. Color attributes................................................................................................................. 157
3.4. Total antioxidant capacity ................................................................................................ 159
3.5. Total phenolics ................................................................................................................. 162
3.6. Total anthocyanins ........................................................................................................... 164
3.7. Browning Index .............................................................................................................. 166
3.8. Correlation analyses ......................................................................................................... 167
4. CONCLUSIONS..................................................................................................................... 167
ACKNOWLEDGEMENTS ........................................................................................................ 168
REFERENCES ........................................................................................................................... 169
CHAPTER FIVE
BIOAVAILABILITY OF ANTIOXIDANTS IN EXTRUDED PRODUCTS PREPARED FROM
PURPLE POTATO AND DRY PEA FLOURS ......................................................................... 175
1. INTRODUCTION .................................................................................................................. 176
2. MATERIALS AND METHODS ............................................................................................ 178
2.1. Chemicals and Reagents .................................................................................................. 178
2.2. Preparation of Samples .................................................................................................... 178
xi
2.3 Extraction of Free Phenolic Compounds .......................................................................... 178
2.4. Extraction of Bound Phenolic Compounds...................................................................... 179
2.5. Determination of Total Phenolics .................................................................................... 179
2.6. Determination of Individual Phenolic Acids ................................................................... 180
2.7. Determination of Total Flavonoids .................................................................................. 181
2.8. Measurement of Antioxidant Activity (ORAC) .............................................................. 182
2.9. Cellular Antioxidant Activity assay ................................................................................. 182
2.10. Statistical Analyses ........................................................................................................ 184
3. RESULTS ............................................................................................................................... 184
3.1. Phenolic Contents ............................................................................................................ 184
3.3. Individual Phenolic Acids ................................................................................................ 187
3.4. Total Antioxidant Activity ............................................................................................... 189
3.5. Cellular Antioxidant Activity .......................................................................................... 192
3.6. Correlation Analyses ........................................................................................................ 191
4. DISCUSSION ......................................................................................................................... 193
ABBREVIATIONS USED ......................................................................................................... 201
ACKNOWLEDGEMENT .......................................................................................................... 201
REFERENCES ........................................................................................................................... 203
CHAPTER SIX
THERMAL DEGRADATION OF ANTHOCYANINS FROM PURPLE POTATOES
(‗PURPLE MAJESTY‘ CV) AND IMPACT ON ANTIOXIDANT CAPACITY. .................... 211
1. INTRODUCTION .................................................................................................................. 211
xii
2. MATERIALS AND METHODS ............................................................................................ 213
2.1 Chemicals .......................................................................................................................... 213
2.2. Materials .......................................................................................................................... 214
2.3. Extraction of anthocyanins .............................................................................................. 214
2.4. Purification of anthocyanins ............................................................................................ 215
2.4. Heat treatment of anthocyanins ....................................................................................... 215
2.5. HPLC Analyses ................................................................................................................ 217
2.7. Measurement of anthocyanins ......................................................................................... 218
2.8. Measurement of Antioxidant Capacity ............................................................................ 219
2.9. Determination of thermal kinetics parameters ................................................................. 220
3. RESULTS ............................................................................................................................... 222
3.1. Purification of anthocyanins ............................................................................................ 222
3.2. Degradation of anthocyanins ........................................................................................... 227
3.3. Antioxidant activity of the degradation compounds ........................................................ 229
4. DISCUSSION ......................................................................................................................... 230
5. CONCLUSIONS..................................................................................................................... 236
REFERENCES ........................................................................................................................... 239
CHAPTER SEVEN
CONCLUSIONS AND RECOMMENDATIONS ..................................................................... 246
1. CONCLUSIONS..................................................................................................................... 246
2. RECOMMENDATIONS ........................................................................................................ 248
APPENDIX ................................................................................................................................. 249
xiii
LIST OF TABLES
Table 2. 1. Major antioxidants in some common fruits, vegetables and grains. .......................... 21
Table 2. 2. In vitro assays used for measuring antioxidant activity of fruits and vegetables ...... 24
Table 2. 3. Comparison of different in vitro total antioxidant capacity assays. .......................... 25
Table 2. 4. Common anthocyanins present in fruits and vegetables. ........................................... 52
Table 2. 5. Degradation kinetic parameters of anthocyanins. ...................................................... 76
Table 3. 1. Total antioxidant capacity by DPPH assay, total phenolics content and total
anthocyanins of selected raw potato cultivars. ........................................................ 126
Table 3. 2. Total antioxidant capacity by DPPH assay, total phenolics and total anthocyanins of
raw, blanched and dried potato flakes from purple cultivars.. ................................. 129
Table 3. 3. Color atttributes of raw potato cultivars .................................................................. 133
Table 3. 4. Color atttributes of purple dehydrated flakes ......................................................... 135
Table 4. 1. Extrusion parameters for preparing extruded products using split yellow pea flours
and white potato flours. ........................................................................................... 146
Table 4. 2. Moisture contents and expansion ratios of the extrudates prepared from yellow pea
and purple potato flours ........................................................................................... 156
Table 4. 3. Flour pasting behaviors of white and purple potato flours. ..................................... 157
Table 4. 4. Color attributes of the extruded products prepared from yellow pea and purple potato
flours . ...................................................................................................................... 159
xiv
Table 5. 1. Percentage contributions of phytochemicals in free and bound extract of ingredients
(purple potato flour and dry pea flour), raw formulations and extruded products to
total phenolics, total antioxidant activity and total flavonoids ............................... 186
Table 5. 2. Quantity of free, bound and total individual phenolic acids present in extracts of
ingredients, raw formulations and extruded products ............................................. 188
Table 5. 3. Correlation analysis of phenolics, antioxidant activity (ORAC) and cellular
antioxidant activity of ingredients, raw formulations and extruded product. .......... 195
Table 6. 1. Experimental design of heat treatments with selected temperature and time
combinations. ........................................................................................................... 216
Table 6. 2. Concentrations of total anthocyanins from purple potato extract upon heating at 100
– 150 °C for 0 – 60 min. .......................................................................................... 226
Table 6. 3. Estimation of the order of anthocyanins degradation by examining r2 from plot of
zero-, half-, first and second order reactions. .......................................................... 227
Table 6. 4. Parameters for first-order kinetics and transition state equations for degradation of
anthocyanins from ‗Purple Majesty‘ potatoes after heat treatment over the
temperature range of 100 – 150 °C. ......................................................................... 227
Table 6. 5. Antioxidant Activity using DPPH radical scavenging assay of purified anthocyanins
from ‗Purple Majesty‘ potatoes upon heating at 100 – 150 °C for 0 – 60 min. ...... 234
Table 6. 6. Antioxidant Activity using ABTS radical scavenging assay of purified anthocyanins
from ‗Purple Majesty‘ potato upon heating at 100 – 150 °C for 0 – 60 min. .......... 235
xv
Table 6. 7. Progression of degradation compounds upon thermal exposure as measured by the
ratio of total antioxidant capacity and total anthocyanins in purified anthocyanins
samples over 100 – 150 °C. ..................................................................................... 237
xvi
LIST OF FIGURES
Figure 2. 1. Classification of Phytochemicals.. ........................................................................... 15
Figure 2. 2. Some of the plant phenols in food with antioxidant capacity. ................................. 16
Figure 2. 3. Generic structure of Flavonoid ................................................................................ 18
Figure 2. 4. Structures of common phenolic acids. ..................................................................... 18
Figure 2. 5. Classification of food antioxidants. ......................................................................... 20
Figure 2. 6. Mechanism of antioxidant activity involving free radicals. lipid radical (R·); peroxy
radical (ROO·); peroxide (ROOH); antioxidant (AH)............................................. 22
Figure 2. 7. Total phenols from daily consumption of fruits and vegetables in the American
diet............................................................................................................................ 26
Figure 2. 8. Heating/energy transfer medium in various processing methods. ........................... 28
Figure 2. 9. Changes in total antioxidant activity in vegetable matrix subjected to different
processing conditions.. ............................................................................................. 35
Figure 2. 10. Common anthocyanins found in flowers, fruits and vegetables. ............................. 50
Figure 2. 11. Spectral characteristics of potato anthocyanins, indication of glycosylation and
acylation patterns. .................................................................................................... 53
Figure 2. 12. Interrelationships between anthocyanin quantity and quality, and various factors
affecting the stability of anthocyanins.. ................................................................... 54
Figure 2. 13. Effect of pH on the possible degradation mechanism of anthocyanin (Malvidin-3-
glucoside).. ............................................................................................................... 55
Figure 2. 14. Enzymatic activity on the polyphenol.. .................................................................... 59
xvii
Figure 2. 15. Possible thermal degradation of non-acylated (Cyanidin-3-glucoside and
Pelargonidin-3-glucoside) and acylated (cyanidin glucosides with coumaric acid)
anthocyanins.. .......................................................................................................... 71
Figure 3. 1. Structure of anthocyanins. .................................................................................... 118
Figure 3. 2. Color of potato flakes; (A) without blanching; (B) with blanching ....................... 128
Figure 3. 3 Correlations between total antioxidant capacity by DPPH assay and total phenolics
in colored (purple, red, yellow) and white potato cultivars. .................................. 133
Figure. 4. 1. Effects of screw speed and feed moisture on the different expansion ratio of
extrudates prepared from white potato and yellow pea flours at 140 °C die
temperature ............................................................................................................ 155
Figure. 4. 2. RVA profile of white and purple potato flours. ..................................................... 158
Figure. 4. 3. Total antioxidant capacities by DPPH assay of raw formulations and extruded
products.. ................................................................................................................ 161
Figure. 4. 4. Total phenolic contents of raw formulations and extruded products.. ................... 163
Figure. 4. 5. Total anthocyanins contents and browning indices of raw formulations and extruded
products.. ................................................................................................................ 165
Figure 5. 1. Phenolic contents of ingredients, raw formulations and extruded products prepared
from raw formulations.. ......................................................................................... 185
Figure 5. 2. Flavonoid contents of ingredients, raw formulations and extruded products prepared
from raw formulations.. ......................................................................................... 187
xviii
Figure 5. 3 Antioxidant activity of ingredients, raw formulations and extruded products
prepared from raw formulations.. .......................................................................... 189
Figure 5. 4. Cellular antioxidant activity and EC50 value of ingredients, raw formulations and
extruded products prepared from raw formulations............................................... 191
Figure 6. 1. Specially designed thermal kinetics test (TKT) cells used for heat treatment of
purified anthocyanins (darker area) from ‗Purple Majesty‘ potatoes over a
temperature range of 100 -150 °C.. ........................................................................ 216
Figure 6. 2. Chromatogram of standards as detected using HPLC ........................................... 218
Figure 6. 3. HPLC-DAD profile of total polyphenols (in crude extract), phenolic acids (in ethyl
acetate portion) and purified anthocyanins (in HCl/Methanol portion) from ‗Purple
Majesty‘ potatoes. .................................................................................................. 223
Figure 6. 4 HPLC-DAD profile of control unheated purified anthocyanins from ‗Purple
Majesty‘ potatoes. .................................................................................................. 224
Figure 6. 5 MALDI mass spectra of the pigments from purified anthocyanins of ‗Purple
Majesty‘ potatoes;.. ................................................................................................ 225
Figure 6. 6. Chromatogram of thermal degradation compounds from purified anthocyanins
heated at 100 °C for 1 – 60 min... .......................................................................... 228
Figure 6. 7. First order plot for the degradation of anthocyanins during heating over a
temperature range of 100 -150 °C.. ........................................................................ 230
Figure 6. 8. Plot of ln (k) versus (1/T) for anthocyanin degradation during heating over the
temperature range of 100 -150 °C. ......................................................................... 231
xix
Dedication
This dissertation is dedicated to my parents who provided
both emotional support and encouragement
1
CHAPTER ONE
INTRODUCTION
1. SUMMARY OF RELATED STUDIES AND PROBLEM STATEMENTS
Phytochemicals derived from fruits and vegetables act effectively in preventing formation of
reactive oxygen, nitrogen, hydroxyl and lipid species either by scavenging free radicals,
repairing or removing damaged molecules (Velioglu et al., 1998). Consumption of antioxidant
rich foods maintains higher antioxidant levels in blood serum (Cao, Booth, Sadowski, & Prior,
1998; Cao, Russell, Lischner, & Prior, 1998). Phenolic antioxidants prevent many of the chronic
diseases associated with cancer, inflammation, atherosclerosis and ageing (Prior et al., 1998;
Zern et al., 2005). The relationship between consumption of fruits and vegetables to occurrence
of lung , colon, breast, cervical, esophageal, oral cavity, stomach, bladder, pancreatic and ovarian
cancers has been reviewed for almost 200 epidemiological studies (Block et al., 1992). The
same investigator reported that consumption of fruit and vegetable has significant protective
effect in reducing certain forms of cancer as demonstrated in 128 – 156 dietary studies.
Potatoes (Solanum tuberosum L.) have traditionally been perceived by consumers as a good
source of energy with additional benefits as they contain low fat and are cholesterol free.
Potatoes are also a good source of potassium, providing 21% of the recommended daily value.
Low sodium content in the diet reduces the risk of high blood pressure and heart attack for many
individuals. Potatoes provide 12% of the recommended daily value of dietary fiber beneficial for
a healthy digestive system and are an excellent source of vitamin C (45% of daily value), vitamin
B6 and iron (USDA National Nutrient Database, 2010). Colored potatoes, in particular, have
attracted the attention of investigators as well as consumers due to their taste, appearance, and
2
high level of antioxidant activities. Antioxidant activities in colored potatoes are associated with
the presence of polyphenols-/- flavonoids, carotenoids, ascorbic acid, tocopherols, alpha-lipoic
acid and selenium (Lachman et al., 2000).
Food processing operations such as drying, cooking and extrusion are necessary steps in
production of convenience foods, but they may affect the retention of antioxidants in food
matrices (Clifford, 2000; Kader et al., 2002; Nicoli et al., 1999; Rossi et al., 2003; Wu et al.,
2004a; Wu et al., 2004b). For example, blanching and drying are two unit operations in
preparing shelf-stable potato flakes as ingredients for commercial production of a wide range of
foods products, including mashed potato and extruded snack foods. Bruising/wounding, boiling,
baking, freeze-drying and microwave cooking of white and colored potatoes have different
effects on the total phenolic content (Brown et al., 2003; Reyes and Cisneros-Zevallos, 2003). It
was reported that potato peels and patatin protein hydrolysate suppressed oxidation of beef
patties (Wang and Xiong, 2005) and peel waste retarded oxidation in radiation processed lamb
meat (Kanatt et al., 2005). Most of time, investigators assume vitamin C as an indicator for
processing effects (Burg and Fraile, 1995; Lathrop and Leung, 1980). However, Eberhardt, Lee,
& Liu (2000) observed that vitamin C contributed less than 0.4% to the total antioxidant activity
in apples indicating the importance of phytochemicals towards antioxidant activity. Other than
vitamin C, there is limited literature available on the effects of drying/thermal treatments on
phytochemicals of potatoes. Bioactive phytochemicals exist in free as well as soluble-conjugated
and bound forms (Adom and Liu, 2002). Bound phytochemicals, mostly in cell wall materials,
are difficult to digest in the upper gastrointestine and may digest in the colon which reduces risk
of colon cancer (Andreasen et al., 2001).
3
According to Tiziani, Schwartz, & Vodovotz (2008), metabolism, absorption and
bioavailability of health-beneficial phytochemicals could be improved by combining the effects
of protein and individual phytochemicals. Liu (2004) reported the additive and synergistic
effects of biologically active compounds from fruits, vegetables and grains on health. Studies
have demonstrated that it is possible to puff pulse flours and pulse-based formulations into
potentially commercial nutritious snack and breakfast cereal-type products (Berrios et al., 2002;
Berrios et al., 2010; Berrios et al., 2004; Patil et al., 2007). Pulses (such as yellow peas) have
high levels of protein, dietary fibers, complex carbohydrates and folate, and are low in fat and
sodium (Madar and Stark, 2002). Therefore, it would be desirable to make commodities such as
colored potatoes and yellow peas into functional foods in the form of extruded snacks and
breakfast cereal-type food products. Understanding the profile and bioavailability of
phytochemicals in the unprocessed (raw formulations) and processed (extruded products) foods
is important for optimal use of these antioxidant compounds. Kinetic studies of natural color
pigments and analyses of degradation compounds will help design optimal processes to retain
maximal antioxidant activity in processed food.
Mishra, Dolan, & Yang (2008) studied the degradation of anthocyanins above 100°C and
hypothesized that anthocyanins could be used as food colorants in high temperature processes
such as for extruded snacks or baked cakes. Similarly, numerous studies have reported on the
degradation of anthocyanins in fruits and vegetables during processing and storage (Rossi et al.,
2003; Sadilova et al., 2006). The investigators either assumed or reported first-order kinetics for
anthocyanin degradation in selected fruits and vegetables (Garzon and Wrolstad, 2001; Kirca et
al., 2007; Reyes and Cisneros-Zevallos, 2007; Yue and Xu, 2008). However, those studies were
carried out either in whole pulp puree/extract. Puree/extract from whole pulp of fruits and
4
vegetables or their powders (hence used as unpurified) contain anthocyanins with other
compounds such as salts, sugars and other colorless non-anthocyanin phenolics that could affect
the stability or degradation kinetics and antioxidant capacity of anthocyanins (Del Pozo-Insfran
et al., 2004). Higher antioxidant activity of anthocyanin degradation compounds than the
unheated anthocyanin was also reported by many investigators (Matsufuji et al., 2007; Sadilova
et al., 2007; Yue and Xu, 2008). However, limited investigations have reported on the effect of
processing on the phytochemicals of colored potatoes, on the bioavailability of products prepared
from potatoes and legumes using extrusion cooking technology, and on the thermal kinetics of
purified anthocyanins and antioxidant potencies of degradation compounds from the purple
potato.
2. OBJECTIVES
The overall objective of this dissertation is to discern a fundamental understanding of thermal
degradation of the phytochemicals present in colored potatoes and value added products prepared
from it. The specific aims were to:
Quantify the total antioxidant capacity, total phenolics, and total anthocyanins in selected
white and colored potato cultivars and to study the effect of blanching and consequent
drying on the retention of these quality parameters in purple potato cultivars;
Produce puffed extrudates from mixtures of colored potato and yellow pea flours, and
evaluate the effect of extrusion conditions on antioxidant capacities, color attributes, and
some physical characteristics of the extrudates;
Investigate the complete phytochemicals that exists in free and bound forms as well as
their contribution to bioactivity in the raw ingredients (purple potato flour and dry pea
5
flour), raw formulations and processed products prepared from the above ingredients
using extrusion cooking; and
Evaluate the thermal degradation kinetics parameters of anthocyanins purified from
purple potatoes (‗Purple Majesty‘ cv) over the temperature range of 100 – 150 °C and
determine antioxidant potencies of degradation compounds from the anthocyanins.
3. DISSERTATION OUTLINES
This dissertation contains seven chapters to address the above objectives.
Chapter One: Introduction. This chapter summarizes related research conducted by various
investigators, problem statements of current research and proposed objectives to address the
problems in the study. It also provides an outline/structure of the dissertation.
Chapter Two: Effect of processing on the phenolic antioxidants of fruits, vegetables and
grains- a review. This chapter compiles and briefly discusses various phenolic antioxidants
present in fruits, vegetables and grains. Previous research results and findings on the effect of
various processing methods including extraction, dehydration/drying, cooking and storing on the
phenolic antioxidants are summarized.
Chapter Three: Colored potatoes (Solanum Tuberosum L.) dried for antioxidant-rich value-
added products. This chapter contains information on the selection of colored potatoes based on
the retention of antioxidant compounds in raw potatoes and dehydrated potato flakes prepared as
potential ingredients for snack foods. Purple, red, yellow and white potatoes are dehydrated
using drum-drying, freeze-drying and refractance window-drying to prepare flakes. Total
antioxidant capacity, phenolics and anthocyanins present in raw and dehydrated flakes are
measured using chemical assays. Color attributes of the raw and dehydrated flakes are also
measured to correlate with the presence of anthocyanins. Contributions of the total phenolics and
6
anthocyanins to total antioxidants in potatoes are analyzed using correlation among the
parameters.
Chapter Four: Effect of extrusion on the antioxidant capacity and color attributes of
expanded extrudates prepared from purple potato and yellow pea flour mixes. In this chapter, the
total antioxidant capacity, total phenolics, total anthocyanins, and color attributes of unprocessed
formulations and processed products using a twin-screw extruder are evaluated. The effect of
extrusion cooking in producing expanded extrudates as well as retaining phenolic antioxidants is
also examined. Color attributes such as brightness, chroma, hue and browning index in extruded
products are evaluated for potential retention of natural color due to anthocyanins, as affected by
high temperature short time extrusion cooking.
Chapter Five: Bioavailability of antioxidants in extruded products prepared from purple
potato and dry pea flours. Bioavailability of phytochemicals in vivo in the extruded products
prepared from purple potatoes (‗Purple Majesty‘ cv) and dry peas are evaluated using a cellular
antioxidant assay using liver cancer HepG2 cells. Total phenolic antioxidants including that
present in free and bound fractions are considered for determining the antioxidant activity of the
processed products. Role of phenolic antioxidants evaluated using a cellular method is compared
with the commonly used chemical assay (oxygen radical absorption capacity) for a possible
correlation and better explanation of their health benefits.
Chapter Six: Thermal degradation of anthocyanins from purple potato (‗Purple Majesty‘ cv)
and impact on antioxidant activities. This chapter provides some explanations for possible
increase in the antioxidant activities of extruded products irrespective of reduction in its natural
color/anthocyanins. Anthocyanins are prepared from purple potatoes (‗Purple Majesty‘ cv) by
removing salts, sugars and colorless phenolics and heat treated (100 – 150 °C) and analyzed for
7
potential antioxidant activity of the degradation compounds using chemical antioxidant assays.
Thermal degradation kinetics parameters including reaction order, activation energy, thermal
death time (D-values) and z-value of the purified anthocyanins are reported.
Chapter Seven: Conclusions and recommendations. Summary of the findings of the present
study and recommendations for future work are illustrated in this chapter.
Some chapters in the dissertation carry the styles of a particular journal, where it is published
or submitted. Full citations of these chapters included in this dissertation are as follows:
Chapter Two
Nayak, Balunkeswar, Berrios, J., Powers, J.R. and Tang, J. Effect of processing on phenolic
antioxidants of fruits and vegetables. Trends in Food Science and Technology (internal
review)
Chapter Three
Nayak, Balunkeswar, Powers, Joseph R., Berrios, J. and Tang, Juming. Potential of colored
potatoes for producing high antioxidant snack foods. Journal of Food Processing and
Preservation (in press).
Chapter Four
Nayak, Balunkeswar, Berrios, J., Powers, J.R., and Tang, J. Effect of extrusion on the
antioxidant capacity and color attributes of expanded products prepared from purple
potatoes and yellow peas. Food Chemistry (internal review)
Chapter Five
Nayak, Balunkeswar, Liu, R.H., Berrios, J., Tang, J., Derito, C. Bioavailability of
antioxidants in extruded products prepared from purple potato and dry pea flours. Journal
of Agricultural & Food Chemistry (internal review)
8
Chapter Six
Nayak, Balunkeswar, Berrios, J., and Tang, J. Thermal degradation of anthocyanins in purple
potato and impact on antioxidant capacity. Journal of Food Engineering (internal review)
9
REFERENCES
Adom, K. K., and Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and
Food Chemistry. 50: 6182-6187.
Andreasen, M. F., Kroon, P. A., Williamson, G., and Garcia-Conesa, M. T. (2001). Intestinal
release and uptake of phenolic antioxidant diferulic acids. Free Radical Biology and
Medicine. 31: 304-314.
Berrios, J. D., Camara, M., Torija, M. E., and Alonso, M. (2002). Effect of extrusion cooking
and sodium bicarbonate addition on the carbohydrate composition of black bean flours.
Journal of Food Processing and Preservation. 26: 113-128.
Berrios, J. D., Morales, P., Camara, M., and Sanchez-Mata, M. C. (2010). Carbohydrate
composition of raw and extruded pulse flours. Food Research International. 43: 531-536.
Berrios, J. D., Wood, D. F., Whitehand, L., and Pan, J. (2004). Sodium bicarbonate and the
microstructure, expansion and color of extruded black beans. Journal of Food Processing
and Preservation. 28: 321-335.
Block, G., Patterson, B., and Subar, A. (1992). Fruit, vegetables, and cancer prevention - a
review of the epidemiologic evidence. Nutrition and Cancer-an International Journal. 18: 1-
29.
Brown, C. R., Wrolstad, R., Durst, R., Yang, C. P., and Clevidence, B. (2003). Breeding studies
in potatoes containing high concentrations of anthocyanins. American Journal of Potato
Research. 80: 241-249.
Burg, P., and Fraile, P. (1995). Vitamin C destruction during the cooking of a potato dish.
Lebensmittel-Wissenschaft und-Technologie. 28: 506-514.
10
Clifford, M. N. (2000). Anthocyanins – nature, occurrence and dietary burden. Journal of the
Science of Food and Agriculture. 80: 1063-1072.
Del Pozo-Insfran, D., Brenes, C. H., and Talcottt, S. T. (2004). Phytochemical composition and
pigment stability of acai (Euterpe oleracea Mart.). Journal of Agricultural and Food
Chemistry. 52: 1539-1545.
Eberhardt, M. V., Lee, C. Y., and Liu, R. H. (2000). Nutrition - Antioxidant activity of fresh
apples. Nature. 405: 903-904.
Garzon, G. A., and Wrolstad, R. E. (2001). The stability of pelargonidin-based anthocyanins at
varying water activity. Food Chemistry. 75: 185-196.
Kader, F., Irmouli, M., Nicolas, J. P., and Metche, M. (2002). Involvement of blueberry
peroxidase in the mechanisms of anthocyanin degradation in blueberry juice. Journal of
Food Science. 67: 910-915.
Kanatt, S. R., Chander, R., Radhakrishna, P., and Sharma, A. (2005). Potato peel extract - a
natural antioxidant for retarding lipid peroxidation in radiation processed lamb meat. Journal
of Agricultural and Food Chemistry. 53: 1499-1504.
Kirca, A., Ozkan, M., and Cemeroglu, B. (2007). Effects of temperature, solid content and pH on
the stability of black carrot anthocyanins. Food Chemistry. 101: 212-218.
Lachman, J., Hamouz, K., Orsak, M., and Pivec, V. (2000). Potato tubers as a significant source
of antioxidants in human nutrition. Rostlinna Vyroba. 46: 231-236.
Lathrop, P. J., and Leung, H. K. (1980). Rates of ascorbic acid degradation during thermal
processing of canned peas. Journal of Food Science. 45: 152-153.
Liu, R. H. (2004). Potential synergy of phytochemicals in cancer prevention: mechanism of
action. J. Nutr. 134: 3479S-3485.
11
Madar, Z., and Stark, A. H. (2002). New legume sources as therapeutic agents. British Journal of
Nutrition. 88: S287-S292.
Matsufuji, H., Kido, H., Misawa, H., Yaguchi, J., Otsuki, T., Chino, M., Takeda, M., and
Yamagata, K. (2007). Stability to light, heat, and hydrogen peroxide at different pH values
and DPPH radical scavenging activity of acylated anthocyanins from red radish extract.
Journal of Agricultural and Food Chemistry. 55: 3692-3701.
Mishra, D. K., Dolan, K. D., and Yang, L. (2008). Confidence intervals for modeling
anthocyanin retention in grape pomace during non-isothermal heating. Journal of Food
Science. 73: E9-E15.
Nicoli, M. C., Anese, M., and Parpinel, M. (1999). Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology. 10: 94-100.
Patil, R. T., Berrios, J. D. J., Tang, J., and Swanson, B. G. (2007). Evaluation of methods for
expansion properties of legume extrudates. Applied Engineering in Agriculture. 23: 777-783.
Prior, R. L., Cao, G. H., Martin, A., Sofic, E., McEwen, J., O'Brien, C., Lischner, N., Ehlenfeldt,
M., Kalt, W., Krewer, G., and Mainland, C. M. (1998). Antioxidant capacity as influenced by
total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. Journal
of Agricultural and Food Chemistry. 46: 2686-2693.
Reyes, L. F., and Cisneros-Zevallos, L. (2003). Wounding stress increases the phenolic content
and antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of
Agricultural and Food Chemistry. 51: 5296-5300.
Reyes, L. F., and Cisneros-Zevallos, L. (2007). Degradation kinetics and colour of anthocyanins
in aqueous extracts of purple- and red-flesh potatoes (Solanum tuberosum L.). Food
Chemistry. 100: 885-894.
12
Rossi, M., Giussani, E., Morelli, R., Lo Scalzo, R., Nani, R. C., and Torreggiani, D. (2003).
Effect of fruit blanching on phenolics and radical scavenging activity of highbush blueberry
juice. Food Research International. 36: 999-1005.
Sadilova, E., Carle, R., and Stintzing, F. C. (2007). Thermal degradation of anthocyanins and its
impact on color and in vitro antioxidant capacity. Molecular Nutrition & Food Research. 51:
1461-1471.
Sadilova, E., Stintzing, F. C., and Carle, R. (2006). Thermal degradation of acylated and
nonacylated anthocyanins. Journal of Food Science. 71: C504-C512.
Tiziani, S., Schwartz, S. J., and Vodovotz, Y. (2008). Intermolecular interactions in
phytochemical model systems studied by NMR diffusion measurements. Food Chemistry.
107: 962-969.
Velioglu, Y. S., Mazza, G., Gao, L., and Oomah, B. D. (1998). Antioxidant activity and total
phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food
Chemistry. 46: 4113-4117.
Wang, L. L., and Xiong, Y. L. L. (2005). Inhibition of lipid oxidation in cooked beef patties by
hydrolyzed potato protein is related to its reducing and radical scavenging ability. Journal of
Agricultural and Food Chemistry. 53: 9186-9192.
Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., and Prior, R. L.
(2004a). Lipophilic and hydrophilic antioxidant capacities of common foods in the United
States. Journal of Agricultural and Food Chemistry. 52: 4026-4037.
Wu, X. L., Gu, L. W., Holden, J., Haytowitz, D. B., Gebhardt, S. E., Beecher, G., and Prior, R.
L. (2004b). Development of a database for total antioxidant capacity in foods: a preliminary
study. Journal of Food Composition and Analysis. 17: 407-422.
13
Yue, X., and Xu, Z. (2008). Changes of anthocyanins, anthocyanidins, and antioxidant activity in
bilberry extract during dry heating. Journal of Food Science. 73: C494-C499.
Zern, T. L., Wood, R. J., Greene, C., West, K. L., Liu, Y. Z., Aggarwal, D., Shachter, N. S., and
Fernandez, M. L. (2005). Grape polyphenols exert a cardioprotective effect in pre- and
postmenopausal women by lowering plasma lipids and reducing oxidative stress. Journal of
Nutrition. 135: 1911-1917.
http://www.nal.usda.gov/fnic/foodcomp/cgi-bin/list_nut_edit.pl. USDA National Nutrient
Database for Standard Reference, Release 23 (2010).
14
CHAPTER TWO
EFFECT OF PROCESSING ON THE PHENOLIC ANTIOXIDANTS OF
FRUITS, VEGETABLES AND GRAINS – A REVIEW
1. INTRODUCTION
Phytochemicals are bioactive non-nutrient plant compounds in fruits, vegetables, grains and
other plant foods thought to promote health. These are broadly classified as carotenoids,
phenolics, alkaloids, nitrogen-containing, and organosulfur compounds (Figure 2.1).
Phytochemicals prevent many of the chronic diseases associated with cancer, inflammation,
atherosclerosis and ageing caused by free radicals and oxygen (Prior et al., 1998; van den Berg,
Haenen, van den Berg, & Bast, 1999; Zern et al., 2005). Consumption of antioxidant rich foods
maintains higher antioxidant levels in blood serum (Cao, Booth, Sadowski, & Prior, 1998; Cao,
Russell, Lischner, & Prior, 1998). The relationship between consumption of fruits and vegetables
to lung, colon, breast, cervical, esophageal, oral cavity, stomach, bladder, pancreatic and ovarian
cancers has been reviewed for almost 200 epidemiological studies (Block, Patterson, & Subar,
1992).
Phenolics are products of secondary metabolism in plants and have antioxidant activity
(Figure 2.2). These compounds are important in plants for protection against pathogens,
parasites and predators besides helping in reproduction and growth (Liu & Felice, 2007). In our
diet, 2/3 of all phenolics consist of flavonoids and 1/3 are derived from phenolic acids (Liu &
Felice, 2007). Flavonoids are found as conjugates in glycosylated or esterified forms in fruits and
vegetables. These types of compounds can also occur as aglycones as a result of food
15
Phytochemicals
PhenolicsCarotenoids Alkaloids Nitrogen-
containingOrganosulfur
FlavonoidsPhenolic acids Stilbenes Coumarins Tannins
FlavonesFlavonols Isoflavonoids Flavanones Flavanols AnthocyanidinsHydroxy-
cinnamic acid
Hydroxy-
benzoic acid
Gallic acid
Protocatechuic
acid
Vannilic acid
Syringic acid
p-Hydroxy-
benzoic acid
P-Coumaric
acid
Caffeic acid
Ferulic acid
Sinapic acid
Apigenin
Chrysin
Luteolin
Quercetin
Kaempferol
Myricetin
Galangin
Fisetin
Genistein
Daidzein
Glycitein
formononetin
Catechin
Epicatechin
Epigallocatechin
Epicatechin-
gallate
Epigallocatechin
-gallate
Flavanones Cyanidin
Palargonidin
Delphidin
Peonidin
Petunidiin
Malvidin
Figure 2. 1. Classification of Phytochemicals. Adapted and modified from Liu & Felice (2007).
16
OH
H3CO
CHO
Vanillin
OH
H3CO
Eugenol
OH
H3CO
O
O
OH OH
OHCOOH
Chlorogenic acid
OH
OH
O
O
COOH OH
OH
Rosmaric acid
OH
OH
O
OH
OH
OH
(+)- Catechin
OH
OH
O
OH
OH
OH
(-)- Epicatechin
OH
OH
O
OH
OH
O
O
rutinose
Rutin
OH
OH
O
OH
OH
Luteolin
Figure 2. 2. Some of the plant phenols in food with antioxidant capacity.
processing (Liu & Felice, 2007). The generic structure of flavonoids consists of 2 benzene rings
(A & B rings) linked by 3 carbons that are usually in an oxygenated heterocycle ring or C ring
(Figure 2.3). Hydroxybezoic acid and hydroxycinnamic acid derivatives are major phenolic
acids (Figure 2.4) present in bound form in plant cells. Hydroxybenzoic acid derivatives occur
as sugar derivatives and organic acids in plant foods besides present in lignins and hydrolyzable
tannins, whereas hydroxycinnamic acid derivatives are present in cellulose, lignin and proteins
through ester bonds. Food processing operations such as thermal processing, fermentation, and
freezing release these bound phenolic acids (Dewanto, Wu, Adom, & Liu, 2002).
17
1.1. Antioxidants and Mechanism
Antioxidants are defined as the substances, when present at low concentrations compared to
those of an oxidizable substrate, which significantly delay or inhibit oxidation (process of losing
electrons) of that substrate (Halliwell & Gutteridge, 1990). A free radical is a species capable of
independent existence that contains one or more unpaired electrons (an unpaired electron being
one that is alone in an orbital) (Halliwell, Gutteridge, & Cross, 1992). For example, superoxide
(an oxygen-centered radical), thiyl (a sulfur-centered radical), trichloromethyl (a carbon-centered
radical), and nitric oxide are created as by-products of various metabolic reactions (Halliwell et
al., 1992). Antioxidants reduce localized oxygen concentration, prevent initiation of oxidation
(Halliwell & Gutteridge, 1990), inhibit radical oxygen species by directly scavenging free
radicals, singlet oxygen quenchers, peroxide decomposers, enzyme inhibitors or synergists such
as metal chelating agents or reducing agents (Namiki, 1990). In addition, both aqueous and lipid
phases exist in food systems with different reaction properties. For example, polar antioxidants,
such as ascorbic acid are dissolved in aqueous phase and react with hydrophilic hydroxyl or
peroxyl free radicals, whereas lipophilic antioxidants, such as tocopherols, dissolve in lipidic
phase reacting with liposoluble free radicals produced during lipid oxidation. Antioxidants,
according to their polarity, can accumulate at the water-oil interface forming an oriented mono-
molecular layer that protects the lipid phase against oxidation by oxygen dissolved in the
aqueous phase (Frankel, Huang, Kanner, & German, 1994). Such behaviors should be considered
while accessing the antioxidant functionality.
Food antioxidants are classified as (i) primary or chain-breaking antioxidants, (ii) synergist,
and (iii) secondary antioxidants (Figure 2.5). Major antioxidants in common fruits, vegetables
and grains are shown in Table 2.1. Primary antioxidants such as phenolic compounds terminate
18
Figure 2. 3. Generic structure of Flavonoid
R1
R2
R3
COOH
Benzoic acid derivatives
R1
R2
R3
CH CH COOH
Cinnamic acid derivatives
Benzoic acid
Derivatives
Substitutions Cinnamic acid
derivatives
Substitutions
R1 R2 R3 R1 R2 R3
Benzoic acid H H H Cinnamic acid H H H
p-Hydroxybenzoic acid H OH H p-Coumaric acid H OH H
Protocatechuic acid H OH OH Caffeic acid OH OH H
Vanillic acid CH3O OH H Ferulic acid CH3O OH H
Syringic acid CH3O OH CH3O Sinapic acid CH3O OH CH3O
Gallic acid OH OH OH
Figure 2. 4. Structures of common phenolic acids.
the free radical chain reaction by donating hydrogen or electrons to free radicals and converting
them to more stable compounds. These antioxidants are effective in very low concentrations,
whereas they become prooxidants at higher concentration levels. Synergistic antioxidants, for
example ascorbic acid, act as hydrogen donators to the phenoxy radical to regenerate and provide
an acidic medium to stabilize the primary antioxidants. Secondary or preventive antioxidants
function by decomposing the lipid peroxides into stable end compounds (Rajalakshmi &
Narasimhan, 1996). The mechanism of antioxidant activity involving free radicals in lipid
oxidation includes three steps of initiation, propagation and termination (Figure 2.6). However,
O
5 4
3
2
6
7
2'
3'
4'
5'6'
8
A C
B
19
an inhibition reaction is also considered that competes with the propagation step yielding more
stable products (Nawar, 1996).
1.2. Measurement of antioxidant activity
A number of methods have been used for measuring antioxidant activity, all of which
consider (i) measuring current state of oxidation in the model systems, and (ii) radical
scavenging assays. The methods include features such as a suitable substrate, an oxidation
initiator, and measurement of end point (Antolovich, Prenzler, Patsalides, McDonald, &
Robards, 2002). Shahidi & Zhong (2005) reviewed the methods for lipid oxidation.
Radical scavenging assays (Table 2.2) require no lipid substrate and directly involve either
measuring (i) hydrogen atom transfer (HAT) i.e. the ability of an antioxidant to quench free
radicals by hydrogen donation, or (ii) single electron transfer (SET) i.e. ability of a potential
antioxidant to transfer one electron to reduce any compound, including metal, carbonyl and
radicals in the assay. However, the end results in these assays remain the same, regardless of
mechanism, even if the kinetics and potential for side reactions vary (Prior, Wu, & Schaich,
2005). The concept of bond dissociation energy and ionization potential of antioxidants are
important to understand the mechanism and efficacy of their action (Wright, Johnson, &
DiLabio, 2001). HAT based methods (Table 2.2) use a free radical generator that generates
stable or short-lived radicals, an oxidizable molecular probe and an antioxidant. The added
antioxidant competes with probes for the radicals and thus, inhibits or retards the oxidation of the
probes. Most commonly used HAT based assays are oxygen radical absorbance capacity
(ORAC) and total radical-trapping antioxidant parameter (TRAP). SET based assay (Table 2.2)
such as ferric reducing antioxidant power (FRAP) involves an electron transfer from the
20
Food
Antioxidants
Primary
Antioxidants
Secondary /Synergistic
Antioxidants
‗Hindred‘
PhenolsPhenols
Miscellaneous
Primary
Antioxidants
Oxygen
Scavengers
Chelating
Agents
Gallates
Hydroquinone
Trihydroxy-
butyrophenone
Nordihydroguairetic
acid
BHA
BHT
TBHQ
Tocopherols
Gum Guaiac
Ionox series
Sulfites
Ascorbic acid
Ascorbyl
palmitate
Erythorbic acid
Ethoxyquin
Anoxomer
Trolox-C
Polyphosphates
EDTA
Tartaric acid
Citric acid
Citrate esters
Phytic acid
Lecithin
Nitrites
Amino acids
Spice extracts
Flavonoids
Vitamin A
Β-Carotene
Tea extracts
Zinc
Selenium
Thiodipropionic
acid
Dilauryl,
Distearyl esters
Secondary
Antioxidants
Miscellaneous
Antioxidants
Figure 2. 5. Classification of food antioxidants. Adapted and modified from Rajalakshmi & Narasimhan (1996)
21
Table 2. 1. Major antioxidants in some common fruits, vegetables and grains. Modified from
Sun & Powers (2007).
Source Major antioxidants
Fruits
Apple Benzoic acid, cinnamic acid, flavan-3-ols, anthocyanidin, flavonols, dihydrochalcones
Berry Anthocyanins, flavonols, flavanols, proanthocyanidins, ellagitannins, gallotannins,
stilbenoinds, phenolic acids
Grape Resveratol, catechin, anthocyanins, gallic acid
Grapefruit Narirulin, hesperetin, hesperidin, ascorbic acid
Vegetables
Asparagus Rutin, chlorogenic acid, ascorbic acid
Bean Quercetin, kaempferol glucoside, ascorbic acid
Beet Ferulic acid
Broccoli Caffeic acid, ferulic acid, quercetin, kaempferol, ascorbic acid
Cabbage Chlorogenic acid, caffeic acid, kaempferol, lutein, ascorbic acid
Carrot Chlorogenic acid, caffeic acid, β-carotene
Cauliflower Cinnamic acid, quercetin, ascorbic acid
Celery Chlorogenic acid, apigenin, apigenin glucoside, luteolin glucoside, α-tocopherol
Corn Caffeic acid, cinnamic acid, coumaric acid
Cucumber Ascorbic acid
Garlic Myricetin, apigenin, ascorbic acid
Lettuce Chlorogenic acid, caffeic acid, quercetin
Mushroom Ferulic acid, ascorbic acid
Onion Quercetin glucoside
Bell Pepper Caffeic acid, quercetin glucoside, luteolin glucoside, α-tocopherol, ascorbic acid
Potato Caffeic acid, cinnamon acid, p-hydroxybenzoic acid, ascorbic acid
Squash Β-carotene
Spinach Quercetin, ascorbic acid, patuletin glucoside, spinacetin glucoside, 5, 3‘-hydroxy-3-
methoxy-6,7-methylenedioxyflavone-4‘-glucronide methyl ester, 5-hydroxy-3,3‘-
dimethoxy-6,7- methylenedioxyflavone-4‘-glucronide methyl ester
Sweet potato Caffeic acid, cinnamic acid, α-tocopherol
Tomato Chlorogenic acid, caffeic acid, quercetin, ascorbic acid
Grains
Soybean Isoflavones, tocopherols, tocotrienols
Wheat Carotenoids, tocopherol, ferulic, vanillic, caffeic, coumaric and syringic acid, phytosterols
Rice bran Tocopherols, γ-oryzanol,
Oat Tocopherols, avenanthramides, p-hydroxybenzoic acid, vanillic acid, phytosterols
Corn Lutein, α- and β-carotene, β-cryptoxanthin, zeaxanthin, tocopherols, phytosterols
22
Figure 2. 6. Mechanism of antioxidant activity involving free radicals. lipid radical (R·); peroxy
radical (ROO·); peroxide (ROOH); antioxidant (AH). Adapted from Shahidi & Zhong (2007)
antioxidants to the probe (oxidants), thus reducing the probe and oxidizing the antioxidants. The
oxidation reaction brings color change in the probe, which is proportional to the antioxidant
concentration and thus estimates the reducing capacity or antioxidant activity of the antioxidant
under investigation (Prior et al., 2005). Although trolox equivalent antioxidant capacity (TEAC)
and DPPH (2, 2-diphenyl-1-picrylhydrazyl) assays are also classified as SET based assays, these
two indicator radicals may be neutralized either by direct reduction via electron transfers or by
radical quenching via hydrogen atom transfer (Jimenez, Selga, Torres, & Julia, 2004). Table 2.3
provides a brief comparison of different antioxidant assays used to measure antioxidant activity
of fruits and vegetables.
2. PHENOLIC ANTIOXIDANTS OF FRUITS, VEGETABLE AND GRAIN
Consumption of foods such as grains, vegetables, and fruits containing antioxidant
compounds (Figure 2.7) may prevent many diseases and promote good health (Temple, 2000).
Vegetable consumption may provide protection against oxidative stress that is a pathogenic
mechanism of both carcinogenesis and atherosclerosis (Ames, Shigenaga, & Hagen, 1993). The
R H R
+ H
ROO
R
+ O2
ROO
+ R H
ROOH
+
R
ROO
+ A H
ROOH
+
A
Initiation
Propagation
Inhibition
Termination ROO
+ A
2 ROO
ROO
+R
R
+ R
Non-radical
products
23
importance of various fruits and vegetables in the American diet has been compiled based on
their total phenolics, antioxidant capacity and daily consumption (Chun et al., 2005). The
mechanism for the protective effect of these phenolic antioxidants has been recently reviewed
(Kinsella, Frankel, German, & Kanner, 1993). Many of these phenols have been found to be
more powerful antioxidants than vitamins C, E, and β-carotene using an in vitro model for heart
disease, namely the oxidation of low density lipoproteins (Vinson, Dabbagh, Serry, & Jang,
1995). For example, antioxidant activities in colored potatoes are mainly due to the presence of
polyphenols / flavonoids, carotenoids, ascorbic acid, tocopherols, alpha-lipoic acid and selenium
(Lachman, Hamouz, & Orsak, 2005). The most common phenolics in potato are flavanols,
cinnamic acid, p-coumaric acid, caffeic acid, chlorogenic acid, ferulic acid, and quinic acid.
Studies on phenolic composition and antioxidant action of 23 vegetables showed highest total
phenol content and antioxidant activity (LDL oxidation) per fresh weight in beans (kidney and
pinto) (Vinson, Hao, Su, & Zubik, 1998). Similarly, Kahkonen et al. (1999) reported total
phenolic contents and antioxidant activities of a number of fruits, vegetables and cereal grains.
Alsaikhan, Howard, & Miller (1995) also reported that antioxidant activity of broccoli using β-
carotene/linoleic acid was highest followed by potato, carrot, onion and bell pepper. The main
antioxidative components in grain are classified as phenolic compounds such as anthocyanins,
tannins, and ferulic acid, and other substances (Martinez-Tome et al., 2004). White & Xing
(1997) reported the presence of p-hydroxybenzoic, protocatechuic, vanillic, trans-p-coumaric, (p-
hydroxyphenyl)- acetic, syringic, trans-sinapic, caffeic, and ferulic acids, with ferulic acid as the
most abundant phenolic acid in oat flour. According to Terao et al. (1993), caffeic acid showed
strong antioxidant activity followed by ferulic acid with moderate activity and p-coumaric acid
in a model solution. Chinese black-grained wheat has higher antioxidant activity compared to
24
Table 2. 2. In vitro assays used for measuring antioxidant activity of fruits and vegetables.
Adapted from Huang, Ou, & Prior (2005)
Assays involving hydrogen atom transfer
reactions
ROO. + AH ROOH + A
.
ROO. + LH ROOH + L
.
1. ORAC (oxygen radical absorbance capacity)
2. TRAP (total radical-trapping antioxidant
parameter)
3. Crocin bleaching assay
4. IOU (inhibition oxygen uptake)
5. Inhibition of linoleic oxidation
6. Inhibition of LDL oxidation
Assays by electron transfer reaction
M (n) + e (from AH) AH.+ + M (n-1)
1. TEAC (Trolox Equivalent Antioxidant
Capacity)
2. FRAP (Ferric Reducing Antioxidant Power )
3. DPPH (2, 2-diphenyl-1-picrylhydrazyl)
4. Copper (II) reduction capacity
5. Total phenols assay by Folin-Ciocalteu reagents
Other assays 1. TOSC (total oxidant scavenging capacity)
2. Inhibition of Briggs-Rauscher oscillation
reaction
3. Chemiluminescence
4. Electrochemiluminescence
LH: substrate; AH: antioxidant; A.: antioxidant radical; ROO
.: peroxyl radical; M: stable radical;
e: electron.
white and blue wheat genotypes (Li, Shan, Sun, Corke, & Beta, 2005).
Genetics, environment (location), growing conditions (moisture, fertilization, pests and
disease burden etc.), processing methods, and storage affect the level of antioxidant activity of
phytochemicals in fruits and vegetables (Blessington, 2005; Dewanto, Wu, Adom et al., 2002;
Reyes, Miller, & Cisneros-Zevallos, 2004). For example, highest intensity of skin and flesh
color in potatoes has reported to be in those grown in sandy soils (Burton, 1989). A significant
interaction between the antioxidant capacity and year-wise genotype of blueberry cultivars is
reported (Connor, Luby, Tong, Finn, & Hancock, 2002). Alsaikhan et al. (1995) found that the
phenolic content and antioxidative activity of four potato cultivars was genotype-dependent and
not related to flesh color.
25
Table 2. 3. Comparison of different in vitro total antioxidant capacity assays. Method ORAC TRAP FRAP TEAC TOSC Crocin DPPH
Reagents AAPH or
ABAP, Trolox,
Fluorescein
AAPH or ABAP,
Trolox,
R-phycoerythrin
TPTZ, FeCl3,
sodium acetate
ABTS, K2SO4,
Trolox
KMBA,
AAPH or
ABAP
Crocin, AAPH
or ABAP,
Trolox
DPPH
Instrument Fluorescent
plate reader
spectrometer
Fluorescent plate
reader
spectrometer
Spectro-
photometer
Spectro-
photometer
Head space
GC
Spectro-
photometer
Spectro-
photometer
Temperature 37 °C 37 °C 37 °C 37 °C 37 °C 37 °C 37 °C
Absorbance
(nm)
Excitation: 485
Emission: 520
Excitation: 495
Emission: 575
593 415 or 734 Depending on
detector type
443 515
pH 7.4 7.4 3.6 7.4 7.4 7.4
Endpoint End of
fluorescence
decay
Lag phase Fixed time
(4 – 10 min)
Fixed time
(4 – 6 min)
End of
production of
ethylene
Fixed time
(10 min)
% remaining
DPPH
Calibration Trolox solution Trolox solution Fe3+
standard
solution
Trolox solution - Trolox
solution
Trolox solution
Calculation of
Results
Area of the
fluorescence
curve decay
Length of the lag
phase
Absorbance of
final reading
minus blank
Absorbance
decrease in
presence of the
sample
Area under the
kinetic curve
Ka/Kc of
antioxidant
divided for
Ka/Kc of
Trolox
Absorbance
decrease in
presence of the
sample
Dimension of
results
µmol of Trolox
equivalent
µmol of Trolox
equivalent
Absorbance of
Fe2+
complex
produced by
antioxidant
reduction of
corresponding
tripyridyltriazine
Fe3+
complex
mM Trolox
equivalent to 1
mM test
substance
µmol of
Trolox
equivalent
% inhibition;
EC50; TEC50;
AE =
(1/ EC50)TEC50
ORAC (oxygen radical absorbance capacity); TRAP (total radical-trapping antioxidant parameter); TEAC (trolox equivalent antioxidant capacity);
FRAP (ferric reducing antioxidant power); DPPH (2, 2-diphenyl-1-picrylhydrazyl); TOSC (total oxidant scavenging capacity); ABTS (2,2'-azino-
bis(3-ethylbenzthiazoline-6-sulphonic acid)); AAPH or ABAP (Azo-bis(2-amidinopropane); TPTZ (2,4,6-tripyridyl-s-triazine); KMBA(α-keto-γ-
methiolbutyric acid); EC50 (concentration to decrease concentration of test free radical by 50%); TEC50 (time to decrease concentration of test free
radical by 50%);AE (antiradical efficiency). Adapted from Antolovich et al.(2002).
26
Figure 2. 7. Total phenols from daily consumption of fruits and vegetables in the American diet.
Adapted from Chun et al. (2005).
Selected and relatively unstable antioxidants of nutritional interest (e.g. ascorbic acid) have
been commonly assessed as indicators of processing damage (Miller, Diplock, & Riceevans,
1995). However, it was reported that vitamin C provides less than 0.4% of total antioxidant
activity in apples (Dewanto, Wu, & Liu, 2002) . Antioxidant activity is correlated with the
occurrence of polyphenols including anthocyanins (Eberhardt, Lee, & Liu, 2000; Prior et al.,
1998). Complex mixtures of phytochemicals in whole foods are responsible for their health
27
benefits and are better than single antioxidants due to a combination of additive and/or
synergistic effects (Eberhardt et al., 2000; Liu, 2002). Processed tomatoes and sweet corn exhibit
higher antioxidant activities than fresh ones due to the increased release of bound phenolic
compounds (Dewanto, Wu, Adom et al., 2002; Dewanto, Wu, & Liu, 2002) despite loss in
vitamin C content.
2.1. Effect of processing on phytochemicals
Processing of foods involve heating with different energy transfer media such as water, air,
oil and electromagnetic waves. In addition, storage can also be classified as passive processing
with no energy applied directly to foods (Figure 2.8). Polyphenolic compounds including
anthocyanins and proanthocyanidins are not completely stable during processing (Talcott,
Brenes, Pires, & Del Pozo-Insfran, 2003). Physical and biological factors such as temperature
increase and enzymatic activity may result in destruction of phenolics such as phenolic acids and
anthocyanins. After harvest these compounds can change during food processing and storage
(Kader, Irmouli, Nicolas, & Metche, 2002; Rossi et al., 2003), which may reduce related
bioactivity. During the processing of foods, various transformations of phenolics occur to
produce yellowish or brownish pigments (Clifford, 2000). Generally, food-processing procedures
are recognized as one of the major factors on the destruction or changes of natural
phytochemicals, which may affect the antioxidant capacity in foods (Nicoli, Anese, & Parpinel,
1999). But recent research has now established that food processing also has some positive
effects that improve the quality and health benefits of foods. These effects are mainly attributed
to the retained availability of some antioxidants. In addition, some compounds with potential
antioxidant activity after processing increases. For example, both hydrophilic and lipophilic-
28
Figure 2. 8. Heating/energy transfer medium in various processing methods.
ORAC values in cooked tomatoes are significantly higher than in their raw forms (Wu, X. et al.,
2004; Wu, X. L. et al., 2004). Significant increases in hydrophilic-ORAC but decreases in
lipophilic-ORAC values are reported in baked russet potatoes compared to raw ones (Wu, X. et
al., 2004; Wu, X. L. et al., 2004). In contrast, lipophilic and hydrophilic-ORAC values of raw
broccoli and carrots are significantly higher than that of cooked forms (Wu, X. et al., 2004; Wu,
X. L. et al., 2004). Interestingly, cooking in boiling water decreases the radical scavenging
activity of peppers, whereas microwave heating without water increase the activity (Chuah,
Yamaguchi, & Matoba, 2005). Antioxidant properties of grains are affected by processing
temperature during thermal processes (Dewanto, Wu, & Liu, 2002). Antioxidant activity of
cereals during processing is important as phenolics are localized in outer layers (husk, pericarp,
Food Boiling
Blanching
Pasteurization
Sterilization
Evaporation
Extrusion cooking
Roasting
Baking
Drying
Microwave heating
Irradiation
Pulsed electric field
Ultrasound
sonication
Processed Food
Water
Air
Electromagnetic
Waves
Shallow and
Deep Fat Frying
Oil
Storage
(air, vacuum, inert gas,
refrigeration, freezing)
29
testa, and aleurone cells) of the grain than in any other component. About 80% of the trans-
ferulic acid in rye and wheat grain was observed in the grain (White & Xing, 1997). Similarly,
freeze-dried fractions from durum wheat (Triticum durum) bran exhibit stronger antioxidant
activity than extracts from other milling fractions (Onyeneho & Hettiarachchy, 1992). Thus,
processing can alter antioxidant activity in both positive and negative ways. In the last decade,
excellent reviews by Nicoli et al. (1999), Klein & Kurilich (1993), and Kaur & Kapoor (2001)
provide brief information on the antioxidant activity as influenced by processing. Thus the
evaluation of processing factors influencing the antioxidant activity is imperative in optimizing
the conditions to increase or retain their activity and availability.
2.1.1. Extraction
Yields of phenolic contents from fruits and vegetables are affected by various extraction
solvents. Methanol has been reported to extract lower molecular weight phenolic compounds
compared to hexane and aqueous acetone in a fruit (Malus domestica) (Guyot, Marnet, Laraba,
Sanoner, & Drilleau, 1998). Extraction solvents have different effects on the peel, flesh and seed
of fruits. For example, phenolic content of pomegranate peel in methanol extract was more than
in ethanol extracts followed by water, whereas water extract of seeds contained high phenolic
content followed by methanol and ethanol extracts (Singh, Murthy, & Jayaprakasha, 2002). The
methanolic extract from pomegranate peel showed varied inhibition capacity to different
antioxidant assays such as 56, 58, and 93.7% in the thiobarbituric acid method, hydroxyl radical
scavenging activity and LDL oxidation, respectively (Singh et al., 2002). In another study,
acidified aqueous methanol effectively extracted more total phenolics and exhibited greater
antioxidant capacity from blueberries than acidified acetonitrile, or an acidified mixture of
methanol and acetone (Kalt et al., 2001). Among six extraction solvents (hexane, chloroform,
30
ethyl acetate, acetone, methanol and water), the methanolic and aqueous extracts of Decalepis
hamiltonee, a tuber, has higher antioxidant activity using DPPH, superoxide and hydroxyl
radicals, and inhibits microsomal lipid peroxidation and exhibits strong reducing power and
metal chelating activity (Srivastava, Harish, & Shivanandappa, 2006). However, the phenolics
content in the methanolic extract of Decalepis hamiltonee was higher than that of the aqueous
extract. Whole oats extracted with 80% methanol resulted in substantially higher levels of total
phenolic compounds and exhibit higher antioxidant capacity than water extracts (Zielinski &
Kozlowska, 2000), while both 80% methanol or ethanol were found to be efficient at extracting
phenolic compounds from barley (Bonoli, Verardo, Marconi, & Caboni, 2004).
Among eight solvent combinations involving ether, diethyl ether, petroleum ether,
chloroform, dichloroethane and methanol it was found that methanol extracts had greatest
antioxidant activity in oat groats and hulls (Duve & White, 1991). Comparison of various
aqueous ethanol, methanol or acetone mixtures showed more phenolics were extracted from
wheat flour and bran using successive acidified methanol/water (50:50, v/v, pH 2) and acetone/
water (70:30, v/v) than 70:30 (v/v) of either ethanol: water or methanol: water (Perez-Jimenez &
Saura-Calixto, 2005). Better extraction efficiency of methanol: HCl and ethanol: HCl could be
attributed to the polarity difference of acidic extracting solvents compared to methanol solvent
(Li, Pickard, & Beta, 2007). In contrast, 95% ethanol: HCl (85:15 v/v) has found to be more
efficient in total phenolic extraction than methanol: HCl (99:1 v/v) followed by 100% methanol
in purple wheat bran (Li et al., 2007). Comparison of four solvent systems including 50%
acetone (v/v), 70% methanol (v/v), 70% ethanol (v/v), and 100% ethanol, 50% acetone (v/v)
showed as a better solvent for extracting phenolic antioxidants from wheat (Zhou & Yu, 2004).
Application of a second component in addition to the extraction solvents also enhanced
31
extractability of phenolic antioxidants. Combination of supercritical carbon dioxide and ethanol
was found to extract greater antioxidant activity from marjoram than with either alone, or in
combination with hexane (Vagi et al., 2005). It was also observed that extraction of phenolic
compounds and antioxidant capacity from oat bran concentrate can be enhanced by microwave-
irradiating at 150 °C for 10 min in 50% ethanol (Stevenson et al., 2008).
2.1.2. Blanching
Blanching is an important processing step applied to soften the product as well as to
inactivate the enzymes that otherwise could cause browning or other possible reactions. The
blanching efficiency is determined by taking into account the complete inactivation of
peroxidase. The most commonly used method for thermal inactivation is heating by steam or hot
water, where the resistance to heat transfer at the surface is negligible compared to the internal
resistance to heat transfer (mechanism of heat penetration is by conduction). Thus, blanching
time depends on the dimension of food matrix. For example, blanching time for small size
products, such as peas, are 1-2 min, while for larger products, such as corn on the cob, is 11 min
(Feinberg, Winter, & Roth, 1968). The longer time required for the temperature rise at the cold
spot or slowest heating point, normally the geometric center, of a relatively large object such as
corn on the cob could damage the quality of the kernels, whereas shortening the length of
blanching time could reduce the degree of enzyme inactivation and thus result in shorter shelf-
life, nutritional and functional value. Thus, optimal blanching time is necessary for a particular
fruit or vegetable to preserve its overall nutritional and health promoting components.
A blanching time of 1 min in boiling water has been recommended for green leaves of sweet
potato to retain high antioxidant activity (Chu, Chang, & Hsu, 2000). Blanching opens up the
cell matrix and therefore, could increase the polyphenols yield during extraction that may either
32
enhance or reduce the antioxidant activity. Blueberry juice has higher recovery of phenolic
compounds and strong radical-scavenging activity to DPPH and hydroxyl radicals after steam
blanching for 3 min than the non-blanched juice (Rossi et al., 2003). In contrast, blanching in
water at 98°C for 2 min diminishes the antioxidant capacity of purple carrots (Uyan, Baysal,
Yurdagel, & El, 2004). Amin & Lee (2005) observed that even 5 – 10 min of blanching in hot
water at 98°C reduced (p < 0.05) antioxidant activities and phenolics content of all vegetables
except for cabbage and mustard cabbage. After 15 min of blanching, the loss of antioxidant
activity (β-carotene bleaching assay) was highest in Chinese cabbage (40%) followed by Chinese
white cabbage (19%), mustard cabbage (9%) and red cabbage (4%). However, the total phenolic
content of Chinese cabbage increased (p < 0.05) after 15 min of blanching compared with other
vegetables.
Mizrahi (1996) observed that 2 min of ohmic blanching of large whole vegetables had similar
effects as 4 min of water blanching. The investigator also reported that the energy dissipated by
the electric current passing through the samples was capable of heating it uniformly and very
quickly regardless of its shape or size. In another study, ohmic blanching (25–40 V/cm) of
artichoke by-product was faster at inactivating the peroxidase enzyme, without producing
blanching waste water compared to hot water blanching at 85°C, thus retaining higher total
phenolic content (Icier, 2010). The same investigator reported that ohmic blanching (40 V/cm) at
85 °C had similar peroxidase inactivation times (310 ± 2 s) as water blanching at 100 °C (300 ±
2 s). Increase in the mass transfer and effective diffusion rate has been reported after ohmic
blanching of strawberries during osmotic dehydration (Allali, Marchal, & Vorobiev, 2010).
Peeling of fruits and vegetables followed by blanching affects phenolic antioxidants. For
example, peach puree containing periderm tissue blanched in boiling water (20 min) and
33
pasteurized (boiling water for 30 min) was 7 – 11% higher in antioxidant activity (β-
carotene/linoleic acid assay) than peeled samples (Talcott, Howard, & Brenes, 2000b). Shorter
blanching of puree with periderm in boiling water (2 min) yielded the lowest initial antioxidant
activity, but the level of retention was greater during storage than that of peeled samples. The
same investigators also reported that total water-soluble phenolic compounds were higher in the
case of longer blanching time than that of shorter blanching time. That was due to increased
tissue softening and enhanced chemical extraction with the additional heat applied prior to
pasteurization.
2.1.3. Cooking
Cooking of vegetables, fruits and grains has a mixed effect on phenolic content and
antioxidant activity. Processing and heating during jam making (at 104 – 105 °C) decreases the
content of total phenolics of some varieties of cherries and plums, whereas no significant change
(p < 0.05) has observed in raspberries and in some varieties of cherries and plums (Kim &
Padilla-Zakour, 2004). However, the same study reported an increase or a decrease in antioxidant
activity (ABTS assay) of cherries, plums and raspberries depending on variety during jam
processing. Canning of raspberries and blueberries increases the phenolic content and antioxidant
activity by 50 and 53% respectively (Sablani et al., 2010). In contrast, the total phenolics has
decreased after dehydration of fresh plums, while antioxidant capacity in dried plums increased
compared to fresh plums (Piga, Del Caro, & Corda, 2003). During baking, the outer layer is
usually heated to over 120 °C, while the inner temperature remains lower than 95 – 100 °C. At
these temperatures, in addition to caramelization (between reducing sugars and ascorbic acid),
Maillard reaction (between reducing sugar and amino acids), Strecker degradation (dicarbonylic
compounds with amino acids), and hydrolysis of esters and glycosides of antioxidants, oxidation
34
of phenolic antioxidants to quinones and their polymers occur. Chemical or enzymatic oxidation
of polyphenols is generally responsible for their loss of antioxidant activity. However, baking of
purple wheat bran at 177 °C for 20 min has not altered the total phenolic content in the processed
samples (Li et al., 2007). By contrast, the total phenolics and total antioxidant activity of sweet
corn has increased by 54 and 44%, respectively, after thermal processing at 100 – 121 °C for 10
– 50 min (Dewanto, Wu, & Liu, 2002). In other studies, antioxidant activities in processed
tomatoes (Dewanto, Wu, Adom et al., 2002; Re, Bramley, & Rice-Evans, 2002) and coffee
(Nicoli, Anese, Parpinel, Franceschi, & Lerici, 1997) were retained or higher than their fresh
equivalents. The increase or retention of antioxidant activities in processed foods is attributed to
the development of new compounds with potential antioxidant capacity (Figure 2.9), although
the content of naturally occurring antioxidants has significantly decreased due to the heat
processing (Anese, Manzocco, Nicoli, & Lerici, 1999; Nicoli et al., 1999; Nicoli et al., 1997).
Antioxidant compounds depletion in thermally treated fruits and vegetables may also be
attributed to consumption of ascorbic acid and polyphenols as reactants in the Maillard reaction
(Kaanane, Kane, & Labuza, 1988). Although a decrease in the antioxidant potential is found for
short heat treatments, a recovery of these properties has been reported during prolonged heat
treatment. For example, Jiratanan & Liu (2004) observed 12% reduction in phenolic content of
the beets at initial application of heat (115 °C for 15 – 30 min), but further processing raised its
content back to the equivalent of unprocessed beets and eventually increased by 14% after
processing at 115 °C for 45 min. Similar results in the antioxidant capacity of aged citrus juice
and orange juice has been reported as they became more discolored (Lee, 1992). The antioxidant
activity of Maillard reaction products can be mainly attributed to the high molecular weight
brown compounds, which are formed in the advanced stages of reaction.
35
Figure 2. 9. Changes in total antioxidant activity in vegetable matrix subjected to different
processing conditions. Adapted and modified from Nicoli et al. (1999).
The initial reduction in the antioxidative activity can be attributed not only to thermal
degradation of naturally occurring antioxidants but also to formation of early MRPs with pro-
oxidant properties. The gain in antioxidant activity coincided with the formation of brown
MRPs.
Phenolic contents, including free and bound, and antioxidant activity during processing also
depend on the type of crop. Either increase or non-significant change in free, bound and total
phenolic content, total flavonoids, and total antioxidant activity of table beets were observed
during heat treatment at 105 – 125 °C for 15 – 45 min (Jiratanan & Liu, 2004). The same
investigators observed reductions in the antioxidant activity, phenolic contents and total
flavonoids (majority comes from free flavonoids) in green beans at similar processing conditions
100 – 121 °C for 10 – 40 min. The authors hypothesized that the processed beets and green beans
would contribute little to release of bound phytochemicals in the colon by intestinal microflora to
induce a site-specific reinforcement of antioxidants (Adom & Liu, 2002; Andreasen, Kroon,
Williamson, & Garcia-Conesa, 2001). After being heated at 90 °C for 147 h, the radical
Heating time
Anti
oxid
ant
acti
vit
y
Total antioxidant activity
Natural antioxidants
New antioxidants
36
scavenging capacity (DPPH) of the roselle pigment model system increased from 18% to 43%,
and FRAP decreased from 980 to 640 mol/l, while TEAC (ABTS assay) remained around 2.2
Trolox (mmol/l) (Tsai & Huang, 2004).
Phenolic antioxidant activity in-vitro sometimes has a different effect on the bioavailability
of the compounds. For example, although heat treatments including moderate heating (IQF and
freeze-drying), high temperature heating (cooking at 100 °C for 5 min, canning and spray-
drying) and jam preparation retain most of the phenolic content and antioxidant activity (FRAP
and DPPH) of wild or cultivated blueberries found in unprocessed whole fruit, processing
diminished antiproliferation activity on heap-1c1c7 cells (Schmidt, Erdman, & Lila, 2005). The
changes in the antiproliferation activity on heap-1c1c7 cells are thought to be the breakdown of
the active proanthocyanidin oligomers ranging from monomeric catechin units to hexamers in
blueberries (Kayano et al., 2003).
Microwave cooking of vegetables did not resulted in a specific trend on phenolic
antioxidants. Microwave cooking (with a power of 800 Watt) either retains or decreases a small
quantity of total phenolic content in cauliflower, peas, spinach and Swiss chard, whereas
significant reduction occurs after boiling (6 – 13 min) (Natella, Belelli, Ramberti, & Scaccini,
2010). The same investigators observed significant increase or retention of total antioxidant
capacity in cauliflower, peas, spinach, Swiss chard, potatoes, tomatoes and carrots. In another
study, researchers investigated the effect of microwave cooking on different radical scavenging
capacity (lipoperoxyl, hydroxyl and ABTS radicals) of vegetables (Jiménez-Monreal, García-
Diz, Martínez-Tomé, Mariscal, & Murcia, 2009). The researchers observed 30 – 50% losses of
lipoperoxyl radical scavenging capacity in most of the vegetables, whereas artichoke, asparagus,
garlic, onion, and spinach retained and eggplant, maize, pepper, and Swiss chard increased their
37
antioxidant activity after microwave cooking. Microwave cooked potatoes retained 55% of the
chlorogenic acids in the potatoes (cv NDA 1725) followed by boiled potatoes (35%), whereas
oven-baked potatoes lost all of them (Dao & Friedman, 1992). Commercially processed french-
fried potatoes, mashed potato flakes, and potato skins contained no chlorogenic acid in the same
study. The nature of the heat and their harshness in different cooking methods is attributed to
relative loss of chlorogenic acid in the potato. The total antioxidant activity of asparagus, using
ABTS assay, is significantly higher (P < 0.05) after refractance window and freeze-drying than
tray-drying, spouted bed, and combined microwave and spouted bed drying compared to raw
material (Nindo, Sun, Wang, Tang, & Powers, 2003). Similar results were observed in combined
microwave and hot air-drying of purple carrots (Uyan et al., 2004). By contrast, microwave
cooking of broccoli severely degrades phenolic content and antioxidant capacity in florets and
stems (Zhang & Hamauzu, 2004), with similar findings for potatoes (Tudela, Cantos, Espin,
Tomas-Barberan, & Gil, 2002). Studies on green vegetables and herbs found phenolic content
and antioxidant capacity increased or remained unchanged after microwave irradiation
(Turkmen, Sari, & Velioglu, 2005). Microwave heating of apple mash from 40 °C to 70 °C
results in an increase in phenolic and flavonoid compounds in the juice with increase in
temperature (Gerard & Roberts, 2004).
2.1.4. Drying/Dehydration
Saskatoon berries (‗Thiessen‘ and ‗Smoky‘ cv.) processed using freeze-drying, vacuum
microwave-drying, air-drying, and a combination of air-drying and vacuum microwave-drying
methods caused reduction (P < 0.05) in total phenolics, antioxidant activities and anthocyanin
contents as compared with fresh frozen berries (Kwok, Hu, Durance, & Kitts, 2004). However,
freeze-drying followed by vacuum microwave-drying render maximum antioxidant activity in
38
the berries. Similar results are observed during drying of cranberries using vacuum microwave,
freeze-drying and air-drying method. Vacuum microwave-drying produce dehydrated product
with higher ORAC antioxidant activity followed by freeze-drying and air-drying (Leusink, Kitts,
Yaghmaee, & Durance, 2010). However, the antioxidant activity (ABTS) of freeze dried samples
was higher than vacuum microwave and air dried samples. One possible explanation for this
discrepancy could be that freeze-drying degraded cranberry antioxidants that contribute to the lag
phase more than vacuum microwave-drying. During microwave-drying, water molecules
transmit and absorb energy because of volumetric heat generation in the wet sample resulting in
higher interior temperature that helps water rapidly reached its boiling point compared to other
convective drying methods (Khraisheh, Cooper, & Magee, 1995). Microwave-drying in
combination with vacuum also reduce the drying temperature, oxygen exposure, and heating
time because of enhanced penetration of heat that provides a constant internal temperature and
lasts until the final stage of drying has been reached (Oliveira & Franca, 2002).
Steaming and flaking oat groats has been reported to decrease tocotrienols, caffeic acid and
some avenanthramides, but increase ferulic acid and vanillin, whereas autoclaving whole grains
increases contents of tocopherols, tocotrienols, and acids of vanillin, ferulic and p-coumaric, but
degrade avenanthramides (Bryngelsson, Dimberg, & Kamal-Eldin, 2002). Drum-drying of whole
meal or rolled oats results in decreases in all tocols and phenolic compounds but
avenanthramides are unaffected (Bryngelsson et al., 2002).
2.1.5. Irradiation
The antioxidant activity decreases in kiwi fruits after treatment with 2 and 3 kGy compared
to the non-irradiated and 1kGy during storage (1 – 3 weeks) (Kim & Yook, 2009). The same
investigators reported that electron-donating ability (DPPH) decreased slightly during storage for
39
control samples; however, samples irradiated at1 and 2 kGy doses were not significantly
different throughout storage time. The total phenolic content and antioxidant capacity (FRAP) of
the irradiated (3 and 5 kGy) carrot juice are higher than that of the non-irradiated control,
whereas antioxidant activity of kale juice decreases after irradiation (5kGy) (Song et al., 2006).
Breitfellner, Solar, & Sontag (2002) studied the effect of gamma radiation (1 – 10 kGy) on
phenolic acids such as 4-hydroxybenzoic acid, gallic acid, cinnamic acid, p-coumaric acid, and
caffeic acid and their hydroxylation products in strawberry. The investigators observed a
significant increase in 4-hydroxybenzoic acid with the gamma radiation doses. Similarly, an
increase in the antioxidant activities due to irradiation was observed in fruit juice (2002) and
alfalfa sprouts (Fan & Thayer, 2001). Free radicals generated during irradiation act as stress
signals and may trigger stress-responses in lettuce (Fan, Toivonen, Rajkowski, & Sokorai, 2003)
resulting in increased antioxidant synthesis. Fan et al. (2003) hypothesized that irradiation
stimulates the synthesis of phenolics but not that of vitamin C, although both phenolics and
vitamin C are antioxidants. Gamma radiation of citrus fruit induces accumulation of 4-(3-methyl-
2-butenoxy) isonitrosoacetophenone that exhibits both antioxidant and antifungal activities
(Dubery, Louw, & van Heerden, 1999). In another study, seeds of kidney bean, cabbage and
beets exposed to ultraviolet (UV) irradiation (460-760 µW/cm2 for 30, 60 and 90 min)
stimulated synthesis of anthocyanins in leaves of kidney bean varieties (3-6%) and white beet
(14-21%) (Kacharava, Chanishvili, Badridze, Chkhubianishvili, & Janukashvili, 2009). In the
same study, low doses of irradiation were also effective (9-20%) in stimulating synthesis of
anthocyanins in cabbage and red beet. Free radicals produced by UV irradiation of seeds change
cell membrane permeability and electric potential, presumably initiating diverse metabolic
40
responses including biosynthesis of antioxidants in addition to some stress factors (Barka,
Kalantari, Makhlouf, & Arul, 2000).
2.1.6. Extrusion
Extrusion cooking increases the phenolic content of oats (Zielinski, Kozlowska, & Lewczuk,
2001), cauliflower by-products (Stojceska, Ainsworth, Plunkett, Ibanoglu, & Ibanoglu, 2008),
and increases the antioxidant activity in sweet potatoes (Shih, Kuo, & Chiang, 2009), cauliflower
by-products (Stojceska et al., 2008) and retains in the fruit powders from blueberries,
cranberries, concord grapes and raspberries (Camire, Dougherty, & Briggs, 2007). The increase
or retention of phenolic compounds and antioxidant activity of the extrudates is the consequence
of the high temperature, water-stress and wounding, (Reyes, Villarreal, & Cisneros-Zevallos,
2007) and partly accounted for the presence of the high molecular weight Maillard reaction
products, which are formed at higher temperatures and act as antioxidants. In contrast, reduction
in phenolic content has also been reported for extruded bean (19–21%), oat cereals (24–46%)
and oat extrudates (50%) (Korus, Gumul, & Czechowska, 2007; Viscidi, Dougherty, Briggs, &
Camire, 2004; Zadernowski, Nowak-Polakowska, & Rashed, 1999). Antioxidant activities
(DPPH) and total phenolics in barley extrudate samples was reduced by 60–68% and 46–60%,
respectively, compared with that of the unprocessed barley flour (Altan, McCarthy, & Maskan,
2009). Similar losses in antioxidant activity during extrusion cooking were observed in sorghum
(Dlamini, Taylor, & Rooney, 2007) and grass peas (Grela, Jensen, & Jakobsen, 1999). The loss
of natural antioxidants during extrusion over 80 °C has been attributed to their low resistance to
heat (Zadernowskl, Nowak-Polakowska, & Rashed, 1999), evaporation and decomposition
(Hamama & Nawar, 1991) at elevated temperatures. It was also reported that high temperature
during extrusion can alter molecular structure of phenolic compounds and either reduce their
41
chemical reactivity or decrease their extractability due to a certain degree of polymerization
(Alonso, Grant, Dewey, & Marzo, 2000) causing loss of antioxidant properties (Zadernowskl et
al., 1999). Interestingly, the effect of screw speed (co-rotating twin screw extruder) did not
follow a specific trend on the losses of antioxidant activity and total phenolics content of
extrudates from barley (Altan et al., 2009) and dry-mix of chick peas, corn, oats, carrot powder
and hazelnuts (Ozer, Herken, Guzel, Ainsworth, & Ibanoglu, 2006). The investigators
hypothesized that the increased shearing effects at increased speed were more dominant than the
effect of residence time on the destruction of antioxidant activity over the extrusion condition
even though increased screw speeds associates with decreased residence times.
2.1.7. Non-thermal processing
Effect of high pressure treatments on fruit and vegetable products are dependent on the food
matrix and processing parameters (pressure, time, and temperature), and antioxidant activity
assay methods (de Ancos, Gonzalez, & Cano, 2000; Fernandez-Garcia, Butz, Bognar, &
Tauscher, 2001; Fernandez-Garcia, Butz, & Tauscher, 2001; Sanchez-Moreno, De Ancos, Plaza,
Elez-Martinez, & Cano, 2009). Slight modification in the nutritional composition (vitamins C, A,
E, B1, B2, and folic acid), bioactive compounds and their antioxidant capacity (vitamins C, A
and E, carotenoid compounds, flavonoids) has been reported in purees (persimmons, tomatoes,
strawberries, kiwifruit), juices (lemon, orange, carrot, apple and broccoli) and a mix of vegetable
soup (gazpacho) (Donsi, Ferrari, & DiMatteo, 1996; Quaglia, Gravina, Paperi, & Paoletti, 1996;
Sanchez-Moreno et al., 2009).
Long time high pressurization (600 MPa/30 min) combined with thermal treatment (60 °C)
causes higher reduction in antioxidant capacity (linoleic acid/β-carotene assay) of freshly
squeezed apple juice than thermal treatment alone (60 °C/30 min) 25 and 10%, respectively,
42
compared to untreated samples (Sanchez-Moreno et al., 2009). Similarly, the antioxidant
capacity of tomato puree (DPPH) is significantly reduced after high pressure treatments (400
MPa/25 °C/15 min), although no difference is observed between pressurized tomato puree and
low and high pasteurized products (70 °C/30 s and 90 °C/1 min). In contrast, the antioxidant
capacity (ABTS) of fruit juices (orange, apple, peach, and citrus) and vegetable purees (carrots
and tomato) has not affected after high pressure treatment (600 MPa/20 °C/60 min) (Butz et al.,
2003). Similar results have been reported on the antioxidant capacity (DPPH) of freshly
squeezed orange juice with combined treatments of high pressure/temperature (100 MPa/60
°C/5min, 350 MPa/30 °C/2.5 min, 400 MPa/40 °C/1 min) (Plaza, Sanchez-Moreno, Elez-
Martinez et al., 2006; Sanchez-Moreno, Plaza, de Ancos, & Cano, 2003).
Pulsed electric field (PEF) treatment (35 kV/cm in bipolar mode, 800 Hz pulse frequency, 4
μs pulse width, 750 μs total treatment time, temperature ≤ 50 °C) of freshly squeezed orange
juice has not altered the contents of naringenin, hesperetin or total flavanone, whereas
pasteurization using heat only (90 °C/1 min) reduces naringenin content of squeezed orange juice
(Sanchez-Moreno et al., 2005). Similarly, apple juice pasteurized using a PEF treatment (35
kV/cm, bipolar pulses of 4 μs 1,200 pulses per second) has a 14.5% reduction in the total
phenolic content, whereas thermal pasteurization (90 °C, 30 s) reduced it by 32.2% (Aguillar-
Rosas, Ballinas-Casarrubias, Nevarez-Moorillon, Martin-Belloso, & Ortega-Rivas, 2007). Plaza,
Sanchez-Moreno, De Ancos, & Cano (2006) reported that a low pasteurization treatment (70 °C,
30 s) or a PEF treatment of 35 kV/cm for 750 μs (4-μs bipolar pulses, 800 Hz) did not
significantly modify the antioxidant activity of treated compared to untreated orange juice.
Ultraviolet (UV) rays induce an increase in enzymes responsible for the biosynthesis of
secondary metabolites such as flavonoids, which act as UV screens preventing UV-induced
43
damage in the genetic material of plant cells (Cantos, Garcia-Viguera, de Pascual-Teresa, &
Tomas-Berberan, 2000). Ultrasound (US) releases enzymes from cells for the secondary
metabolite biosynthesis due to mechanical stresses and microstreaming induced by acoustic
cavitations at low US intensity levels (Lin, Wu, Ho, & Qi, 2001). Prolonged US pre-treatment
combined with the same air-drying conditions decreases total phenolics, flavonoid contents and
the antioxidant capacity of dried apples (Opalic et al., 2009). The same investigator observed that
drying time had no significant effect on the contents of total phenolics, flavonoid or antioxidant
activity. However, the samples dried without the ultrasound pre-treatment was most sensory
acceptable. In contrast, ultraviolet (UV) rays and ultrasound increase the total phenolics, total
antioxidant capacity (TEAC and ORAC) of peanuts and US is more effective than UV in
increasing total antioxidants (Sales & Resurreccion, 2010).
2.1.8. Storage
The effects of storage on the polyphenols have been reported in apples (Price, Prosser,
Richetin, & Rhodes, 1999), broccoli (Price, Casuscelli, Colquhoun, & Rhodes, 1998), berries
(Hakkinen, Karenlampi, Mykkanen, & Torronen, 2000) and onions (Price, Bacon, & Rhodes,
1997). Hakkinen et al. (2000) observed that domestic processing and storage (–20 °C for 3 – 9
months) decreased myrecitin and kaempferol more than quercetin. The ORAC values of black
raspberries remained stable after IQF and throughout 3 months storage and increased by 18% at
6 months. The ORAC values of berries canned in syrup remained stable during storage, while
values for berries canned-in-water remained stable up to 1 month of storage and increased by
32% and 27% after 3 and 6 months storage. The ORAC values of puree, non-clarified and
clarified juices remained stable over the 6 months storage (Hager, Howard, Prior, &
Brownmiller, 2008). Similar results were observed in the storage of processed blackberries
44
(Hager, Howard, & Prior, 2008). During the storage (at 10 °C for 1 – 3 days) of irradiated (3 and
5 kGy) kale juice, the antioxidant capacity decreases in spite of increase in total phenolic content
(Song et al., 2006). However, the antioxidant activity and total phenolic content of carrots
increase during the same storage period. The lack of decrease in the antioxidant activity in
carrots is attributed to the presence of β-carotene and its synergistic effect with other antioxidants
to protect against oxidation. In contrast, the same investigators hypothesized that presence of
vitamin C in kale juice contributes more to its antioxidant activity, which decreases during
irradiation, than the irradiation-induced phenolic compounds (Wrona, Korytowski, Rózanowska,
Sarna, & Truscott, 2003; Yun-Zhong, Sheng, & Guoyao, 2002). The total flavonoids content
remained constant during storage in both air dried and fresh spinach stored in modified
atmosphere (Gil, Ferreres, & Tomas-Barberan, 1999). The ORAC and percent polymeric color
values of canned cherries increase after 5 months storage at 22 °C (Chaovanalikit & Wrolstad,
2004a). This suggests that polymeric compounds form during storage compensated for the loss
of antioxidant capacity due to degradation of monomeric anthocyanins. Friedman (1997)
observed greater polyphenol content at lower temperature storage of potatoes and attributed to
less polyphenol oxidase (PPO) activity at lower temperatures. The investigator further
hypothesized that enzymatic activity is indirectly related to monomeric polyphenol content,
which means greater the enzymatic activity, the greater the transformation of monomeric
polyphenols to polymeric ones. The higher PPO activity at the higher storage temperature may
account for the greater discoloration due to transformation of polyphenols to polymeric pigments
at that temperature. In another study, the DPPH radical scavenging ability in new mulberry wine
was 71% and increased after a year‘s storage to 78%. At the same time, the FRAP reducing
power decreased from 5720 m mol/L to 4630 m mol/L (Tsai, Huang, & Huang, 2004). These
45
changes support the hypotheses of Friedman (1997) on the conversion of monomeric
anthocyanins to the copigmented and polymeric forms during storage. Light has affect on the
phenolic acid content during storage. Chlorogenic acid levels of dark-stored potatoes increases,
but less compared to light-stored potatoes (Friedman, 1997; Griffiths, Bain, & Dale, 1995).
Gamma irradiation increases 0.21 units µg TE/g FW in antioxidant activity (DPPH) for each
day in storage, whereas 0.17 units in antioxidant activity (DPPH) with 1 unit increase of dosage
in potato (Atlantic cv). Increase in the phenolic content with storage has reported to be 1.98 units
per day and 0.16 units for 1 Gy increase in radiation (Blessington et al., 2007). The gain in
phenolic content may be due to dehydration, leading to concentration of solids at the end of the
storage period. Additionally, stimulation of synthesis of both antioxidants and polyphenols is
known to occur with stress, which may have increased at the end of the storage period due to
dehydration (Friedman, 1997; Kang & Saltveit, 2002). For example, the activity of PAL
(phenylalanine ammonia lyase), which produces a precursor to phenolic compounds, has been
reported to increase under stressful conditions, and this is associated with the accumulation and
synthesis of phenolic compounds (Kang & Saltveit, 2002).
Processing or storage can promote or enhance the stability and antioxidant properties of food
because of its complex matrix with lots of components mixed together in aqueous and lipid
phase. The antioxidant activity differs depending on measurement duration and mode,
temperature, oxygenation and medium in the model systems used. The behavior of antioxidants
also depends on the mediums such as bulk lipid, emulsified and aqueous, which is called the
polar paradox. According to the polar paradox, polar antioxidants are more effective in bulk
lipids, whereas non-polar antioxidants are effective in emulsified media (Cuvelier, Bondet, &
Berset, 2000; Porter, Levasseur, & Henick, 1977). The overall antioxidant activity would depend
46
on the redox reactions occurring between different natural antioxidants and lipid oxidation
products (Halliwell, Murcia, Chirico, & Aruoma, 1995; Mortensen & Skibsted, 1997; Namiki,
1990). For example, when a small amount of olive oil is mixed with tomato puree, ascorbic acid
decreased after a few hours of storage. This is because of the ability of ascorbate compounds to
reduce the radical forms compared to α-tocopherol contained in the lipid matrix (Nicoli et al.,
1999). The investigators hypothesized that the above mechanism is due to the lower standard
redox potential value of the ascorbyl radical forms of the α-tocopherol radical. The interaction
between the vegetable matrix and the lipid fraction becomes more evident when heated. Thus, in
addition to study the antioxidant properties of complex foods, it must be taken into account that
water-soluble antioxidants could protect lipid soluble antioxidants, because of the polar paradox
(Porter et al., 1977).
There is not a particular trend in the effect of non-thermal treatment and storage on the
antioxidant capacity of fruits and vegetables. During storage at 4 °C of treated orange juice (100
MPa/60 °C/5min, 350 MPa/30 °C/2.5 min, 400 MPa/40 °C/1 min), the antioxidant capacity was
not significantly different from freshly squeezed orange juice after 10 days, whereas a 17 – 23 %
decrease was reported after 40 days (Plaza, Sanchez-Moreno, Elez-Martinez et al., 2006;
Sanchez-Moreno et al., 2003). In contrast, no significant differences in antioxidant capacity
(ABTS) of treated orange juice were observed at more extreme conditions of 500 MPa and 800
MPa for 5 min at 20 °C and stored for 21 days at 4 °C (Fernandez-Garcia, Butz, Bognar et al.,
2001). In another study, there was no difference (P > 0.05) in the antioxidant capacity of
combined treated apple juice (600 MPa/60 °C/30 min) and non treated samples after 1 month
storage at 4 °C (Sanchez-Moreno et al., 2009). Antioxidant capacity of carrot and tomato juices
was unaltered after treatment (250 MPa/35 °C/15 min) and storage for 30 days at 4 °C (Dede,
47
Alpas, & Bayindirli, 2007). On the contrary, the antioxidant capacity of a tomato product known
as ―gazpacho‖ in Spain was not affected after high pressure treatments (150MPa/60◦C/15 min
and 350 MPa/60◦C/15 min) but decreased 39 – 46% after storage for 40 days at 4 °C (Plaza,
Sanchez-Moreno, De Ancos et al., 2006). However, reports on high pressure treatments for
whole fruits and vegetables are limited. Doblado, Frias, & Vidal-Valverde (2007) observed HPP
had no effect on antioxidant activity for broccoli, whereas a decrease in carrots and an increase
in green beans were observed when treated at 400 MPa/25 °C/2 min.
2.1.9. Enzymatic/Chemical Oxidation
Although chemical or enzymatic oxidations have been widely proven to cause a progressive
decrease in polyphenol antioxidant properties, processing or prolonged storage times can
promote or enhance the rate oxidation of phenolic compounds depending on intrinsic properties
of the food matrix as well as on processing conditions such as water activity, pH, time,
temperature, and oxygen availability (Nicoli et al., 1999). For example, thermal processing and
storage at high temperature (40 °C for 4 weeks) significantly increase the antioxidant activity of
carrot puree depending on the concentration and oxidation of the phenolic acids (Talcott,
Howard, & Brenes, 2000a). Similarly, the antioxidant properties of pasteurized (105 °C for 20
min) and air-bottled tea extracts increased during 30 day storage at 20 °C (Manzocco, Anese, &
Nicoli, 1998). The same investigators further observed that green tea extracts showed higher
phenol content and chain-breaking activity than those obtained from black tea leaves.
Additionally, reduction in the oxygen-scavenging ability of tea extracts after pasteurization and
storage is correlated with a progressive increase in the redox potential value for black tea extracts
indicating the gain in radical scavenging activity is associated with a corresponding decrease in
the reducing power of the tea extracts. The modifications in chain-breaking activity and oxygen
48
uptake detected in tea extracts is ascribable to the progressive oxidation of polyphenols, a
process leading to the formation of macromolecular compounds with stronger radical scavenging
power. Catechin when subjected to a progressive enzymatic oxidation shows a remarkable
increase in its chain breaking activity prior to the formation of brown macromolecular
compounds, whereas a subsequent loss in the antioxidant properties has found for advanced
enzymatic oxidation steps (Cheigh, Um, & Lee, 1995). Enzymatic oxidation of the polyphenol
fraction causes a decrease in the chain-breaking activity, whereas chemical oxidation has found
to have the opposite effect (Manzocco et al., 1998; Nicoli et al., 1999). The above mechanism is
explained as different pathways of enzymatic and chemical oxidation to polyphenols, which
leads to the formation of compounds having markedly contrasting radical scavenging capacities.
The investigators hypothesized that chemical proceeds much slower than enzymatic oxidation
and the compounds formed during pasteurization and storage would have an intermediate
oxidation status when compared to those formed by enzymatic oxidation. Since the oxidation
products of phenolic compounds still retain antioxidant activity (Yen, Chen, & Peng, 1997), the
increased stability of partially oxidized polyphenols gained in chain-breaking efficiency during
the processing of the beverages. Thus, polyphenols with an intermediate oxidation state exhibit
higher radical scavenging efficiency than non-oxidized ones. The higher antioxidant properties
of partially oxidized polyphenols is attributed to their increased ability to donate a hydrogen
atom from the aromatic hydroxyl group to a free radical and/or to the capacity of their aromatic
structures to support the unpaired electron through delocalization around the π- electron system
(Nicoli et al., 1999).
49
3. ANTHOCYANINS
Anthocyanins are water-soluble pigments responsible for the orange red through deep purple
colors produced by chemical combination of its C6-C3-C6 structure with glycosides, acyl
groups, and other molecules in flowers, fruits and vegetables (Figure 2.10). Mostly,
anthocyanins occur as glycosides such as 3-monoglycosides and 3, 5-diglycosides in nature.
Table 2.4 shows common anthocyanins present in fruits, vegetable and grains. In addition, many
anthocyanins have acylations (i.e. ester bonds between sugars and organic acids) with coumaric,
caffeic, ferulic, p-hydroxybenzoic, synapic, malonic, acetic, succinic, oxalic, and malic acids
(Francis, 1989). Substitution of hydroxyl and methoxyl groups influences the color of the
anthocyanins. For example, more hydroxyl groups provide bluish color, whereas methoxyl
groups increase the redness of the anthocyanins. Anthocyanins are present in the vacuole of the
plant cell and the molecules are protected by unique mechanisms such as self-association and
copigmentation in the cell. Self-association is to form helical stacks through the hydrophobic
attraction and hydrogen bonding between the flavylium nuclei. The stacking protects the
chromophores behind the sugar groups from the hydration reaction (Hoshino, Matsumoto, &
Goto, 1981). Copigmentation occurs through hydrogen bonding of the phenolic groups between
anthocyanin and flavone molecules (Francis, 1989). Flavonols, amino acids, benzoic acids,
coumaric and cinnamic acids also reacts with anthocyanins due to interaction known as
intermolecular copigmentation. Intramolecular copigmentation occurs in the anthocyanins
because of the acylation by organic acids. Acylation of the molecule is believed to improve
anthocyanin stability by protecting it from hydration (Patras, Brunton, O'Donnell, & Tiwari,
2010). A spectral characteristic of glycosylation and acylation in potato anthocyanin is shown in
Figure 2.11.
50
O+
OH
OH
OH
O G
Pelargonidin-3-glucoside
O+
OH
OH
OH
O G
OH
Cyanidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
OCH3
Malvidin-3-glucoside
O+
OH
OH
OH
O G
OH
OH
Delphidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
Petunidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
Peonidin-3-glucoside
OH
Figure 2. 10. Common anthocyanins found in flowers, fruits and vegetables.
Antioxidant activity of anthocyanins in-vitro and in-vivo has been well documented (Prior,
2003). Antioxidant activity of the anthocyanins depends on the number of free hydroxyl groups
attached to the structure. For example, petunidin glucoside (Figure 2. 10) in purple potatoes has
high antioxidant activity compared to others. However, total antioxidant activity depends on the
amount of phenolic acid and anthocyanins in the tuber (Hamouz, Lachman, Vokal, & Pivec,
1999; Lachman, Hamouz, Orsak, & Pivec, 2000). The same investigators also reported that
purple /blue coloration of the purple potato is due to acylation of cinnamic acid to anthocyanidin
giving high antioxidant activities, whereas glycosidic attachment at position 3 and 5 reduces
antioxidant activities. In another study, Wang, Cao, & Prior (1997) reported that cyanidin
glycosides tend to have higher antioxidant capacity than peonidin or malvidin glycosides because
of the free hydroxyl groups on the 3‘ and 4‘ positions.
Anthocyanins have many applications for regulatory authorities to check adulteration as well
51
as in food industries. For example, prune juice adulterated with other fruit juices will show
increased levels of anthocyanins compared to prune juice alone (Van Gorsel, Li, Kerbel, Smits,
& Kader, 1992). Adulteration of blackberry jams with strawberries can be detected with the
profile analysis of pelargonidin and cyanidin-3-glucoside (Garcıia-Viguera, Zafrilla, & Tomás-
Barberán, 1997). In red raspberry juices, it was found that under-processed samples had higher
levels of polymeric color instead of the monomeric anthocyanin pigments, which is considered
as a poor quality product (Altamirano, Drdak, Simon, Smelik, & Simko, 1992). Other important
applications include fungicidal properties of anthocyanins that might block potato blight from
reaching tubers underground (Lachman et al., 2005). Cultivation of blueberries (Camire,
Chaovanalikit, Dougherty, & Briggs, 2002), grape (Camire et al., 2002), red and purple fleshed
potatoes give alternative sources of natural colorants from their rich anthocyanins (Lachman et
al., 2005; Rodriguez-Saona, Giusti, & Wrolstad, 1998). Potato peel powders have high
antioxidant activities (Singh & Rajini, 2004) and extracts from peels could be used with oils, fats
and other food products to suppress lipid oxidation (Rehman, Habib, & Shah, 2004). However,
lack of comprehensive knowledge on various factors affecting the stability of anthocyanins limits
their use.
The factors affecting the stability and interrelationship between the quality and quantity of
the anthocyanins have been reported in plants or food systems (Figure 2.12) (Jackman et al.,
1987). The stability of anthocyanins has also been related to the presence of number of hydroxyl
groups and methoxyl groups attributed to the flavylium structure besides glycosylation (Francis,
1989). The investigator also reported that diglucosides are more stable than monoglucosides.
However, presence of more sugar molecules in the diglucosides accelerates browning in the food
matrix. In another thermal degradation of anthocyanin study, Tanchev & Yoncheva (1974)
52
Table 2. 4. Common anthocyanins present in fruits and vegetables. Fruit/veg/grain Major anthocyanin Minor anthocyanins Reference
Blackberry Cyanidin-3-glucoside Cyanidin-3-rutinoside; Cyanidin-3-dioxalylglucoside;
Cyanidin-3-xyloside Cyanidin-3-malonylglucoside
Fan-Chiang & Wrolstad (2005);
Rommel, Wrolstad, & Heatherbell
(1992)
Strawberries Pelargonidin-3-glucoside Cyanidin-3-glucoside Jackman et al. (1987)
Litchi pericarp Cyanidin-3-rutinoside Malvidin-3-acetylglucoside;
Quercetin-3-rutinoside; Cyanidin glucoside; Quercetin
glucoside
Lee & Wicker (1991); Sarni-
Manchado, Le Roux, Le Guerneve,
Lozano, & Cheynier (2000)
Blood orange Cyanidin-3-glucoside Delphinidin-3,5-diglucoside; Cyanidin-3,5-diglucoside;
Delphinidin-3-glucoside; Peonidin-3,5-diglucoside; Cyanidin-
3-(acetyl)-glucoside; Cyanidin-3-(feruloyl)-glucoside
Krifi et al. (2000)
Acai Cyanidin-3-glucoside Pelargonidin-3-glycoside Del Pozo-Insfran et al. (2004)
Bilberry Cyanidin galactoside Delphidin arabinoside; Cyanidin glucoside; Delphidin
galactoside; Delphidin glucoside; Petunidin glucoside;
Malvidin galactoside; Cyanidin arabinoside; Peonidin
glucoside; Malvidin arabinoside
Yue & Xu (2008)
Elderberry Cyanidin-3-sambubioside Cyanidin-3-glucoside;
Cyanidin 3,5-diglucoside;
Cyanidin-3-sambubioside-5-glucoside
Stintzing, Stintzing, Carle, Frei, &
Wrolstad (2002)
Red radish Pelargonidin-3-sophoroside-5-
glucoside
Matsufuji et al. (2007)
Black carrot Cyanidin-3-xylosylgalactoside Cyanidin-3-xylosylglucosyl-galactoside Stintzing et al. (2002)
Cherry Cyanidin-3- glucosyl-rutinoside Cyanindin-3-glucoside; Cyanidin-3-rutinoside; Peonidin-3-
rutinoside
Kim & Padilla-Zakour (2004)
Purple carrot Cyanidin 3-(sinapoylxylosyl-
glucosylgalactoside)
Malvidin 3-monoglucoside;
Peonidin 3-monoglucoside
Glassgen, Wray, Strack, Metzger,
& Seitz (1992)
Red cabbage Cyanidin-3-sophoroside-5-
glucoside
Cyanidin-3,5-diglucoside Giusti, Rodriguez-Saona, Griffin,
& Wrolstad (1999)
Purple-fleshed
potato
Petunidin-3-(p-coumaroyl-
rutinoside)-5-glucoside
Malvidin-3-(p-coumaroyl-rutinoside)-5-glucoside Lewis, Walker, Lancaster, &
Sutton (1998)
Red-fleshed
potato
Pelagonidin-3-(p-coumaroyl-
rutinoside)-5-glucoside
Peonidin-3-(p-coumaroyl-
rutinoside)-5-glucoside
Lewis et al. (1998)
53
Figure 2. 11. Spectral characteristics of potato anthocyanins, indication of glycosylation and
acylation patterns. Modified from Rodriguez-Saona, Giusti, & Wrolstad (1998).
reported that the existence of three hydroxyl groups leads to more rapid degradation of
delphidin-3-rutinoside than malvidin-3-glucoside. Sadilova, Stintzing, & Carle (2006) reported
that methoxylation of the acyl moiety improves the structural integrity towards heat. The
equilibrium between the colored cationic form and colorless pseudobase and degradation is
directly influenced by pH (Figure 2.13). Anthocyanins are stable at pH (< 4) and become
colorless at high pH (Francis, 1989). In slightly alkaline solutions (pH 8 to 10) highly colored
ionized anhydro bases are formed. At pH 12, these ionized anhydro bases hydrolyze rapidly to
fully ionized chalcones (Bridle & Timberlake, 1997). Acylated anthocyanins retain more color at
54
the higher pH than non-acylated anthocyanins in addition to their resistance to heat, light and
SO2. Giusti & Wrolstad (1996a) observed that the C-5 acylation of anthocyanins in red radish
provide high stability to the compounds. In another study on Vitis vinifera wines, substitution of
vitisins at C-4 position of anthocyanin structure provided resistance to color loss with SO2, at
higher pH values (Bakker & Timberlake, 1997). Acylation of anthocyanins with ferulic, sinapic
Figure 2. 12. Interrelationships between anthocyanin quantity and quality, and various factors
affecting the stability of anthocyanins. Adapted from Jackman et al.(1987).
and coumaric acids in black carrots prolongs their half-life compared with non-acylated
derivatives from strawberry and elderberry isolates (Sadilova, Carle, & Stintzing, 2007).
Acylation has not been shown to increase the stability of anthocyanins in some model systems.
For example, anthocyanins from grape, red cabbage and Ajuga reptans in pH 3.5 citrate buffer
solutions has not showed any difference in their degradation rate in spite of their different
Quantity of
anthocyanin
pigment
Quality of anthocyanin
B-ring structrure
Glycosidic substitution
Acylation
Hue
pKA
Rate of reactivity
Reaction with other
compounds
Leucoanthocyanins
Flavonols
Acetaldehyde
Acid
Polyphenoloxidase
Ascorbic acid
Proteins
Oxygen
Hydrogen peroxide
Metal ions
Sulfur dioxide
Browning reactions
Maillard
Enzymatic
Ascorbic acid
55
+H+ OCH3
O+
OH
OH
OH
O G
OCH3
OCH3
OCH3
O+
OH
OH
OH
OH
OCH3
O
OH
OH
OH
OH
OH
OCH3
OCH3
O
OH
OH
O G
OCH3
O
OCH3
OCH3
O
O
OH
OH
O
OH
OH
OCH3
OCH3
OH
O
OH
OH
OHO
O
H2OH2O
OCH3
OH
HOOC
OCH3
OH
OH OH
CHO +
PhloroglucinaldehydeProtocatechuic acid
Malvidin-3-glucoside (blue)Malvidin-3-glucoside (red)
Quinoidal base or Anhydrobase
Deglycosylation
Malvidin
- Diketone
Figure 2. 13. Effect of pH on the possible degradation mechanism of anthocyanin (Malvidin-3-
glucoside). Adapted from Wong (1989).
56
chemical configurations (Baublis, Spomer, & Berberjimenez, 1994). Grape anthocyanins include
mono and diglucosides of five different monoacylated aglycones (Lea, 1988), red cabbage
contains diacylated triglucosides of cyanidin (Mazza & Miniati, 1993), and Ajuga reptans
contains glucosylated cyanidin, acylated with p-hydroxycinnamic acid, ferulic acid, and malonic
acid (Callebaut, Hendrickx, Voets, & Motte, 1990). Similar findings have been reported on the
acylated aglycones of pelargonidin from strawberry juice or concentrate (Garzon & Wrolstad,
2002) and a model system (Garzón & Wrolstad, 2001).
3.1. Effect of processing on Anthocyanins
Processing can change the anthocyanin content and color in fruits and vegetables. The overall
color changes may be due to a number of factors such as pigment degradation, pigment
polymerization, reactions with other components of the formulation, nonenzymatic browning,
oxidation of tannins, and other reactions completely unrelated to the added colorant (Francis,
1989).
3.1.1. Extraction
Solvents to be used for extraction of anthocyanin pigments from fruits and vegetables
without disturbing the acylation, structure of anthocyanin and interfering with unwanted
compounds are reviewed in the literature (Jackman et al., 1987). Extraction procedures have
generally involved the use of acidic solvents, which denature the membranes of cell tissue and
simultaneously dissolve the pigments followed by ether precipitation. Acidified methanol
(HCl/methanol) has been the most commonly used extraction solvent, where HCl maintains low
pH for favoring the formation of flavylium chloride salt and methanol allows easier
concentration of extracted material. Lipid materials and unwanted polyphenols can be separated
57
by using petroleum ether, diethyl ether or ethyl acetate. Many researchers have suggested using
alcohol with very low acid concentration or without acid to minimize the possible changes to the
acylated pigments during extraction (Jackman et al., 1987). In addition, use of neutral solvents
such as 60 % methanol, acetone/methanol/water mixtures, cold acetone, n-butanol or boiling
water has also been recommended (Jackman et al., 1987). However, application of external
technologies in addition to solvents for extraction of anthocyanin pigments has not been
discussed in detail. Optimal extraction of anthocyanins from grape skin was reported to be
carried out by formic acid/methanol solvent (5/95, v/v), with ultrasonication for 10 min,
extraction temperature at 25 °C, and extraction time for 1.5 hr (Li, Pan, Cui, & Duan, 2010). In
another study, yield of anthocyanins from red cabbage using high pressure CO2 (HPCD)
increased compared to conventional acidified water (CAW) (Xu et al., 2010). Fractionated high
pressure extractions using CO2 supercritical fluid extraction followed by CO2/ethanol-H2O
mixtures (1 – 100%, v/v) at 313 K and 20 MPa extracted higher anthocyanin contents from dry
and raw elderberry pomace. Presence of water both in the raw material and in the solvent
mixture accelerates higher extraction of anthocyanin from elderberry pomace (Seabra, Braga,
Batista, & de Sousa, 2010).
3.1.2. Blanching
Anthocyanins are degraded by a number of enzymes found in plant tissue such as
glycosidases, polyphenoloxidases (PPO), and peroxidases. Glycosidases produce anthocyanidins
and sugars, and anthocyanidins are very unstable and rapidly degraded. PPO catalyzes the
oxidation of o-dihydrophenols to o-quinones that further react to brown polymers (Figure 2.14).
Kader, Irmouli, Zitouni, Nicolas, & Metche (1999) proposed that Cyanidin 3-glucoside (o-
diphenolic) is degraded by a mechanism of coupled oxidation involving the enzymatically
58
generated o-quinone with partial regeneration of the o-diphenolic co-substrate confirming the
role of PPO in anthocyanin degradation. In another study, pelargonidin-3-glucoside, were
degraded by a mechanism involving a reaction between the o-quinone and/or secondary products
of oxidation formed from the quinone and the anthocyanin pigment (Kader, Irmouli, Nicolas, &
Metche, 2001). The destruction of anthocyanins by enzymatic activity could be important in the
design of an extraction procedure and perhaps in the final formulation in a food (Francis, 1989;
Rossi et al., 2003). Blanching of fruits and vegetables inactivate enzymes and improves the color
by retaining anthocyanins in the processed foods. Addition of 30 ppm SO2 inhibits phenolase
activity in sour cherry juice (Cemeroglu, Velioglu, & Isik, 1994). Juice prepared from blanched
blueberry-pulp extract degrades anthocyanins, whereas unblanched extract cause 50% loss of
anthocyanins (Skrede, Wrolstad, & Durst, 2000). Similarly, blanching (steam for 3 min) of
blueberry fruits induced higher anthocyanin retention i.e. 23% instead of 12% (unblanched)
when processed into juice (Rossi et al., 2003). Anthocyanin content of blanched (98 °C for 2
min) purple carrots increase 27% compared with the fresh sample (Uyan et al., 2004). Increase in
the anthocyanin retention in fruits is attributed to two main factors; (1) reduction of enzyme
mediated anthocyanin degradation i.e. complete inactivation of native PPO, and (2) greater
extraction yield linked to the increase of fruit skin permeability caused by the heat treatment
(Kalt, McDonald, & Donner, 2000). Introduction of the blanching step has also a positive effect
on the recovery of individual anthocyanin. For example, in blueberry juice processing, the
percent recovery increase by 71 – 2672 % for monoglucosides of cyanidin, malvidin, petunidin,
peonidin and delphidin (Rossi et al., 2003). Heat inactivation by blanching (boiling water for 10
sec) retain or increase anthocyanin content of apple peels after oven-drying (Wolfe & Liu, 2003).
Similarly, steam blanching of purple- and red-fleshed potatoes reduces the peroxidase activity
59
OH
OH
Enzyme
O2 + OH2 +
O
O
O-diphenol O-benzoquinone
O
O
O-benzoquinone
+ AnthocyaninOxidized
anthocyanin+
Degradation
Product
Figure 2. 14. Enzymatic activity on the polyphenol. Adapted from Wong (1989).
by 98 – 99% to retain the anthocyanins (Reyes & Cisneros-Zevallos, 2007). Interestingly, heat
and SO2 treatments of high bush blueberries increase the recovery of anthocyanins but not of
other polyphenols in the juices (Lee, Durst, & Wrolstad, 2002). Blanching (95 °C for 3 min) in
combination with pasteurization during preparation of blueberry purees reduce 43% total
monomeric anthocyanins compared to fresh fruit (Brownmiller, Howard, & Prior, 2008). Volden
et al. (2008) observed 59% loss in anthocyanin content of red cabbage after blanching.
3.1.3. Heating
At high temperatures, the structure of anthocyanin is opened to form chalcone, which is
degraded further to brown products (Francis, 1989). However, it has been observed that optimal
conditions permit the regaining of color on cooling if there is sufficient time (several hours) for
the reconversion. Processing of black raspberries in canned-in-water or syrup (87.8 – 93.3 °C for
4 min) losses 42 and 51% of total anthocyanin, respectively (Hager, A. et al., 2008), whereas
blackberry products losses 17.8 and 10.5%, respectively, in similar condition (Hager, T. J. et al.,
2008). In another study, only 50% of the total anthocyanin content of elderberry was lost after 3
60
h of heating 95 °C (Sadilova et al., 2006). Degradation of anthocyanins occurs in strawberry
(Garzon & Wrolstad, 2002), raspberry (Kim & Padilla-Zakour, 2004) and sour cherry
(Cemeroglu et al., 1994) as the fruits are processed into juice, concentrate or jam and continues
during storage. The investigators also reported that the degradation of anthocyanins in
concentrates was greater compared to juices. Patras, Brunton, Da Pieve, & Butler (2009)
reported significant loss (P < 0.05) in anthocyanin content of blackberry (3%) and strawberry
(28%) puree processed at 70 °C for 2 min. Anthocyanin degradation in processed berry products
is attributed to indirect oxidation by phenolic quinones generated by polyphenol oxidase and
peroxidase (Kader, Rovel, Girardin, & Metche, 1997; Skrede et al., 2000). In contrast,
anthocyanins in Bing cherries increase slightly after canning (100 °C for 12 min) (Chaovanalikit
& Wrolstad, 2004b). The increase in the anthocyanin content is attributed to the increased
extraction efficiency in the softened fruits. Increase in membrane permeability at high
temperatures in the macerated peel tissue facilitate phenolic extraction (Spanos, Wrolstad, &
Heatherbell, 1990) and release of bound phenolic compounds by breakdown of the cellular
constituents was also reported in the literature (Dewanto, Wu, Adom et al., 2002).
Boiling and steaming cause 41% and 29% losses in anthocyanin content of red cabbage,
respectively (Volden et al., 2008). Similar losses were reported in blueberry products (Lee et al.,
2002). In contrast, Kirca, Ozkan, & Cemeroglu (2007) reported that anthocyanins from black
carrots were reasonably stable during heating at 70 – 80 °C due to di-acylation of anthocyanin
structure. According to the same investigators, the stability of monomeric anthocyanins in black
carrot juice and concentrates also depends on temperature, solids content and pH. Similarly,
cyanidin-3-rutinoside has the highest stability to the effect of thermal treatment at 95 °C in
blackcurrants (Rubinskiene, Viskelis, Jasutiene, Viskeliene, & Bobinas, 2005). Greater thermal
61
stability (25 – 80 °C) of anthocyanins present in red cabbage compared to blackcurrants, grape
skins and elderberries in a soft drink model system is attributed to the protection of flavylium
system through co-pigmentation (Dyrby, Westergaard, & Stapelfeldt, 2001). In another study,
purple-fleshed potato and grape extracts showed lower color stability than red-fleshed potatoes
and purple carrots at 98 °C (Reyes & Cisneros-Zevallos, 2007). Maccarone, Maccarrone, &
Rapisarda (1985) studied the stabilization of anthocyanins in blood orange juice and found that
microwave pasteurization and addition of tartaric acid and glutathione improved the stability.
The investigators reported that complexation of anthocyanins with rutin and caffeic acid
provided the highest stability.
Extrusion cooking of corn meal with grape juice and blueberry concentrate at a die
temperature of 130 °C degrades 74% of anthocyanin compared to the unheated formulation
(Camire et al., 2002). Similarly, extrusion cooking of corn meal with dehydrated fruit powder
from blueberry, cranberry, concord grape and raspberry at the similar die temperature reduce up
to 90% of anthocyanin content for all the dehydrated fruits used except raspberries (Camire et
al., 2007). There is no difference (P > 0.05) observed in the total anthocyanin content of purple
wheat bran heated at 177 °C for 20 min compared to unheated bran (Li et al., 2007). However,
baking of muffins prepared from purple wheat bran with other ingredients at a similar
temperature for 7 – 12 min has not retained any anthocyanin.
3.1.4. Drying/Dehydration
Depending on the severity of heat application, dehydration reduces moisture content of fruits
and vegetables, which is important to maintaining the equilibrium media for stability of
anthocyanins. The rate of evaporation of water might have influence on the water soluble
anthocyanin pigments. Air-drying and freeze-drying increase the anthocyanin content of apple
62
peels, whereas oven-drying has similar amount compared to fresh peels (Wolfe & Liu, 2003).
Interestingly, the blanched freeze dried peels contain more anthocyanins than the fresh apple
peels in the same study (Wolfe & Liu, 2003). The retention of total anthocyanins in Saskatoon
berries is higher (~ 30%) when dried by combining the air with microwave vacuum method than
with air-drying (15%) compare to fresh berries (Kwok et al., 2004). Better retention of
anthocyanin in berries is attributed to the reduced time of microwave vacuum-drying. The same
investigators reported that the combination of heat and atmospheric oxygen in air-drying favored
enzymatic browning activity of polyphenol oxidase, whereas less enzymatic browning was
observed for vacuum microwave-drying, because of reduced heat and oxygen exposure. Similar
results were observed during drying of cranberry using microwave vacuum, freeze-drying and
air-drying methods. Microwave vacuum-drying and freeze-drying produce dehydrated products
that contain a relatively higher level of total anthocyanins per gram dry solid compared to air-
drying (Leusink et al., 2010). Uyan et al. (2004) observed a 2-fold increase in anthocyanin
content during hot air dehydration of purple carrots, whereas combining microwave and hot air-
drying increased it 1.75-fold. However, jam processing (105 °C) reduces (P < 0.05) 21 – 89%
anthocyanin contents of cherries, plums and raspberries (Kim & Padilla-Zakour, 2004). Ersus &
Yurdagel (2007) studied microencapsulation of black carrot anthocyanins by spray-drying
(drying air inlet: 160 – 200 °C; outlet: 107 – 131 °C) using different maltodextrins as carrier and
coating agents. The investigators observed an increase in anthocyanin contents of powders (7.9 –
36.83%) with different maltodextrins at a constant air inlet with varying outlet temperatures,
whereas higher inlet temperatures (>160 – 180 °C) caused more anthocyanin losses.
63
3.1.5. CO2 treatment
Reduction in red color intensity of the internal tissue in some fruits (Kader, 1986) and change
in external color from red to red-purple in strawberries with CO2 treatment (Ke, Goldstein,
Omahony, & Kader, 1991) has been reported. No significant difference (P > 0.05) in external
anthocyanin content of fresh strawberries stored under CO2 treated atmosphere compared to the
initial fruit, whereas internal anthocyanin content decreases significantly (P < 0.05)(Gil,
Holcroft, & Kader, 1997). The discrepancy of opposite behavior of anthocyanin in external and
internal tissues is attributed to higher concentrations of cyanidin 3-glucoside in external tissue
providing higher color stability, while pelargonidin glycosides are present in the internal tissue.
In addition, the accumulation of phenolic compounds was greater in external tissue which
provides more stability to the color.
3.1.6. Addition of external ingredients
Methods including addition of external ingredients have been proposed to retain
anthocyanins to reach higher color intensity. In frozen strawberries, it is shown that sugar
addition has a stabilizing effect on the total monomeric anthocyanins, enhancing the shelf life of
colored products (Wrolstad, Skrede, Lea, & Enersen, 1990). Similarly, litchi is a tropical fruit
and within 2 to 3 days after harvest its pericarp becomes desiccated and turns brown. To reduce
the color degradation, litchis are coated with chitosan (1.0 to 2.0%) and stored (4 ºC/90% relative
humidity). The use of chitosan delay changes of contents of anthocyanin, flavonoid, and total
phenolics. Interestingly, the activities of polyphenol oxidase and peroxidase, which have been
involved in anthocyanin degradation, are inhibited (Zhang & Quantick, 1997). The same
investigators also suggested that a plastic coating forms a protective barrier on the surface of the
64
fruit and reduces the supply of oxygen for enzymatic oxidation of phenolics.
3.1.6.1. Effect of light
In general, it is considered that light has deleterious effects on anthocyanin stability and
exposure of natural colored beverages to light must be avoided. It has been reported that
presence of other flavonoids, flavone, isoflavone, and aurone sulfonates increase the
photostability of anthocyanins (Francis, 1989). On the other hand, light intensity has a profound
effect on apple color, because the light-exposed peel contains twice as much anthocyanin as a
shaded peel (Ju, Liu, & Yuan, 1995). Results similar to that of apple peel was observed in
anthocyanin extract heated at 43 °C for 160 min in a model system (Kearsley & Rodriguez,
1981). Interestingly, there is no difference (P > 0.05) in the color changes of raspberries, sweet
and sour cherries during light or dark storage, although the reaction rate of color degradation in
light is higher than that in dark storage (Ochoa, Kesseler, De Michelis, Mugridge, & Chaves,
2001). Light intensity has no affect (P > 0.05) on the stability of freeze dried elderberry
anthocyanin stored at different water activities (BrØnnum-Hansen & Flink, 1985).
3.1.6.2. Effect of oxygen and metal
Oxygen has a negative effect on anthocyanin stability (Markakis, 1982) depending on the
media conditions. Additionally, it has been suggested that flavonoids act as free radical
scavengers to protect anthocyanin molecules (Sarma, Sreelakshmi, & Sharma, 1997). The
presence of oxygen can accelerate the degradation of anthocyanins either through a direct
oxidative mechanism and/or through the action of oxidizing enzymes (Jackman et al., 1987). In
contrast, the presence or absence of oxygen has no influence on the stability of freeze dried
elderberry anthocyanins during storage at lower moisture content (BrØnnum-Hansen & Flink,
65
1985). Anthocyanins are very reactive toward metals, and they form stable complexes with tin,
copper, and iron; it has been proposed that metal complexes could be used as colorants (Sarma et
al., 1997).
3.1.7. Non-thermal processing
Conventional heat processing of fruits and vegetables remains the most widely accepted
technology for food safety and shelf-life. However, in the last decade non-thermal technologies
such as high hydrostatic pressure, pulsed electric field and ultrasound have emerged as
alternative techniques for minimizing the degradation of anthocyanin content of fruit juices. For
example, more than 80% anthocyanin content of strawberry juice processed with high intensity
pulsed electric field (HIPEF) is retained (Odriozola-Serrano, Soliva-Fortuny, & Martin-Belloso,
2009a). However, Zhang et al. (2007) observed that processing of cyanidin-3-glucoside in
methanolic solution by pulsed electric field (at 1.2, 2.2 and 3.0 kV/cm, 300 numbers of pulses,
temperature ≤ 47 °C) degraded anthocyanin to colorless chalcones. It has been well documented
that electric field strength, pulse width, pulse frequency, pulse polarity, treatment time or pulse
shape are among the most important HIPEF processing parameters affecting microbial,
enzymatic inactivation, antioxidant activity and level of anthocyanins (Elez-Martínez & Martín-
Belloso, 2007; Marsellés-Fontanet & Martín-Belloso, 2007; Odriozola-Serrano et al., 2009a).
Odriozola-Serrano et al. (2009a) reported higher anthocyanin content of strawberry juice
submitted to bipolar pulses than in monopolar mode. Zabetakis, Leclerc, & Kajda (2000)
reported high hydrostatic pressure processing (200 – 800 MPa for 15 min) had minimal effect on
anthocyanin content of strawberry juice. Cano, Hernandez, & De Ancos (1997) reported that
peroxidase activity was decreased with high pressure up to 300 MPa for a treatment carried out
under 20 °C for 15 min, whereas above 300 MPa the activity increased slightly at this
66
temperature. However, complete loss of enzymatic activity was achieved at 900 MPa. The same
investigators also observed strongly diminishing polyphenol oxidase activity with high-pressure
treatments up to 400 MPa. On the contrary, β-glucosidase has highest activity after a high-
pressure treatment at 400 MPa, and the activity decreased as the pressure treatment increased up
to 800 MPa (Zabetakis, Koulentianos, Orruño, & Boyes, 2000). Application of sonication using
ultrasound decreases anthocyanin content of blackberry juice by 5% (Tiwari, O'Donnell, &
Cullen, 2009). Degradation of compounds responsible for color and anthocyanins during
ultrasound processing is attributed to (i) oxidation reaction that is promoted by the interaction
with free radicals formed during sonication (Portenlanger & Heusinger, 1992); (ii) extreme
physical conditions occur during sonication (temperature up to 5000 K and pressure up to 500
MPa at micro-scale) (Suslick, 1988); and (iii) sonochemical reactions including generation of
free radicals, enhancement of polymerization/depolymerization reactions and other reactions
(Floros & Liang, 1994).
3.1.8. Storage
Proper storage of raw and/or processed foods is important for increasing shelf-life without
altering its color attributes, nutritional value and functionality. Formation of new anthocyanins
by the reaction of malvidin 3- monoglucoside and procyanidin B2 in the presence of
acetaldehyde (15 ºC for 4 months) has observed in a model system imitating wine. Three new
pigments have observed and their visible spectra show a bathochromic shift in relation to the
anthocyanin by the condensation of these compounds. Interestingly, these compounds have
improved stability (Francia-Aricha, Guerra, Rivas-Gonzalo, & Santos-Buelga, 1997).
Individual quick freezing (–70 °C/12 h) and storage (–20 °C/6 months) of black raspberries
increase the total anthocyanins (Hager, A. et al., 2008). Retention of anthocyanins in red
67
raspberries has also observed during frozen storage (Mullen et al., 2002). The retention or
increase in the anthocyanin contents is attributed to moisture loss and enhanced extraction of
anthocyanins due to tissue softening. However, anthocyanin content of raspberries declines
linearly during storage (25 °C/6 months) with losses of 76% and 75% in canned-in water or
syrup, respectively (Hager, A. et al., 2008). Similarly, 65.8, 60.6, and 58.4% losses have
observed in blackberry canned-in-syrup products, canned-in-water products, and purees,
respectively, stored at 25°C for 6 months. In another study, total anthocyanins in strawberries
canned in 20 °Brix syrup declined 69% over 2 months at room temperature (Ngo, Wrolstad, &
Zhao, 2007). Residual polyphenoloxidase (PPO) activity is hypothesized to cause a decrease in
color characteristics in partially processed peach puree during storage (Bian, Gonzalez, &
Asleage, 1994), while nonenzymatic browning in pasteurized peach purees has attributed to
reducing the extent of sugar degradation and HMF formation (Garza, Ibarz, Pagan, & Giner,
1999). During storage of canned products, pelargonidin- 3-glucoside, the main anthocyanin in
strawberries, is hydrolyzed by acid to pelargonidin and further breaks into hydroxybenzoic acid
(Stintzing & Carle, 2004). Losses of anthocyanins and increase in percent polymeric color in
fruits are attributed to residual enzyme activity and/or condensation reactions of anthocyanins
with other phenolics (Brownmiller et al., 2008; Chaovanalikit & Wrolstad, 2004b; Hager, A. et
al., 2008; Hager, T. J. et al., 2008; Ngo et al., 2007). Ersus & Yurdagel (2007) observed 33 and
11% loss of anthocyanins in encapsulated black carrot anthocyanin powders with different
maltodextrins at 25 and 4 °C, respectively stored for 64 days. In contrast, phenolic acids such as
ferulic and syringic acid have also been shown to complex with anthocyanins in strawberry and
raspberry juices to improve the color (Rein & Heinonen, 2004). Condensation reactions have
been reported by the reaction of acetaldehyde, anthocyanins, and flavan-3-ols, producing the
68
increment of color (up to seven times). It is believed that acetaldehyde forms a bridge between
the two flavonoids, and consequently condensation reactions could proceed and contribute to the
polymeric color (Francis, 1989).
Higher stability of anthocyanins can be achieved by using lower temperature and short-time
heating during processing and storage (Krifi et al., 2000; Rodriguez-Saona, Giusti, & Wrolstad,
1999). For example, cyanidin and delphinidin-rutinosides in blood orange juice are the most
stable anthocyanins found during storage for 12 months at -18 °C, whereas transformations at 4
°C under nitrogen storage induced a slow degradation process of anthocyanins (Krifi et al.,
2000). The investigators proposed that the condensation reactions in the presence of carbonyl
derivatives formed by sugar and ascorbic acid degradations under acidic conditions are
responsible for the transformation of anthocyanins into high molecular weight brown
compounds.
In model systems of red radish and red-fleshed potato anthocyanins, radish extracts have
higher stability during storage (2 °C and 25 °C for 65 weeks) than potato extracts (Rodriguez-
Saona et al., 1999). The presence of diacylation in red radish anthocyanin as compared to
monoacylated anthocyanins in red-potatoes is responsible for its enhanced stability. Diacylated
anthocyanins are stabilized by a sandwich-type stacking caused by hydrophobic interactions
between the planar aromatic residues of the acyl groups and the positively charged pyrylium
nucleus, which prevents the addition of nucleophiles such as water to the C2 and C4 position of
the anthocyanin, reducing the formation of pseudobase (Brouillard, 1981; Goto & Kondo, 1991).
In case of monoacylated anthocyanins, only one side of the pyrylium ring can be protected
against the nucleophilic attack (Brouillard, 1983).
69
Water activity is another important factor influencing the stability of anthocyanins during
storage. Markaris, Livingston, & Fellers (1957) observed that strawberry anthocyanins are stable
when stored in dry crystalline form or on dry paper chromatograms. Bronnum-Hansen & Flink
(1985) observed that at water activity (aw) ≤ 0.31, water uptake had no effect on the anthocyanin
extracts from freeze dried elderberries, whereas at aw ≥ 0.5, a significant increase in anthocyanin
degradation rate during storage occurred. The investigators also reported that storage at high
temperature (50 °C) and high water activity (0.5 aw) had a most pronounced effect on the
stability of elderberry anthocyanins i.e. half-life was 2 months. Thakur & Arya (1989) found
that an increase in aw produced loss of grape anthocyanins adsorbed onto microcrystalline
cellulose. In contrast, degradation of strawberry anthocyanin increases with increasing aw in fruit
and in a model system (Erlandson & Wrolstad, 1972; Garzón & Wrolstad, 2001). In another
study, anthocyanin extract in a model system heated at 43 °C for 160 min was relatively stable at
different water activities (Kearsley & Rodriguez, 1981).
3.2. Degradation mechanism and products
Degradation of phenolic compounds is primarily caused by oxidation, cleavage of covalent
bonds or enhanced oxidation reactions due to thermal processing. There are two major pathways
for the degradation of anthocyanins (Markakis & Jurd, 1974). The first proceeds through the
carbinol pseudobase to the chalcone and coumarin glycoside, whereas second pathway involves
hydrolysis of the glycosidic bond as the first step in anthocyanin degradation to form the
anthocyanidin. The aglycon, which is more unstable than its glycosides, proceeds through a
highly unstable α-diketone intermediate to form aldehydes and benzoic acid derivatives. At first,
Markakis et al. (1957) hypothesized that opening of the heterocycle pyrylium ring and chalcone
formation is a first step of anthocyanin degradation and heating shifts the equilibrium toward the
70
chalcone while the chalcone-flavylium reversion is very slow. Hrazdina (1971) reported that
heating decomposed anthocyanin into a chalcone structure, the latter being further transformed
into a coumarin glucoside derivative with a loss of the B-ring. Adams (1973) proposed
hydrolysis of sugar moiety and aglycone formation as initial degradation step possibly due to the
formation of cyclic- adducts. Tanchev & Ioncheva (1976) identified quercetin,
phloroglucinaldehyde and protocatechuic acid in addition to four other compounds by paper
chromatography following thermal degradation of anthocyanins.
Several studies have been reported on the thermal degradation of various anthocyanins and
their degradation products (Jackman & Smith, 1992; Patras et al., 2010; Sadilova et al., 2007;
Sadilova et al., 2006; Seeram, Bourquin, & Nair, 2001). Von Elbe & Schwartz (1996) suggested
that coumarin 3, 5-diglycosides are common thermal degradation products of anthocyanin 3, 5-
diglycosides. In another study on thermal degradation of strawberries, elderberries and black
carrots at pH 1, deglycosylation was proposed as the first step of anthocyanin degradation into
their respective aglycones yielding a phenolic acid (protocatechuic acid) and a phenolic aldehyde
(phloroglucinaldehyde) (Sadilova et al., 2006). However, the same investigator reported that
opening of the pyrylium ring and chalcone glycoside formation was the first step rather than
deglycosylation during the thermal degradation at pH 3.5 for the same anthocyanins (Sadilova et
al., 2007). A proposed mechanism of thermal degradation of acylated and non-acylated
anthocyanin is shown in Figure 2.15. High temperatures in combination with high pH caused
degradation of cherry anthocyanins resulting in three different benzoic acid derivatives (Seeram
et al., 2001). Sarni, Fulcrand, Souillol, Souquet, & Cheynier (1995) studied oxidative
degradation of o-diphenolic cyanidin-3-glucoside and non-o-diphenolic malvidin-3-glucoside in
the presence of caffeoyltartaric acid and grape polyphenoloxidase in model solutions. The same
71
OH
OH
O+
O
OH
OH
glc
A C
B
Cyanidin-3-glucoside
Deglycosylation
OH
OH
O+
OH
OH
OH
A C
B
Cyanidin
COOH
OH
OH
OH
CHO
OHOH
ClevageC
leva
ge
OH
OH
O+
O
OH
glc
A C
B
Pelargonidin-3-glucoside
Deglycosylation
OH
OH
O+
OH
OH
Pelargonidin
Cle
vage
Cle
vage
A C
B
AB COOH
OH
OH
B
Cle
vage
4-Hydroxybenzoic acid
PhloroglucinaldehydeProtocatechuic acid
OH
OH
O+
O
OH
OH
gal glc coum
OH
OH
O+
OH
OH
OH
Cyanidin
Clevage
Cle
vage
Cle
vage
ClevageCoum-gal-glu
Deglycosylation
O
OH
OH
Cyanidin-3-gal-glc-coum
Coumaric acid
Figure 2. 15. Possible thermal degradation of non-acylated (Cyanidin-3-glucoside and
Pelargonidin-3-glucoside) and acylated (cyanidin glucosides with coumaric acid) anthocyanins.
Modified from Sadilova et al. (2007; 2006).
72
investigators reported that both the anthocyanins reacted with the enzymatically generated
caffeoyltartaric acid o-quinone. They also indicated that cyanidin-3- glucoside was degraded
mostly by coupled oxidation, whereas malvidin-3-glucoside formed adducts with caffeoyltartaric
acid quinone. Jam making of cherries (Kim & Padilla-Zakour, 2004) and plums (Donovan,
Meyer, & Waterhouse, 1998) generated 5-(hydroxymethyl) furfural as a Maillard reaction
product.
3.3. Degradation kinetics of anthocyanins
Over the past few decades, there has been an increased concern for nutritional values of
foods in addition to preservation and microbiological safety. Application of kinetic models in
thermal processing of foods is important to assessing and predicting the influence of
operations/processing on critical quality parameters to minimize the undesirable changes and to
optimize quality of specific foods. Thermal degradation of anthocyanins has been studied for
various fruits such as strawberries (Garzón & Wrolstad, 2001; Markaris et al., 1957), raspberries
(Ochoa et al., 2001; Tanchev, 1972), Concord grapes (Calvi & Francis, 1978), plums (Ahmed,
Shivhare, & Raghavan, 2004), pomegranates (Marti, Perez-Vicente, & Garcia-Viguera, 2002),
sour cherries (Cemeroglu et al., 1994), radishes (Giusti & Wrolstad, 1996b), and red cabbage
(Dyrby et al., 2001). Table 2.5 provides detailed degradation kinetics and estimated parameters
of various fruits and vegetables. A varied number of possible inactivation/degradation reactions
in food matrices occur during processing, which may involve several reaction mechanisms. The
rate of inactivation/degradation is reflected by the numerical values of the kinetic parameter
estimates such as order of the reaction, rate constants, activation energy, D-values and z-value.
The order of the reaction in the thermal degradation of anthocyanins, which is dependent on the
concentration and time, could be predicted by the following relationship (Eq.1):
73
nCkdt
dC)( (1)
where k is the rate constant, n is the reaction order, C is the concentration of the total
anthocyanins and t is the reaction time.
Order of reaction is determined by comparing the coefficient of regression, where exponent n
in eq. 1 was set to zero, half, one and two in zero-, half-, 1st and 2
nd order reactions, respectively.
The integrated form of zero-, half-, 1st and 2
nd order kinetic models is given in Eq. (2) – (5).
Zero-order: ktCCt 0 (2)
Half-order: ktCCt 02 (3)
First-order: ktC
C
o
t ln (4)
Second-order: ktCCt
0
11 (5)
Degradation of anthocyanins under isothermal conditions are reported to follow a first order
kinetics (Eq. 4) in strawberries, elderberries and black carrots at pH 3.5 (Sadilova et al., 2007),
purple-fleshed potatoes, red-fleshed potatoes, grapes and purple carrots (Reyes & Cisneros-
Zevallos, 2007) and delphidin-3-rutinoside isolates (from eggplant skin) and malvidin-3-
glucoside (from grapes) (Tanchev & Yoncheva, 1974). However, degradation of anthocyanins
from red radishes and red-fleshed potatoes follows a quadratic model during storage at 25 °C for
65 weeks (Rodriguez-Saona et al., 1999). For first order degradation kinetics, estimation of half-
lives is important. The half-lives (t1/2) of the anthocyanins were calculated as:
k
t2ln
21 (6)
74
Acylation of anthocyanins in black carrots prolongs their half-life (t1/2 ~ 2.8 h) compared
with nonacylated derivatives from strawberry (t1/2 ~ 1.9 h) and elderberry (t1/2 ~ 1.9 h) isolates at
pH 3.5 (Sadilova et al., 2007). However, higher half-life values of 4.1 and 3.2 h are observed for
black carrots and strawberries, respectively, at similar temperature conditions (Sadilova et al.,
2006). In vegetables, diacylated anthocyanins have higher half-lives compared to monoacylated
anthocyanin. For example, red radish juice with diacylated anthocyanin has half-lives of 16
weeks compared to red-fleshed potato juice with a half-life of 10 weeks at pH 3.5 (Rodriguez-
Saona et al., 1999). The same researchers reported that the half-lives of prepared anthocyanin
extracts from red radishes (24 weeks) were higher than those of red-fleshed potatoes (11 weeks)
at pH 3.5.
Thermal processing (blanching, pasteurization, sterilization) and storage of foods involve
temperature as the major extrinsic factor for food safety and nutritional quality. Parameters to
estimate the influence of temperature on the quality of food such as activation energy (Ea), z-
value and Q10 (Eq. 7 – 10) which have been reported for acai (Del Pozo-Insfran et al., 2004),
blood orange juice (Cisse, Vaillant, Acosta, Dhuique-Mayer, & Dornier, 2009; Kirca &
Cemeroglu, 2003), blackberry juice, roselle extract (Cisse et al., 2009) and black carrot juice
(Kirca et al., 2007). The thermal death time method (D-z model) is used to estimate the decimal
reduction time (D-value) i.e. heating time required to reduce the anthocyanins concentration by
90% and z-value i.e. temperature change necessary to alter the thermal death time by one log
cycle (Holdsworth, 2000). Q10 is the factor by which the reaction rate is increased if the
temperature is raised by 10 °C.
kD
10ln (7)
75
TTR
Ekk
ref
aref
11exp (8)
zTTDD refref /)()/log( (9)
T
T
k
kQ
)10(
10
(10)
)exp( ktCC
CC
eqo
eqt
(11)
where Dref is the D-value at temperature Tref, Ea is the activation energy (kJmol-1
), T is the
absolute temperature (K), R is the universal constant (8.135 Jmol-1
K-1
), kT is the reaction rate at
temperature T and k(T+10) is the reaction rate at temperature T+10, and Ceq is the final equilibrium
value of the concentration. Ochoa et al. (2001) used the concept of fractional conversion (Eq. 11)
to study the effect of light and room temperature on the kinetics of color change in raspberries,
sweet and sour cherries.
Calculation of activation energy using the two-step procedure has resulted in a relatively
large standard deviation and particularly with a large confidence interval caused by the small
number of degree of freedom (Arabshahi & Lund, 1985). Ochoa et al. (2001) used a non-linear
model (Eq. 12 & 13) to increase the degree of freedom, thus narrowing the confidence interval
for kinetics of color in fruits.
)exp( )((0),( ) ijTTTt tkCCiiiij
(12)
i
aijoTTt
RT
EtkCC
iiijexpexp
)(0),( (13)
where ),( iij TtC is the concentration of C at time tij and temperature Ti, and )(0 iTC is the initial
concentration at time zero for temperature Ti.
76
Table 2. 5. Degradation kinetic parameters of anthocyanins.(d:days; h:hour) Fruit/vegetable Processing condition Degradation
kinetics
Kinetic parameter Reference
Acai 10 – 30 °C;
H2O2 (0- 30 mmol/L)
1st order k = (7.7 – 13.9) x 10
3 min
-1;
t1/2 = 90 – 50 min;
Q10 = 1.5 (10 – 20 °C);
Q10 = 1.2 (20 – 30 °C)
Del Pozo-Insfran et al.
(2004)
Grapes 25 – 98 °C;
pH 3.0
1st order k = 0.0006 – 0.2853 h
-1;
t1/2 = 47 d – 2.4 h;
D-value = 157 d – 8.1 h;
z-value = 28 °C; Q10 = 2.28;
Ea = 75.03 kJ/mol;
Reyes & Cisneros-Zevallos
(2007)
Grape skin
(in McIlvaine buffer
(B) and carbonated
soft drink (SD))
25 – 80 °C for 0.25 – 6 h;
pH 3.0
1st order k = 3.6 – 15 x 10
-3 h
-1 (B);
k = 2.4 – 320 x 10-3
h-1
(SD);
Ea = 58 kJ/mol (B);
Ea = 77 kJ/mol (SD)
Dyrby et al. (2001)
Concord grapes
pomace
Retort (126.7 °C) for >30 min Non-linear regression
(non-isothermal)
k110 °C = 0.0607 min-1
;
Ea = 65.32 kJ/mol
Mishra et al. (2008)
Raspberries 4 – 40 °C for 5 months Non-linear regression k = (2.00 – 7.10) x 103 d
-1;
Ea = 26 kJ/mol
Ochoa et al. (2001)
Sweet cherries 4 – 40 °C for 5 months Non-linear regression k = (1.28 – 6.95) x 103 d
-1;
Ea = 32.49 kJ/mol
Ochoa et al. (2001)
Sour cherries 4 – 40 °C for 5 months Non-linear regression k = (1.10 – 5.37) x 103 d
-1;
Ea = 34.8 kJ/mol
Ochoa et al. (2001)
Plum puree 50 – 90 °C for 0 – 20 min 1st order Ea = 37.48 kJ/mol Ahmed et al. (2004)
Blood orange juice 5 – 37 °C & 70 – 90 °C 1st order Ea = 73.2 – 89.5 kJ/mol (11.2 – 69 °Brix) Kirca & Cemeroglu (2003)
Blood orange juice 30 – 90 °C 1st order D-value = (13 – 158) x 10
3 s;
Z-value = 36 °C;
Ea = 66 kJ/mol;
∆H = 63 kJ/mol; ∆S = -149 J/mol-K
Cisse et al. (2009)
Blueberry juice 40 – 80 °C 1st order k = (0.064 – 2.25) x 10
3 min
-1;
t1/2 = 180.5 – 5.11 h;
Q10 = 4.27 (40 – 50 °C);
Q10 = 1.67 (50 – 60 °C);
Q10 = 2.95 (60 – 70 °C);
Q10 = 1.67 (70 – 80 °C);
∆H = 77.8 kJ/mol; ∆S = -43.07 J/mol-K
Kechinski et al. (2010)
Strawberries Stored at 25 °C 1st order t1/2 = 56 – 934 d Garzon & Wrolstad (2001)
77
Strawberries (fresh-
cut)
5 – 20 °C with 80 kPa O2
flushing
Non-linear regression
(Weibull model)
kα = 4.4 x 10-3
– 4.4 x 10-2
d-1
Odriozola-Serrano et al.
(2009b)
Strawberry
concentrate
95 °C for 6 – 7 h 1st order t1/2 = 1.95 h (pH 3.5)
t1/2 = 3.2 h (pH 1.0)
Sadilova et al. (2007);
Sadilova et al. (2006)
Elderberry
concentrate
95 °C for 6 – 7 h 1st order t1/2 = 1.96 h (pH 3.5)
t1/2 = 1.9 h (pH 1.0)
Sadilova et al. (2007);
Sadilova et al. (2006)
Elderberries
(in McIlvaine buffer
(B) and carbonated
soft drink (SD))
25 – 80 °C for 0.25 – 6 h;
pH 3.0
1st order k = 31 – 90 x 10
-3 h
-1 (B);
k = 5.5 – 180 x 10-3
h-1
(SD);
Ea = 89 kJ/mol (B);
Ea = 56 kJ/mol (SD)
Dyrby et al. (2001)
Blackcurrant juice
(model system)
Heating
(4 – 100 °C)
1st order k = (0.16 – 310) x 10
-3 h
-1;
t1/2 = 180 d – 2.18 h;
Ea = 73 kJ/mol (21 – 100 °C)
Harbourne et al. (2008)
110 °C for 1 – 30 min Non-linear regression
(non-isothermal)
k110 °C = 1.02 h-1
;
Ea = 81.5 kJ/mol
Harbourne et al. (2008)
110 – 140 °C for 1 min Non-linear regression
(non-isothermal)
k = 9.954 h-1
;
Ea = 91.09 kJ/mol
Harbourne et al. (2008)
Blackcurrants
(in McIlvaine buffer
(B) and carbonated
soft drink (SD))
25 – 80 °C for 0.25 – 6 h;
pH 3.0
1st order k = 2.4 – 43 x 10
-3 h
-1 (B);
k = 4.5 – 87 x 10-3
h-1
(SD);
Ea = 69 kJ/mol (B);
Ea = 50 kJ/mol (SD)
Dyrby et al. (2001)
Blackberry juice &
concentrate
60 – 90 °C
(for 8.9 °Brix)
1st order k = 0.69 – 3.94 x 10
3 min
-1;
t1/2 = 16.7 h – 2.9 h;
Ea = 58.95 kJ/mol
Wang & Xu (2007)
Blackberry juice (65
°Brix)
5 – 37 °C 1st order k = 2.0 – 59.1 x 10
3 min
-1;
t1/2 = 330.1 h – 11.7 h;
Ea = 75.5 kJ/mol
Wang & Xu (2007)
Blackberry
concentrate
(65 °Brix)
5 – 37 °C 1st order k = 5.2 – 89.9 x 10
3 min
-1;
t1/2 = 133.3 h – 7.7 h;
Ea = 65.06 kJ/mol
Wang & Xu (2007)
Blackberry juice 100 – 180 °C Non-linear regression
(non-isothermal)
Ea = 92 ± 8 kJ/mol (100 – 140 °C);
Ea = 44 ± 40 kJ/mol (140 – 180 °C);
Jimenez et al. (2010)
Blackberry juice 100 – 180 °C 1st order (isothermal) Ea = 74 kJ/mol Jimenez et al. (2010)
Blackberry juice 30 – 90 °C 1st order D-value = (30 – 341) x 10
3 s
Z-value = 56 – 57 °C
Ea = 37 kJ/mol
∆H = 34 kJ/mol; ∆S = -232 to -233 J/mol-K
Cisse et al. (2009)
78
Roselle extract 30 – 90 °C 1st order D-value = (30 – 2280) x 10
3 s
Z-value = 34 – 44 °C
Ea = 47 – 61 kJ/mol
∆H = 44 – 58 kJ/mol;
∆S = -205 to -165 J/mol-K
Cisse et al. (2009)
Black Carrot juice 70 – 90 °C;
pH 4.3 and pH 6.0;
11 – 64 °Brix
1st order Ea = 62.5 – 95.1 kJ/mol;
Q10 = 1.7 – 2.8 (70 – 80 °C);
Q10 = 2.0 – 2.2 (80 – 90 °C)
Kirca et al. (2007)
70 – 90 °C; pH 2.5 – 7.0 1st order Ea = 78.1 – 47.4 kJ/mol; Kirca et al. (2007)
Storage at 4 – 37 °C;
pH 4.3
1st order Ea = 62.1 – 86.2 kJ/mol;
Q10 = 2.3 – 3.1 (4 – 20 °C);
Q10 = 2.5 – 3.6 (20 – 37 °C)
Kirca et al. (2007)
Black carrot
concentrate
95 °C for 6 – 7 h 1st order t1/2 = 2.81 h (pH 3.5)
t1/2 = 4.1 h (pH 3.5)
Sadilova et al. (2007);
Sadilova et al. (2006)
Red radish juice
concentrate
Stored (2 or 25 °C for 65
weeks)
2nd
order t1/2 = 16 weeks (25 °C)
t1/2 > 65 weeks (2 °C)
Rodriguez-Saona et al.
(1999)
Red-fleshed potato
juice concentrate
Stored (2 or 25 °C for 65
weeks)
2nd
order t1/2 = 10 weeks (25 °C)
t1/2 = 60 weeks (2 °C)
Rodriguez-Saona et al.
(1999)
Purple-fleshed
potatoes
25 – 98 °C;
pH 3.0
1st order k = 0.0007 – 0.3259 h
-1;
t1/2 = 41 d – 2.1 h;
D-value = 137 d – 7.1 h;
z-value = 28.4 °C; Q10 = 2.25;
Ea = 72.49 kJ/mol;
Reyes & Cisneros-Zevallos
(2007)
Red-fleshed potatoes 25 – 98 °C;
pH 3.0
1st order k = 0.0003 – 0.0725 h
-1;
t1/2 = 89 d – 9.6 h;
D-value = 297 d – 32 h;
z-value = 31.5 °C; Q10 = 2.08;
Ea = 66.7 kJ/mol;
Reyes & Cisneros-Zevallos
(2007)
Purple carrots 25 – 98 °C;
pH 3.0
1st order k = 0.0001 – 0.1004 h
-1;
t1/2 = 216 d – 6.9 h;
D-value = 717 d – 23 h; z-value = 26 °C
Q10 = 2.44; Ea = 81.34 kJ/mol;
Reyes & Cisneros-Zevallos
(2007)
Red cabbage
(in McIlvaine buffer
(B) and carbonated
soft drink (SD))
80 °C;
pH 3.0
1st order k = 9.0 x 10
-3 h
-1 (B);
k = 3.6 x 10-3
h-1
(SD)
Dyrby et al. (2001)
79
The isothermal methods work well for kinetic studies of samples with rapid heat transfer rate,
and where sufficient concentration remains when lag time ends. For example, for 1st order
isothermal reactions, ―sufficient concentration‖ for accurate estimation of k is C/C0 ≥ 25% (Back
& Arnold, 1977). Processing of solid or semi-solid foods enriched with anthocyanins at high
temperature may be non-isothermal because of their varying water content. In addition the come-
up-time i.e. time to reach ± 0.5 °C of the set temperature, will be different depending on the
sample matrix. For a non-isothermal process, temperature of an individual sample changes with
time. The non-isothermal model (Eq. 14 & 15) involves thermal history (β) combining both
individual time and temperature. The kinetic parameters such as reaction rate constant (k) and
activation energy (Ea) can be estimated by minimizing the sum of square errors (Dolan, 2003).
Recently, Mishra, Dolan, & Yang (2008) and Harbourne, Jacquier, Morgan, & Lyng (2008)
studied the non-isothermal kinetic degradation of anthocyanins of grape pomace and
blackcurrants, respectively, in model systems.
tk
o
t eC
C (14)
t
ref
a
TTR
E
0
11exp (15)
The Weibull model (Eq. 16) has the potential to describe chemical degradation kinetics in
addition to microbial and enzymatic kinetics. Recently, Oms-Oliu, Odriozola-Serrano, Soliva-
Fortuny, & Martín-Belloso (2009) used the Weibull model and accurately described the kinetics
of phenolics and antioxidant changes (DPPH) in fresh-cut watermelon stored at 5 – 20 °C for 14
days. In another study, Odriozola-Serrano, Soliva-Fortuny, & Martin-Belloso (2009b) reported
the accuracy of the Weibull model to describe the changes in anthocyanins and antioxidant
80
activity (DPPH) of fresh-cut strawberries stored at similar conditions under a high oxygen
atmosphere (80 kPa).
).(exp[0 ktAOXAOX (16)
where AOX is the percentage relative antioxidant property, AOX0 is the intercept of the curve, t
(days) is the storage time, kα (per day) is the kinetic constant, which is the inverse of the scale
factor (α) and γ is the shape parameter. Mean storage time (tm) i.e. the time for 100% depletion of
the antioxidant property and reaction rate constant (k) is expressed as Eq. 17 & 18, respectively:
)1
1()1
(
k
tm (17)
m
cTTck )}](exp{1ln[ (18)
where Ґ is the gamma function, T (K) is the storage temperature, Tc (K) is the marker of the
temperature range where the changes accelerate, c (per K), and m are dimensional constants.
The enthalpy of activation (∆H) and entropy of activation (∆S) of anthocyanins has been
reported for blueberry juice (Kechinski et al., 2010) and blood oranges, blackberries, and roselle
(Cisse et al., 2009) using the Eyring-Polanyi model based on transition state theory:
RT
STH
b Teh
kk
(19)
where T is the absolute temperature (K), kb is the Boltzman constant (1.381 x 10-23
J/K), h is the
Planck constant (6.626 x 10-34
Js) and R is the gas constant (8.31 J/mol K).
4. SUMMARY
Most investigations in the literature on the phenolic antioxidants in fruits, vegetables, and
grains have shown that high temperature treatments can reduce the antioxidant activity, whereas
some studies reported to have increased in the antioxidant activity of processed foods. Some
81
investigators also emphasized the intrinsic properties of the food matrix responsible for the
mixed behavior of the antioxidant compounds during processing studies of fruits and vegetables.
Thermal degradation of anthocyanins in fruits and vegetables has been extensively conducted
and reported in the model systems, juices and concentrates. Degradation kinetics of
anthocyanins, mostly studied under 100 °C, follows the first order reaction in these matrices.
However, the responses of antioxidant compounds depend on the specific fruit or vegetable.
Based on the available knowledge, it will be biased to predict the effect of thermal treatments on
phenolic antioxidants retention. There is a need to analyze these compounds on a case-by-case
basis. Therefore, the current study aimed to investigate the presence of phenolic antioxidants in
colored potatoes, their retention/degradation, bioavailability and kinetics in the processed (at >
100 °C) value-added products used in canning, baking and extrusion cooking. The results could
provide valuable information to farmers, researchers and industry.
82
REFERENCES
Adams, J. B. (1973). Thermal degradation of anthocyanins with particular reference to the 3-
glycosides of cyanidin. I. In acidified aqueous solution at 100 °C. Journal of the Science of
Food and Agriculture, 24, 747-762.
Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and
Food Chemistry, 50, 6182-6187.
Aguillar-Rosas, S. F., Ballinas-Casarrubias, M. L., Nevarez-Moorillon, G. V., Martin-Belloso,
O., & Ortega-Rivas, E. (2007). Thermal and pulsed electric fields pasteurization of apple
juice: Effects on physicochemical properties and flavour compounds. Journal of Food
Engineering, 83, 41-46.
Ahmed, J., Shivhare, U. S., & Raghavan, G. S. V. (2004). Thermal degradation kinetics of
anthocyanin and visual colour of plum puree. European Food Research and Technology,
218, 525-528.
Allali, H., Marchal, L., & Vorobiev, E. (2010). Blanching of strawberries by ohmic heating:
effects on the kinetics of mass transfer during osmotic dehydration. Food and Bioprocess
Technology, 3, 406-414.
Alonso, R., Grant, G., Dewey, P., & Marzo, F. (2000). Nutritional assessment in vitro and in
vivo of raw and extruded peas (Pisum sativum L.). Journal of Agricultural and Food
Chemistry, 48, 2286-2290.
Alsaikhan, M. S., Howard, L. R., & Miller, J. C. (1995). Antioxidant activity and total phenolics
in different genotypes of potato (Solanum tuberosum, L). Journal of Food Science, 60, 341-
347.
83
Altamirano, R. C., Drdak, M., Simon, P., Smelik, A., & Simko, P. (1992). Stability of red beet
pigment concentrate in maize starch. Journal of the Science of Food and Agriculture, 58,
595-596.
Altan, A., McCarthy, K. L., & Maskan, M. (2009). Effect of extrusion process on antioxidant
activity, total phenolics and beta-glucan content of extrudates developed from barley-fruit
and vegetable by-products. International Journal of Food Science and Technology, 44, 1263-
1271.
Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the
degenerative diseases of aging. Proceedings of the National Academy of Sciences of the
United States of America, 90, 7915-7922.
Amin, I., & Lee, W. Y. (2005). Effect of different blanching times on antioxidant properties in
selected cruciferous vegetables. Journal of the Science of Food and Agriculture, 85, 2314-
2320.
Andreasen, M. F., Kroon, P. A., Williamson, G., & Garcia-Conesa, M. T. (2001). Intestinal
release and uptake of phenolic antioxidant diferulic acids. Free Radical Biology and
Medicine, 31, 304-314.
Anese, M., Manzocco, L., Nicoli, M. C., & Lerici, C. R. (1999). Antioxidant properties of
tomato juice as affected by heating. Journal of the Science of Food and Agriculture, 79, 750-
754.
Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods
for testing antioxidant activity. Analyst, 127, 183-198.
Arabshahi, A., & Lund, D. B. (1985). Considerations in calculating kinetic parameters from
experimental data. Journal of Food Process Engineering, 7, 239-251.
84
Back, J. V., & Arnold, K. J. (1977). Parameter estimation in engineering and science. New
York, NY: John Wiley & Sons.
Bakker, J., & Timberlake, C. F. (1997). Isolation, identification, and characterization of new
color-stable anthocyanins occurring in some red wines. Journal of Agricultural and Food
Chemistry, 45, 35-43.
Barka, E. A., Kalantari, S., Makhlouf, J., & Arul, J. (2000). Effects of UV-C irradiation on lipid
peroxidation markers during ripening of tomato (Lycopersicon esculentum L.) fruits.
Australian Journal of Plant Physiology, 27, 147-152.
Baublis, A., Spomer, A., & Berberjimenez, M. D. (1994). Anthocyanin pigments - comparison of
extract stability. Journal of Food Science, 59, 1219-&.
Bian, Y., Gonzalez, A. R., & Asleage, J. M. (1994). Effects of pasteurization or sodium benzoate
on long-term storage stability of peach puree. Journal of Food Quality, 17, 299-310.
Blessington, A., Miller, J. C., Nzaramba, M. N., Hale, A. L., Redivari, L., Scheuring, D. C., &
Hallman, G. J. (2007). The effects of low-dose gamma irradiation and storage time on
carotenoids, antioxidant activity, and phenolics in the potato cultivar Atlantic. American
Journal of Potato Research, 84, 125-131.
Blessington, T. (2005). The effects of cooking, storage and ionizing irradiation on carotenoids,
antioxidant activity and phenolics in potato. Texas A&M, College station.
Block, G., Patterson, B., & Subar, A. (1992). Fruit, vegetables, and cancer prevention - a review
of the epidemiologic evidence. Nutrition and Cancer-an International Journal, 18, 1-29.
Bonoli, M., Verardo, V., Marconi, E., & Caboni, M. F. (2004). Phenols in barley (Hordeum
vulgare L.) flour: Comparative spectrophotometric study among extraction methods of free
85
and bound phenolic compounds. Journal of Agricultural and Food Chemistry, 52, 5195-
5200.
Breitfellner, F., Solar, S., & Sontag, G. (2002). Effect of gamma-irradiation on phenolic acids in
strawberries. Journal of Food Science, 67, 517-521.
Bridle, P., & Timberlake, C. F. (1997). Anthocyanins as natural food colours - Selected aspects.
Food Chemistry, 58, 103-109.
BrØnnum-Hansen, K., & Flink, J. M. (1985). Anthocyanin colourants from elderberry
(Sambucus nigra L.). 3. Storage stability of the freeze dried product. International Journal of
Food Science & Technology, 20, 725-733.
Brouillard, R. (1981). Origin of the exceptional colour stability of the Zebrina anthocyanin.
Phytochemistry, 20, 143-145.
Brouillard, R. (1983). The in vivo expression of anthocyanin colour in plants. Phytochemistry,
22, 1311-1323.
Brownmiller, C., Howard, L. R., & Prior, R. L. (2008). Processing and storage effects on
monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed
blueberry products. Journal of Food Science, 73, H72-H79.
Bryngelsson, S., Dimberg, L. H., & Kamal-Eldin, A. (2002). Effects of commercial processing
on levels of antioxidants in oats (Avena sativa L.). Journal of Agricultural and Food
Chemistry, 50, 1890-1896.
Burton, W. G. (1989). The Potato. New York, NY: Wiley & Sons.
Butz, P., FernandezGarcia, A., Lindauer, R., Dieterich, S., Bognar, A., & Tauscher, B. (2003).
Influence of ultra high pressure processing on fruit and vegetable products. Journal of Food
Engineering, 56, 233-236.
86
Callebaut, A., Hendrickx, G., Voets, A. M., & Motte, J. C. (1990). Anthocyanins in cell-cultures
of Ajuga-Reptans. Phytochemistry, 29, 2153-2158.
Calvi, J., & Francis, F. (1978). Stability of concord grape (V. Labrusca) anthocyanins in model
systems. Journal of Food Science, 43, 1448-1456.
Camire, M. E., Chaovanalikit, A., Dougherty, M. P., & Briggs, J. (2002). Blueberry and grape
anthocyanins as breakfast cereal colorants. Journal of Food Science, 67, 438-441.
Camire, M. E., Dougherty, M. P., & Briggs, J. L. (2007). Functionality of fruit powders in
extruded corn breakfast cereals. Food Chemistry, 101, 765-770.
Cano, M. P., Hernandez, A., & De Ancos, B. (1997). High pressure and temperature effects on
enzyme inactivation in strawberry and orange products. Journal of Food Science, 62, 85-88.
Cantos, E., Garcia-Viguera, C., de Pascual-Teresa, S., & Tomas-Berberan, F. A. (2000). Effect
of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table
grapes. Journal of Agricultural and Food Chemistry, 48, 4606-4612.
Cao, G. H., Booth, S. L., Sadowski, J. A., & Prior, R. L. (1998). Increases in human plasma
antioxidant capacity after consumption of controlled diets high in fruit and vegetables.
American Journal of Clinical Nutrition, 68, 1081-1087.
Cao, G. H., Russell, R. M., Lischner, N., & Prior, R. L. (1998). Serum antioxidant capacity is
increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women.
Journal of Nutrition, 128, 2383-2390.
Cemeroglu, B., Velioglu, S., & Isik, S. (1994). Degradation kinetics of anthocyanins in sour
cherry juice and concentrate. Journal of Food Science, 59, 1216-1218.
Chaovanalikit, A., & Wrolstad, R. E. (2004a). Anthocyanin and polyphenolic composition of
fresh and processed cherries. Journal of Food Science, 69, C73-C83.
87
Chaovanalikit, A., & Wrolstad, R. E. (2004b). Total anthocyanins and total phenolics of fresh
and processed cherries and their antioxidant properties. Journal of Food Science, 69, C67-
C72.
Cheigh, H. S., Um, S. H., & Lee, C. Y. (1995). Antioxidant characteristics of melanin-related
products from enzymatic browning reaction of catechin in a model system. Enzymatic
Browning and Its Prevention, 600, 200-208.
Chu, Y. H., Chang, C. L., & Hsu, H. F. (2000). Flavonoid content of several vegetables and their
antioxidant activity. Journal of the Science of Food and Agriculture, 80, 561-566.
Chuah, A. M., Yamaguchi, H. T., & Matoba, T. (2005) 2nd International conference on
polyphenols and health (pp. 168). University of California, Davis, CA.
Chun, O. K., Kim, D. O., Smith, N., Schroeder, D., Han, J. T., & Lee, C. Y. (2005). Daily
consumption of phenolics and total antioxidant capacity from fruit and vegetables in the
American diet. Journal of the Science of Food and Agriculture, 85, 1715-1724.
Cisse, M., Vaillant, F., Acosta, O., Dhuique-Mayer, C., & Dornier, M. (2009). Thermal
degradation kinetics of anthocyanins from blood orange, blackberry, and roselle using the
Arrhenius, Eyring, and Ball models. Journal of Agricultural and Food Chemistry, 57, 6285-
6291.
Clifford, M. N. (2000). Anthocyanins – nature, occurrence and dietary burden. Journal of the
Science of Food and Agriculture, 80, 1063-1072.
Connor, A. M., Luby, J. J., Tong, C. B. S., Finn, C. E., & Hancock, J. F. (2002). Genotypic and
environmental variation in antioxidant activity, total phenolic content, and anthocyanin
content among blueberry cultivars. Journal of the American Society for Horticultural
Science, 127, 89-97.
88
Cuvelier, M.-E., Bondet, V., & Berset, C. (2000). Behavior of phenolic antioxidants in a
partitioned medium: structure—Activity relationship. Journal of the American Oil Chemists'
Society, 77, 819-824.
Dao, L., & Friedman, M. (1992). Chlorogenic acid content of fresh and processed potatoes
determined by ultraviolet spectrophotometry. Journal of Agricultural and Food Chemistry,
40, 2152-2156.
de Ancos, B., Gonzalez, E., & Cano, M. P. (2000). Effect of high-pressure treatment on the
carotenoid composition and the radical scavenging activity of persimmon fruit purees.
Journal of Agricultural and Food Chemistry, 48, 3542-3548.
Dede, S., Alpas, H., & Bayindirli, A. (2007). High hydrostatic pressure treatment and storage of
carrot and tomato juices: Antioxidant activity and microbial safety. Journal of the Science of
Food and Agriculture, 87, 773-782.
Del Pozo-Insfran, D., Brenes, C. H., & Talcottt, S. T. (2004). Phytochemical composition and
pigment stability of acai (Euterpe oleracea Mart.). Journal of Agricultural and Food
Chemistry, 52, 1539-1545.
Dewanto, V., Wu, X. Z., Adom, K. K., & Liu, R. H. (2002). Thermal processing enhances the
nutritional value of tomatoes by increasing total antioxidant activity. Journal of Agricultural
and Food Chemistry, 50, 3010-3014.
Dewanto, V., Wu, X. Z., & Liu, R. H. (2002). Processed sweet corn has higher antioxidant
activity. Journal of Agricultural and Food Chemistry, 50, 4959-4964.
Dlamini, N. R., Taylor, J. R. N., & Rooney, L. W. (2007). The effect of sorghum type and
processing on the antioxidant properties of African sorghum-based foods. Food Chemistry,
105, 1412-1419.
89
Doblado, R., Frias, J., & Vidal-Valverde, C. (2007). Changes in vitamin C content and
antioxidant capacity of raw and germinated cowpea (Vigna sinensis var. carilla) seeds
induced by high pressure treatment. Food Chemistry, 101, 918-923.
Dolan, K. D. (2003). Estimation of kinetic parameters for nonisothermal food processes. Journal
of Food Science, 68, 728-741.
Donovan, J. L., Meyer, A. S., & Waterhouse, A. L. (1998). Phenolic composition and antioxidant
activity of prunes and prune juice (Prunus domestica). Journal of Agricultural and Food
Chemistry, 46, 1247-1252.
Donsi, G., Ferrari, G., & DiMatteo, M. (1996). High pressure stabilization of orange juice:
Evaluation of the effects of process conditions. Italian Journal of Food Science, 8, 99-106.
Dubery, I. A., Louw, A. E., & van Heerden, F. R. (1999). Synthesis and evaluation of 4-(3-
methyl-2-butenoxy) isonitrosoacetophenone, a radiation-induced stress metabolite in Citrus.
Phytochemistry, 50, 983-989.
Duve, K. J., & White, P. J. (1991). Extraction and identification of antioxidants in oats. Journal
of the American Oil Chemists Society, 68, 365-370.
Dyrby, M., Westergaard, N., & Stapelfeldt, H. (2001). Light and heat sensitivity of red cabbage
extract in soft drink model systems. Food Chemistry, 72, 431-437.
Eberhardt, M. V., Lee, C. Y., & Liu, R. H. (2000). Nutrition - antioxidant activity of fresh
apples. Nature, 405, 903-904.
Elez-Martínez, P., & Martín-Belloso, O. (2007). Effects of high intensity pulsed electric field
processing conditions on vitamin C and antioxidant capacity of orange juice and gazpacho, a
cold vegetable soup. Food Chemistry, 102, 201-209.
90
Erlandson, J. A., & Wrolstad, R. E. (1972). Degradation of anthocyanins at limited water
concentration. Journal of Food Science, 37, 592-595.
Ersus, S., & Yurdagel, U. (2007). Microencapsulation of anthocyanin pigments of black carrot
(Daucuscarota L.) by spray drier. Journal of Food Engineering, 80, 805-812.
Fan-Chiang, H. J., & Wrolstad, R. E. (2005). Anthocyanin pigment composition of blackberries.
Journal of Food Science, 70, C198-C202.
Fan, X. T., & Thayer, D. W. (2001). Quality of irradiated alfalfa sprouts. Journal of Food
Protection, 64, 1574-1578.
Fan, X. T., & Thayer, D. W. (2002). Gamma-radiation influences browning, antioxidant activity,
and malondialdehyde level of apple juice. Journal of Agricultural and Food Chemistry, 50,
710-715.
Fan, X. T., Toivonen, P. M. A., Rajkowski, K. T., & Sokorai, K. J. B. (2003). Warm water
treatment in combination with modified atmosphere packaging reduces undesirable effects of
irradiation on the quality of fresh-cut iceberg lettuce. Journal of Agricultural and Food
Chemistry, 51, 1231-1236.
Feinberg, B., Winter, F., & Roth, T. L. (1968). The preparation for freezing and freezing of
vegetables. In D. K. Tressler, W. B. Van-Arsdel & M. J. Copley (Eds.), The freezing
preservation of foods. Westport, Connecticut: Avi publishing Co.
Fernandez-Garcia, A., Butz, P., Bognar, A., & Tauscher, B. (2001). Antioxidative capacity,
nutrient content and sensory quality of orange juice and an orange-lemon-carrot juice product
after high pressure treatment and storage in different packaging. European Food Research
and Technology, 213, 290-296.
91
Fernandez-Garcia, A., Butz, P., & Tauscher, B. (2001). Effects of high-pressure processing on
carotenoid extractability, antioxidant activity, glucose diffusion, and water binding of tomato
puree (Lycopersicon esculentum Mill.). Journal of Food Science, 66, 1033-1038.
Floros, J. D., & Liang, H. H. (1994). Acoustically assisted diffusion through membranes and
biomaterials. Food Technology, 48, 79-84.
Francia-Aricha, E. M., Guerra, M. T., Rivas-Gonzalo, J. C., & Santos-Buelga, C. (1997). New
anthocyanin pigments formed after condensation with flavanols. Journal of Agricultural and
Food Chemistry, 45, 2262-2266.
Francis, F. J. (1989). Food colorants - Anthocyanins. Critical Reviews in Food Science and
Nutrition, 28, 273-314.
Frankel, E. N., Huang, S. W., Kanner, J., & German, J. B. (1994). Interfacial phenomena in the
evaluation of antioxidants - bulk oils vs emulsions. Journal of Agricultural and Food
Chemistry, 42, 1054-1059.
Friedman, M. (1997). Chemistry, biochemistry, and dietary role of potato polyphenols. A review.
Journal of Agricultural and Food Chemistry, 45, 1523-1540.
Garcıia-Viguera, C., Zafrilla, P., & Tomás-Barberán, F. A. (1997). Determination of authenticity
of fruit jams by HPLC analysis of anthocyanins. Journal of the Science of Food and
Agriculture, 73, 207-213.
Garza, S., Ibarz, A., Pagan, J., & Giner, J. (1999). Non-enzymatic browning in peach puree
during heating. Food Research International, 32, 335-343.
Garzon, G. A., & Wrolstad, R. E. (2002). Comparison of the stability of pelargonidin-based
anthocyanins in strawberry juice and concentrate. Journal of Food Science, 67, 1288-1299.
92
Garzón, G. A., & Wrolstad, R. E. (2001). The stability of pelargonidin-based anthocyanins at
varying water activity. Food Chemistry, 75, 185-196.
Gerard, K. A., & Roberts, J. S. (2004). Microwave heating of apple mash to improve Juice yield
and quality. Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology, 37,
551-557.
Gil, M. I., Ferreres, F., & Tomas-Barberan, F. A. (1999). Effect of postharvest storage and
processing on the antioxidant constituents (flavonoids and vitamin C) of fresh-cut spinach.
Journal of Agricultural and Food Chemistry, 47, 2213-2217.
Gil, M. I., Holcroft, D. M., & Kader, A. A. (1997). Changes in strawberry anthocyanins and
other polyphenols in response to carbon dioxide treatments. Journal of Agricultural and
Food Chemistry, 45, 1662-1667.
Giusti, M. M., Rodriguez-Saona, L. E., Griffin, D., & Wrolstad, R. E. (1999). Electrospray and
tandem mass spectroscopy as tools for anthocyanin characterization. Journal of Agricultural
and Food Chemistry, 47, 4657-4664.
Giusti, M. M., & Wrolstad, R. E. (1996a). Characterization of red radish anthocyanins. Journal
of Food Science, 61, 322-326.
Giusti, M. M., & Wrolstad, R. E. (1996b). Radish anthocyanin extract as a natural red colorant
for maraschino cherries. Journal of Food Science, 61, 688-694.
Glassgen, W. E., Wray, V., Strack, D., Metzger, J. W., & Seitz, H. U. (1992). Anthocyanins from
cell-suspension cultures of Daucus-Carota. Phytochemistry, 31, 1593-1601.
Goto, T., & Kondo, T. (1991). Structure and molecular stacking of anthocyanins - flower color
variation. Angewandte Chemie-International Edition in English, 30, 17-33.
93
Grela, E. R., Jensen, S. K., & Jakobsen, K. (1999). Fatty acid composition and content of
tocopherols and carotenoids in raw and extruded grass pea (Lathyrus sativus L). Journal of
the Science of Food and Agriculture, 79, 2075-2078.
Griffiths, D. W., Bain, H., & Dale, M. F. B. (1995). Photoinduced changes in the total
chlorogenic acid content of potato (Solanum tuberosum) tubers. Journal of the Science of
Food and Agriculture, 68, 105-110.
Guyot, S., Marnet, N., Laraba, D., Sanoner, P., & Drilleau, J. F. (1998). Reversed-phase HPLC
following thiolysis for quantitative estimation and characterization of the four main classes of
phenolic compounds in different tissue zones of a French cider apple variety (Malus
domestica var. Kermerrien). Journal of Agricultural and Food Chemistry, 46, 1698-1705.
Hager, A., Howard, L. R., Prior, R. L., & Brownmiller, C. (2008). Processing and storage effects
on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed
black raspberry products. Journal of Food Science, 73, H134-H140.
Hager, T. J., Howard, L. R., & Prior, R. L. (2008). Processing and storage effects on monomeric
anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry
products. Journal of Agricultural and Food Chemistry, 56, 689-695.
Hakkinen, S. H., Karenlampi, S. O., Mykkanen, H. M., & Torronen, A. R. (2000). Influence of
domestic processing and storage on flavonol contents in berries. Journal of Agricultural and
Food Chemistry, 48, 2960-2965.
Halliwell, B., & Gutteridge, J. M. C. (1990). The antioxidants of human extracellular fluids.
Archives of Biochemistry and Biophysics, 280, 1-8.
Halliwell, B., Gutteridge, J. M. C., & Cross, C. E. (1992). Free radicals, antioxidants, and human
disease - Where are we now. Journal of Laboratory and Clinical Medicine, 119, 598-620.
94
Halliwell, B., Murcia, M. A., Chirico, S., & Aruoma, O. I. (1995). Free radicals and antioxidants
in food and in vivo - What they do and how they work. Critical Reviews in Food Science and
Nutrition, 35, 7-20.
Hamama, A. A., & Nawar, W. W. (1991). Thermal decomposition of some phenolic
antioxidants. Journal of Agricultural and Food Chemistry, 39, 1063-1069.
Hamouz, K., Lachman, J., Vokal, B., & Pivec, V. (1999). Influence of environmental conditions
and way of cultivation on the polyphenol and ascorbic acid content in potato tubers.
Rostlinna Vyroba, 45, 293-298.
Harbourne, N., Jacquier, J. C., Morgan, D. J., & Lyng, J. G. (2008). Determination of the
degradation kinetics of anthocyanins in a model juice system using isothermal and non-
isothermal methods. Food Chemistry, 111, 204-208.
Holdsworth, S. D. (2000). Thermal processing of packaged foods. New York: Blackie Academic
& Professional.
Hoshino, T., Matsumoto, U., & Goto, T. (1981). Self-association of some anthocyanins in neutral
aqueous solution. Phytochemistry, 20, 1971-1976.
Hrazdina, G. (1971). Reactions of the anthocyanidin-3,5-diglucosides: Formation of 3,5-di-(O-
[beta]--glucosyl)-7-hydroxy coumarin. Phytochemistry, 10, 1125-1130.
Huang, D. J., Ou, B. X., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays.
Journal of Agricultural and Food Chemistry, 53, 1841-1856.
Icier, F. (2010). Ohmic blanching effects on drying of vegetable byproduct. Journal of Food
Process Engineering, 33, 661-683.
Jackman, R. L., & Smith, J. L. (1992). Anthocyanins and betalains. In G. A. F. Hendry & J. D.
Houghton (Eds.), Natural food colorants (pp. 183 - 241). London: Blackie and Son Ltd.
95
Jackman, R. L., Yada, R. Y., & Tung, M. A. (1987). A review - Separation and chemical
properties of anthocyanins used for their qualitative and quantitative analysis. Journal of
Food Biochemistry, 11, 279-308.
Jiménez-Monreal, A. M., García-Diz, L., Martínez-Tomé, M., Mariscal, M., & Murcia, M. A.
(2009). Influence of cooking methods on antioxidant activity of vegetables. Journal of Food
Science, 74, H97-H103.
Jimenez, A., Selga, A., Torres, J. U., & Julia, L. (2004). Reducing activity of polyphenols with
stable radicals of the TTM series. Electron transfer versus H-abstraction reactions in flavan-
3-ols. Organic Letters, 6, 4583-4586.
Jimenez, N., Bohuon, P., Lima, J., Dornier, M., Vaillant, F., & Perez, A. M. (2010). Kinetics of
anthocyanin degradation and browning in reconstituted blackberry juice treated at high
temperatures (100 - 180 °C). Journal of Agricultural and Food Chemistry, 58, 2314-2322.
Jiratanan, T., & Liu, R. H. (2004). Antioxidant activity of processed table beets (Beta vulgaris
var, conditiva) and green beans (Phaseolus vulgaris L.). Journal of Agricultural and Food
Chemistry, 52, 2659-2670.
Ju, Z. G., Liu, C. L., & Yuan, Y. B. (1995). Activities of chalcone synthase and udpgal -
Flavonoid-3-o-glycosyltransferase in relation to anthocyanin synthesis in apple. Scientia
Horticulturae, 63, 175-185.
Kaanane, A., Kane, D., & Labuza, T. (1988). Time and temperature effect on stability of
Moroccan processed orange juice during storage. Journal of Food Science, 53, 1470-1473.
Kacharava, N., Chanishvili, S., Badridze, G., Chkhubianishvili, E., & Janukashvili, N. (2009).
Effect of seed irradiation on the content of antioxidants in leaves of Kidney bean, Cabbage
and Beet cultivars. Australian Journal of Crop Science, 3, 137-145.
96
Kader, A. A. (1986). Biochemical and physiological-basis for effects of controlled and modified
atmospheres on fruits and vegetables. Food Technology, 40, 99 -100 and 102 - 104.
Kader, F., Irmouli, M., Nicolas, J. P., & Metche, M. (2001). Proposed mechanism for the
degradation of pelargonidin 3-glucoside by caffeic acid o-quinone. Food Chemistry, 75, 139-
144.
Kader, F., Irmouli, M., Nicolas, J. P., & Metche, M. (2002). Involvement of blueberry
peroxidase in the mechanisms of anthocyanin degradation in blueberry juice. Journal of
Food Science, 67, 910-915.
Kader, F., Irmouli, M., Zitouni, N., Nicolas, J. P., & Metche, M. (1999). Degradation of cyanidin
3-glucoside by caffeic acid o-quinone. Determination of the stoichiometry and
characterization of the degradation products. Journal of Agricultural and Food Chemistry,
47, 4625-4630.
Kader, F., Rovel, B., Girardin, M., & Metche, M. (1997). Mechanism of browning in fresh
highbush blueberry fruit (Vaccinium corymbosum L). Role of blueberry polyphenol oxidase,
chlorogenic acid and anthocyanins. Journal of the Science of Food and Agriculture, 74, 31-
34.
Kahkonen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J. P., Pihlaja, K., Kujala, T. S., &
Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds.
J. Agric. Food Chem., 47, 3954-3962.
Kalt, W., McDonald, J. E., & Donner, H. (2000). Anthocyanins, phenolics, and antioxidant
capacity of processed lowbush blueberry products. Journal of Food Science, 65, 390-393.
Kalt, W., Ryan, D. A. J., Duy, J. C., Prior, R. L., Ehlenfeldt, M. K., & Vander Kloet, S. P.
(2001). Interspecific variation in anthocyanins, phenolics, and antioxidant capacity among
97
genotypes of highbush and lowbush blueberries (Vaccinium section cyanococcus spp.).
Journal of Agricultural and Food Chemistry, 49, 4761-4767.
Kang, H. M., & Saltveit, M. E. (2002). Antioxidant capacity of lettuce leaf tissue increases after
wounding. Journal of Agricultural and Food Chemistry, 50, 7536-7541.
Kaur, C., & Kapoor, H. C. (2001). Antioxidants in fruits and vegetables - the millennium's
health. International Journal of Food Science & Technology, 36, 703-725.
Kayano, S., Yamada, N. F., Suzuki, T., Ikami, T., Shioaki, K., Kikuzaki, H., Mitani, T., &
Nakatani, N. (2003). Quantitative evaluation of antioxidant components in prunes (Prunus
domestica L.). Journal of Agricultural and Food Chemistry, 51, 1480-1485.
Ke, D. Y., Goldstein, L., Omahony, M., & Kader, A. A. (1991). Effects of short-term exposure to
low O2 and high CO2 atmospheres on quality attributes of strawberries. Journal of Food
Science, 56, 50-54.
Kearsley, M. W., & Rodriguez, N. (1981). The stability and use of natural colours in foods:
anthocyanin, β-carotene and riboflavin. International Journal of Food Science &
Technology, 16, 421-431.
Kechinski, C. P., Guimaraes, P. V. R., Norena, C. P. Z., Tessaro, I. C., & Marczak, L. D. F.
(2010). Degradation kinetics of anthocyanin in blueberry juice during thermal treatment.
Journal of Food Science, 75, C173-C176.
Khraisheh, M. A. M., Cooper, T. J. R., & Magee, T. R. A. (1995). Investigation and modeling of
combined microwave and air-drying. Food and Bioproducts Processing, 73, 121-126.
Kim, D. O., & Padilla-Zakour, O. I. (2004). Jam processing effect on phenolics and antioxidant
capacity in anthocyanin-rich fruits: Cherry, plum, and raspberry. Journal of Food Science,
69, S395-S400.
98
Kim, K. H., & Yook, H. S. (2009). Effect of gamma irradiation on quality of kiwifruit (Actinidia
deliciosa var. deliciosa cv. Hayward). Radiation Physics and Chemistry, 78, 414-421.
Kinsella, J. E., Frankel, E., German, B., & Kanner, J. (1993). Possible mechanisms for the
protective role of antioxidants in wine and plant foods. Food Technology, 47, 85-89.
Kirca, A., & Cemeroglu, B. (2003). Degradation kinetics of anthocyanins in blood orange juice
and concentrate. Food Chemistry, 81, 583-587.
Kirca, A., Ozkan, M., & Cemeroglu, B. (2007). Effects of temperature, solid content and pH on
the stability of black carrot anthocyanins. Food Chemistry, 101, 212-218.
Korus, J., Gumul, D., & Czechowska, K. (2007). Effect of extrusion on the phenolic composition
and antioxidant activity of dry beans of Phaseolus vulgaris L. Food Technology and
Biotechnology, 45, 139-146.
Krifi, B., Chouteau, F., Boudrant, J., & Metche, M. (2000). Degradation of anthocyanins from
blood orange juices. International Journal of Food Science and Technology, 35, 275-283.
Kwok, B. H. L., Hu, C., Durance, T., & Kitts, D. D. (2004). Dehydration techniques affect
phytochemical contents and free radical scavenging activities of Saskatoon berries
(Amelanchier alnifolia Nutt.). Journal of Food Science, 69, S122-S126.
Lachman, J., Hamouz, K., & Orsak, M. (2005). Red and purple potatoes - A significant
antioxidant source in human nutrition. Chemicke Listy, 99, 474-482.
Lachman, J., Hamouz, K., Orsak, M., & Pivec, V. (2000). Potato tubers as a significant source of
antioxidants in human nutrition. Rostlinna Vyroba, 46, 231-236.
Lea, A. G. H. (1988). HPLC of natural pigments in foodstuffs. In R. Macrae (Ed.), HLPC in
Food Analysis (pp. 277-333). San Diego,
CA: Academic Press Inc.
99
Lee, H. S. (1992). Antioxidative activity of browning reaction products isolated from storage-
aged orange juice. Journal of Agricultural and Food Chemistry, 40, 550-552.
Lee, H. S., & Wicker, L. (1991). Anthocyanin pigments in the skin of lychee fruit. Journal of
Food Science, 56, 466-&.
Lee, J., Durst, R. W., & Wrolstad, R. E. (2002). Impact of juice processing on blueberry
anthocyanins and polyphenolics: Comparison of two pretreatments. Journal of Food Science,
67, 1660-1667.
Leusink, G. J., Kitts, D. D., Yaghmaee, P., & Durance, T. (2010). Retention of antioxidant
capacity of vacuum microwave dried cranberry. Journal of Food Science, 75, C311-C316.
Lewis, C. E., Walker, J. R. L., Lancaster, J. E., & Sutton, K. H. (1998). Determination of
anthocyanins, flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum
tuberosum L. Journal of the Science of Food and Agriculture, 77, 45-57.
Li, W., Shan, F., Sun, S., Corke, H., & Beta, T. (2005). Free radical scavenging properties and
phenolic content of Chinese black-grained wheat. Journal of Agricultural and Food
Chemistry, 53, 8533-8536.
Li, W. D., Pickard, M. D., & Beta, T. (2007). Effect of thermal processing on antioxidant
properties of purple wheat bran. Food Chemistry, 104, 1080-1086.
Li, Z., Pan, Q. H., Cui, X. Y., & Duan, C. Q. (2010). Optimization on anthocyanins extraction
from wine grape skins using orthogonal test design. Food Science and Biotechnology, 19,
1047-1053.
Lin, L. D., Wu, J. Y., Ho, K. P., & Qi, S. Y. (2001). Ultrasound-induced physiological effects
and secondary metabolite (Saponin) production in Panax ginseng cell cultures. Ultrasound in
Medicine and Biology, 27, 1147-1152.
100
Liu, R. H. (2002). Supplement quick fix fails to deliver. Food Technology International, 1, 71 -
72.
Liu, R. H., & Felice, D. L. (2007). Antioxidants and whole food phytochemicals for cancer
prevention. In F. Shahidi & C.-T. Ho (Eds.), Antioxidant Measurement and Application.
Washington DC: American Chemical Society.
Maccarone, E., Maccarrone, A., & Rapisarda, P. (1985). Stabilization of anthocyanins of blood
orange fruit juice. Journal of Food Science, 50, 901-904.
Manzocco, L., Anese, M., & Nicoli, M. C. (1998). Antioxidant properties of tea extracts as
affected by processing. Food Science and Technology-Lebensmittel-Wissenschaft &
Technologie, 31, 694-698.
Markakis, P. (1982). Stability of anthocyanins in foods. In P. Markakis (Ed.), Anthocyanins as
Food Colors (pp. 163 - 180 ). New York, NY: Academic Press.
Markakis, P., & Jurd, L. (1974). Anthocyanins and their stability in foods. CRC Critical Reviews
in Food Technology, 4, 437 - 456.
Markaris, P., Livingston, G. E., & Fellers, C. R. (1957). Quantitative aspects of strawberry
pigment degradation. Journal of Food Science, 22, 117-130.
Marsellés-Fontanet, Á. R., & Martín-Belloso, O. (2007). Optimization and validation of PEF
processing conditions to inactivate oxidative enzymes of grape juice. Journal of Food
Engineering, 83, 452-462.
Marti, N., Perez-Vicente, A., & Garcia-Viguera, C. (2002). Influence of storage temperature and
ascorbic acid addition on pomegranate juice. Journal of the Science of Food and Agriculture,
82, 217-221.
101
Martinez-Tome, M., Murcia, M. A., Frega, N., Ruggieri, S., Jimenez, A. M., Roses, F., & Parras,
P. (2004). Evaluation of antioxidant capacity of cereal brans. Journal of Agricultural and
Food Chemistry, 52, 4690-4699.
Mazza, G., & Miniati, E. (1993). Anthocyanins in fruits, vegetables and grains. Boca Raton, FL:
CRC Press.
Miller, N. J., Diplock, A. T., & Riceevans, C. A. (1995). Evaluation of the total antioxidant
activity as a marker of the deterioration of apple juice oil storage. Journal of Agricultural and
Food Chemistry, 43, 1794-1801.
Mishra, D. K., Dolan, K. D., & Yang, L. (2008). Confidence intervals for modeling anthocyanin
retention in grape pomace during non-isothermal heating. Journal of Food Science, 73, E9-
E15.
Mizrahi, S. (1996). Leaching of soluble solids during blanching of vegetables by ohmic heating.
Journal of Food Engineering, 29, 153-166.
Mortensen, A., & Skibsted, L. H. (1997). Importance of carotenoid structure in radical-
scavenging reactions. Journal of Agricultural and Food Chemistry, 45, 2970-2977.
Mullen, W., Stewart, A. J., Lean, M. E. J., Gardner, P., Duthie, G. G., & Crozier, A. (2002).
Effect of freezing and storage on the phenolics, ellagitannins, flavonoids, and antioxidant
capacity of red raspberries. Journal of Agricultural and Food Chemistry, 50, 5197-5201.
Namiki, M. (1990). Antioxidants antimutagens in food. Critical Reviews in Food Science and
Nutrition, 29, 273-300.
Natella, F., Belelli, F., Ramberti, A., & Scaccini, C. (2010). Microwave and traditional cooking
methods: effect of cooking on antioxidant capacity and phenolic compounds content of seven
vegetables. Journal of Food Biochemistry, 34, 796-810.
102
Nawar, W. W. (1996). Lipids. In R. O. Fennema (Ed.), Food Chemistry (pp. 225 - 320). New
York, NY: Marcel Dekker, Inc.
Ngo, T., Wrolstad, R. E., & Zhao, Y. (2007). Color quality of Oregon strawberries - Impact of
genotype, composition, and processing. Journal of Food Science, 72, C25-C32.
Nicoli, M. C., Anese, M., & Parpinel, M. (1999). Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology, 10, 94-100.
Nicoli, M. C., Anese, M., Parpinel, M. T., Franceschi, S., & Lerici, C. R. (1997). Loss and/or
formation of antioxidants during food processing and storage. Cancer Letters, 114, 71-74.
Nindo, C. I., Sun, T., Wang, S. W., Tang, J., & Powers, J. R. (2003). Evaluation of drying
technologies for retention of physical quality and antioxidants in asparagus (Asparagus
officinalis, L.). Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology,
36, 507-516.
Ochoa, M. R., Kesseler, A. G., De Michelis, A., Mugridge, A., & Chaves, A. R. (2001). Kinetics
of colour change of raspberry, sweet (Prunus avium) and sour (Prunus cerasus) cherries
preserves packed in glass containers: light and room temperature effects. Journal of Food
Engineering, 49, 55-62.
Odriozola-Serrano, I., Soliva-Fortuny, R., & Martin-Belloso, O. (2009a). Impact of high-
intensity pulsed electric fields variables on vitamin C, anthocyanins and antioxidant capacity
of strawberry juice. Lwt-Food Science and Technology, 42, 93-100.
Odriozola-Serrano, I., Soliva-Fortuny, R., & Martin-Belloso, O. (2009b). Influence of storage
temperature on the kinetics of the changes in anthocyanins, vitamin C, and antioxidant
capacity in fresh-cut strawberries stored under high-oxygen atmospheres. Journal of Food
Science, 74, C184-C191.
103
Oliveira, M. E. C., & Franca, A. S. (2002). Microwave heating of foodstuffs. Journal of Food
Engineering, 53, 347-359.
Oms-Oliu, G., Odriozola-Serrano, I., Soliva-Fortuny, R., & Martín-Belloso, O. (2009). Use of
Weibull distribution for describing kinetics of antioxidant potential changes in fresh-cut
watermelon. Journal of Food Engineering, 95, 99-105.
Onyeneho, S. N., & Hettiarachchy, N. S. (1992). Antioxidant activity of durum-wheat bran.
Journal of Agricultural and Food Chemistry, 40, 1496-1500.
Opalic, M., Domitran, Z., Komes, D., Belscak, A., Horzic, D., & Karlovic, D. (2009). The effect
of ultrasound pre-treatment and air-drying on the quality of dried apples. Czech Journal of
Food Sciences, 27, S297-S300.
Ozer, E. A., Herken, E. N., Guzel, S., Ainsworth, P., & Ibanoglu, S. (2006). Effect of extrusion
process on the antioxidant activity and total phenolics in a nutritious snack food.
International Journal of Food Science and Technology, 41, 289-293.
Patras, A., Brunton, N. P., Da Pieve, S., & Butler, F. (2009). Impact of high pressure processing
on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of
strawberry and blackberry purees. Innovative Food Science & Emerging Technologies, 10,
308-313.
Patras, A., Brunton, N. P., O'Donnell, C., & Tiwari, B. K. (2010). Effect of thermal processing
on anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends in Food
Science & Technology, 21, 3-11.
Perez-Jimenez, J., & Saura-Calixto, F. (2005). Literature data may underestimate the actual
antioxidant capacity of cereals. Journal of Agricultural and Food Chemistry, 53, 5036-5040.
104
Piga, A., Del Caro, A., & Corda, G. (2003). From plums to prunes: Influence of drying
parameters on polyphenols and antioxidant activity. Journal of Agricultural and Food
Chemistry, 51, 3675-3681.
Plaza, L., Sanchez-Moreno, C., De Ancos, B., & Cano, M. P. (2006). Carotenoid content and
antioxidant capacity of Mediterranean vegetable soup (gazpacho) treated by high-
pressure/temperature during refrigerated storage. European Food Research and Technology,
223, 210-215.
Plaza, L., Sanchez-Moreno, C., Elez-Martinez, P., de Ancos, B., Martin-Belloso, O., & Cano, M.
P. (2006). Effect of refrigerated storage on vitamin C and antioxidant activity of orange juice
processed by high-pressure or pulsed electric fields with regard to low pasteurization.
European Food Research and Technology, 223, 487-493.
Portenlanger, G., & Heusinger, H. (1992). Chemical reactions induced by ultrasound and gamma
rays in aqueous solutions of L-ascorbic acid. Carbohydrate Research, 232, 291-301.
Porter, W. L., Levasseur, L. A., & Henick, A. S. (1977). Evaluation of some natural and
synthetic phenolic antioxidants in linoleic acid monolayers on silica. Journal of Food
Science, 42, 1533-1535.
Price, K. R., Bacon, J. R., & Rhodes, M. J. C. (1997). Effect of storage and domestic processing
on the content and composition of flavonol glucosides in onion (Allium cepa). Journal of
Agricultural and Food Chemistry, 45, 938-942.
Price, K. R., Casuscelli, F., Colquhoun, I. J., & Rhodes, M. J. C. (1998). Composition and
content of flavonol glycosides in broccoli florets (Brassica olearacea) and their fate during
cooking. Journal of the Science of Food and Agriculture, 77, 468-472.
105
Price, K. R., Prosser, T., Richetin, A. M. F., & Rhodes, M. J. C. (1999). A comparison of the
flavonol content and composition in dessert, cooking and cider-making apples; distribution
within the fruit and effect of juicing. Food Chemistry, 66, 489-494.
Prior, R. L. (2003). Fruits and vegetables in the prevention of cellular oxidative damage. Am J
Clin Nutr, 78, 570S-578.
Prior, R. L., Cao, G. H., Martin, A., Sofic, E., McEwen, J., O'Brien, C., Lischner, N., Ehlenfeldt,
M., Kalt, W., Krewer, G., & Mainland, C. M. (1998). Antioxidant capacity as influenced by
total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. Journal
of Agricultural and Food Chemistry, 46, 2686-2693.
Prior, R. L., Wu, X. L., & Schaich, K. (2005). Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural
and Food Chemistry, 53, 4290-4302.
Quaglia, G. B., Gravina, R., Paperi, R., & Paoletti, F. (1996). Effect of high pressure treatments
on peroxidase activity, ascorbic acid content and texture in green peas. Food Science and
Technology-Lebensmittel-Wissenschaft & Technologie, 29, 552-555.
Rajalakshmi, D., & Narasimhan, S. (1996). Food antioxidants: sources and methods of
evaluation. In Food Antioxidants: Technological, Toxicological, and Health Persepectives.
(pp. 65 - 157). New York, NY: Marcel Dekker, Inc.
Re, R., Bramley, P. M., & Rice-Evans, C. (2002). Effects of food processing on flavonoids and
lycopene status in a Mediterranean tomato variety. Free Radical Research, 36, 803-810.
Rehman, Z. U., Habib, F., & Shah, W. H. (2004). Utilization of potato peels extract as a natural
antioxidant in soy bean oil. Food Chemistry, 85, 215-220.
106
Rein, M. J., & Heinonen, M. (2004). Stability and enhancement of berry juice color. Journal of
Agricultural and Food Chemistry, 52, 3106-3114.
Reyes, L. F., & Cisneros-Zevallos, L. (2007). Degradation kinetics and colour of anthocyanins in
aqueous extracts of purple- and red-flesh potatoes (Solanum tuberosum L.). Food Chemistry,
100, 885-894.
Reyes, L. F., Miller, J. C., & Cisneros-Zevallos, L. (2004). Environmental conditions influence
the content and yield of anthocyanins and total phenolics in purple- and red-flesh potatoes
during tuber development. American Journal of Potato Research, 81, 187-193.
Reyes, L. F., Villarreal, J. E., & Cisneros-Zevallos, L. (2007). The increase in antioxidant
capacity after wounding depends on the type of fruit or vegetable tissue. Food Chemistry,
101, 1254-1262.
Rodriguez-Saona, L. E., Giusti, M. M., & Wrolstad, R. E. (1998). Anthocyanin pigment
composition of red-fleshed potatoes. Journal of Food Science, 63, 458-465.
Rodriguez-Saona, L. E., Giusti, M. M., & Wrolstad, R. E. (1999). Color and pigment stability of
red radish and red-fleshed potato anthocyanins in juice model systems. Journal of Food
Science, 64, 451-456.
Rommel, A., Wrolstad, R. E., & Heatherbell, D. A. (1992). Blackberry juice and wine -
processing and storage effects on anthocyanin composition, color and appearance. Journal of
Food Science, 57, 385-391.
Rossi, M., Giussani, E., Morelli, R., Lo Scalzo, R., Nani, R. C., & Torreggiani, D. (2003). Effect
of fruit blanching on phenolics and radical scavenging activity of highbush blueberry juice.
Food Research International, 36, 999-1005.
107
Rubinskiene, M., Viskelis, P., Jasutiene, I., Viskeliene, R., & Bobinas, C. (2005). Impact of
various factors on the composition and stability of black currant anthocyanins. Food
Research International, 38, 867-871.
Sablani, S. S., Andrews, P. K., Davies, N. M., Walters, T., Saez, H., Syamaladevi, R. M., &
Mohekar, P. R. (2010). Effect of thermal treatments on phytochemicals in conventionally and
organically grown berries. Journal of the Science of Food and Agriculture, 90, 769-778.
Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyanins and its
impact on color and in vitro antioxidant capacity. Molecular Nutrition & Food Research, 51,
1461-1471.
Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Thermal degradation of acylated and
nonacylated anthocyanins. Journal of Food Science, 71, C504-C512.
Sales, J. M., & Resurreccion, A. V. A. (2010). Maximizing phenolics, antioxidants and sensory
acceptance of UV and ultrasound-treated peanuts. Lwt-Food Science and Technology, 43,
1058-1066.
Sanchez-Moreno, C., De Ancos, B., Plaza, L., Elez-Martinez, P., & Cano, M. P. (2009).
Nutritional approaches and health related properties of plant foods processed by high
pressure and pulsed electric fields. Critical Reviews in Food Science and Nutrition, 49, 552-
576.
Sanchez-Moreno, C., Plaza, L., de Ancos, B., & Cano, M. P. (2003). Effect of high-pressure
processing on health-promoting attributes of freshly squeezed orange juice (Citrus sinensis
L.) during chilled storage. European Food Research and Technology, 216, 18-22.
Sanchez-Moreno, C., Plaza, L., Elez-Martinez, P., De Ancos, B., Martin-Belloso, O., & Cano,
M. P. (2005). Impact of high pressure and pulsed electric fields on bioactive compounds and
108
antioxidant activity of orange juice in comparison with traditional thermal processing.
Journal of Agricultural and Food Chemistry, 53, 4403-4409.
Sarma, A. D., Sreelakshmi, Y., & Sharma, R. (1997). Antioxidant ability of anthocyanins against
ascorbic acid oxidation. Phytochemistry, 45, 671-674.
Sarni-Manchado, P., Le Roux, E., Le Guerneve, C., Lozano, Y., & Cheynier, V. (2000). Phenolic
composition of litchi fruit pericarp. Journal of Agricultural and Food Chemistry, 48, 5995-
6002.
Sarni, P., Fulcrand, H., Souillol, V., Souquet, J. M., & Cheynier, V. (1995). Mechanisms of
anthocyanin degradation in grape must-like model solutions. Journal of the Science of Food
and Agriculture, 69, 385-391.
Schmidt, B. M., Erdman, J. W., & Lila, M. A. (2005). Effects of food processing on blueberry
antiproliferation and antioxidant activity. Journal of Food Science, 70, s389-s394.
Seabra, I. J., Braga, M. E. M., Batista, M. T. P., & de Sousa, H. C. (2010). Fractioned high
pressure extraction of anthocyanins from elderberry (Sambucus nigra L.) pomace. Food and
Bioprocess Technology, 3, 674-683.
Seeram, N. P., Bourquin, L. D., & Nair, M. G. (2001). Degradation products of cyanidin
glycosides from tart cherries and their bioactivities. Journal of Agricultural and Food
Chemistry, 49, 4924-4929.
Shahidi, F., & Zhong, Y. (2005)F. Shahidi (Ed.), Bailey's industrial oil and fat products (Vol. 1,
pp. 357 - 386). Hobiken, NY: John Wiley & Sons Ltd.
Shahidi, F., & Zhong, Y. (2007). Measurement of antioxidant activity in food and biological
systems. In Shahidi. F & Ho. C (Ed.), Antioxidant Measurement and Applications (pp. 36-
66): American Chemical Society, Wasington DC.
109
Shih, M.-C., Kuo, C.-C., & Chiang, W. (2009). Effects of drying and extrusion on colour,
chemical composition, antioxidant activities and mitogenic response of spleen lymphocytes
of sweet potatoes. Food Chemistry, 117, 114-121.
Singh, N., & Rajini, P. S. (2004). Free radical scavenging activity of an aqueous extract of potato
peel. Food Chemistry, 85, 611-616.
Singh, R. P., Murthy, K. N. C., & Jayaprakasha, G. K. (2002). Studies on the antioxidant activity
of pomegranate (Punica granatum) peel and seed extracts using in vitro models. Journal of
Agricultural and Food Chemistry, 50, 81-86.
Skrede, G., Wrolstad, R. E., & Durst, R. W. (2000). Changes in anthocyanins and polyphenolics
during juice processing of highbush blueberries (Vaccinium corymbosum L.). Journal of
Food Science, 65, 357-364.
Song, H. P., Kim, D. H., Jo, C., Lee, C. H., Kim, K. S., & Byun, M. W. (2006). Effect of gamma
irradiation on the microbiological quality and antioxidant activity of fresh vegetable juice.
Food Microbiology, 23, 372-378.
Spanos, G. A., Wrolstad, R. E., & Heatherbell, D. A. (1990). Influence of processing and storage
on the phenolic composition of apple juice. Journal of Agricultural and Food Chemistry, 38,
1572-1579.
Srivastava, A., Harish, S. R., & Shivanandappa, T. (2006). Antioxidant activity of the roots of
Decalepis hamiltonii (Wight & Arn.). LWT - Food Science and Technology, 39, 1059-1065.
Stevenson, D. G., Inglett, G. E., Chen, D., Biswas, A., Eller, F. J., & Evangelista, R. L. (2008).
Phenolic content and antioxidant capacity of supercritical carbon dioxide-treated and air-
classified oat bran concentrate microwave-irradiated in water or ethanol at varying
temperatures. Food Chemistry, 108, 23-30.
110
Stintzing, F. C., & Carle, R. (2004). Functional properties of anthocyanins and betalains in
plants, food, and in human nutrition. Trends in Food Science & Technology, 15, 19-38.
Stojceska, V., Ainsworth, P., Plunkett, A., Ibanoglu, E., & Ibanoglu, S. (2008). Cauliflower by-
products as a new source of dietary fibre, antioxidants and proteins in cereal based ready-to-
eat expanded snacks. Journal of Food Engineering, 87, 554-563.
Sun, T., & Powers, J. R. (2007). Antioxidants and antioxidant activities of vegetables. In F.
Shahidi. & C. T. Ho (Eds.), Antioxidant Measurement and Applicaitons (pp. 160 - 183).
Washington, DC: American Chemical Society.
Suslick, K. S. (1988). Ultrasounds: its chemical, physical and biological effects. New York, NY:
VHC Publishers.
Talcott, S. T., Brenes, C. H., Pires, D. M., & Del Pozo-Insfran, D. (2003). Phytochemical
stability and color retention of copigmented and processed muscadine grape juice. Journal of
Agricultural and Food Chemistry, 51, 957-963.
Talcott, S. T., Howard, L. R., & Brenes, C. H. (2000a). Antioxidant changes and sensory
properties of carrot puree processed with and without periderm tissue. Journal of
Agricultural and Food Chemistry, 48, 1315-1321.
Talcott, S. T., Howard, L. R., & Brenes, C. H. (2000b). Contribution of periderm material and
blanching time to the quality of pasteurized peach puree. Journal of Agricultural and Food
Chemistry, 48, 4590-4596.
Tanchev, S., & Ioncheva, N. (1976). Products of thermal degradation of the anthocyanins
cyanidin-3-glucoside, cyanidin-3-rutinoside and cyanidin-3-sophoroside. Food / Nahrung,
20, 889-893.
111
Tanchev, S. S. (1972). Kinetics of the thermal degradation of anthocyanins of the raspberry.
Zeitschrift für Lebensmitteluntersuchung und -Forschung A, 150, 28-30.
Tanchev, S. S., & Yoncheva, N. (1974). Kinetics of thermal degradation of the anthocyanins
delphinidin-3-rutinoside and malvidin-3-glucoside. Food / Nahrung, 18, 747-752.
Temple, N. J. (2000). Antioxidants and disease: More questions than answers. Nutrition
Research, 20, 449-459.
Terao, J., Karasawa, H., Arai, H., Nagao, A., Suzuki, T., & Takama, K. (1993). Peroxyl radical
scavenging activity of caffeic acid and its related phenolic compounds in solution. Bioscience
Biotechnology and Biochemistry, 57, 1204-1205.
Thakur, B. R., & Arya, S. S. (1989). Studies on stability of blue grape anthocyanins.
International Journal of Food Science and Technology, 24, 321-326.
Tiwari, B. K., O'Donnell, C. P., & Cullen, P. J. (2009). Effect of sonication on retention of
anthocyanins in blackberry juice. Journal of Food Engineering, 93, 166-171.
Tsai, P. J., & Huang, H. P. (2004). Effect of polymerization on the antioxidant capacity of
anthocyanins in Roselle. Food Research International, 37, 313-318.
Tsai, P. J., Huang, H. P., & Huang, T. C. (2004). Relationship between anthocyanin patterns and
antioxidant capacity in mulberry wine during storage. Journal of Food Quality, 27, 497-505.
Tudela, J. A., Cantos, E., Espin, J. C., Tomas-Barberan, F. A., & Gil, M. I. (2002). Induction of
antioxidant flavonol biosynthesis in fresh-cut potatoes. Effect of domestic cooking. Journal
of Agricultural and Food Chemistry, 50, 5925-5931.
Turkmen, N., Sari, F., & Velioglu, Y. S. (2005). The effect of cooking methods on total
phenolics and antioxidant activity of selected green vegetables. Food Chemistry, 93, 713-
718.
112
Uyan, S. E., Baysal, T., Yurdagel, O., & El, S. N. (2004). Effects of drying process on
antioxidant activity of purple carrots. Nahrung-Food, 48, 57-60.
Vagi, E., Rapavi, E., Hadolin, M., Peredi, K. V., Balazs, A., Blazovics, A., & Simandi, B.
(2005). Phenolic and triterpenoid antioxidants from Origanum majorana L. herb and extracts
obtained with different solvents. Journal of Agricultural and Food Chemistry, 53, 17-21.
van den Berg, R., Haenen, G. R. M. M., van den Berg, H., & Bast, A. (1999). Applicability of an
improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant
capacity measurements of mixtures. Food Chemistry, 66, 511-517.
Van Gorsel, H., Li, C., Kerbel, E. L., Smits, M., & Kader, A. A. (1992). Compositional
characterization of prune juice. Journal of Agricultural and Food Chemistry, 40, 784-789.
Vinson, J. A., Dabbagh, Y. A., Serry, M. M., & Jang, J. H. (1995). Plant flavonoids, especially
tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease.
Journal of Agricultural and Food Chemistry, 43, 2800-2802.
Vinson, J. A., Hao, Y., Su, X., & Zubik, L. (1998). Phenol antioxidant quantity and quality in
foods: vegetables. Journal of Agricultural and Food Chemistry, 46, 3630-3634.
Viscidi, K. A., Dougherty, M. P., Briggs, J., & Camire, M. E. (2004). Complex phenolic
compounds reduce lipid oxidation in extruded oat cereals. Lebensmittel-Wissenschaft Und-
Technologie-Food Science and Technology, 37, 789-796.
Volden, J., Borge, G. I. A., Bengtsson, G. B., Hansen, M., Thygesen, I. E., & Wicklund, T.
(2008). Effect of thermal treatment on glucosinolates and antioxidant-related parameters in
red cabbage (Brassica oleracea L. ssp capitata f. rubra). Food Chemistry, 109, 595-605.
Von Elbe, J. H., & Schwartz, S. J. (1996). Colorants. In O. R. Fennema (Ed.), Food Chemistry
(Third ed., pp. 651 - 722). New York, NY: Marcel Dekker, Inc.
113
Wang, H., Cao, G. H., & Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins.
Journal of Agricultural and Food Chemistry, 45, 304-309.
Wang, W. D., & Xu, S. Y. (2007). Degradation kinetics of anthocyanins in blackberry juice and
concentrate. Journal of Food Engineering, 82, 271-275.
White, P. J., & Xing, Y. (1997). Antioxidants from cereals and legumes. In F. Shahidi (Ed.),
Natural Antioxidants: Chemistry, Health Effects, and Applications. Champaign, IL: AOCS
Press.
Wolfe, K. L., & Liu, R. H. (2003). Apple peels as a value-added food ingredient. Journal of
Agricultural and Food Chemistry, 51, 1676-1683.
Wong, D. W. S. (1989). Mechanism and theory in food chemistry. New York: Van Nostrand
Reinhold.
Wright, J. S., Johnson, E. R., & DiLabio, G. A. (2001). Predicting the activity of phenolic
antioxidants: Theoretical method, analysis of substituent effects, and application to major
families of antioxidants. Journal of the American Chemical Society, 123, 1173-1183.
Wrolstad, R. E., Skrede, G., Lea, P., & Enersen, G. (1990). Influence of Sugar on Anthocyanin
Pigment Stability in Frozen Strawberries. Journal of Food Science, 55, 1064-&.
Wrona, M., Korytowski, W., Rózanowska, M., Sarna, T., & Truscott, T. G. (2003). Cooperation
of antioxidants in protection against photosensitized oxidation. Free Radical Biology and
Medicine, 35, 1319-1329.
Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., & Prior, R. L. (2004).
Lipophilic and hydrophilic antioxidant capacities of common foods in the United States.
Journal of Agricultural and Food Chemistry, 52, 4026-4037.
114
Wu, X. L., Gu, L. W., Holden, J., Haytowitz, D. B., Gebhardt, S. E., Beecher, G., & Prior, R. L.
(2004). Development of a database for total antioxidant capacity in foods: a preliminary
study. Journal of Food Composition and Analysis, 17, 407-422.
Xu, Z., Wu, J., Zhang, Y., Hu, X., Liao, X., & Wang, Z. (2010). Extraction of anthocyanins from
red cabbage using high pressure CO2. Bioresource Technology, 101, 7151-7157.
Yen, G. C., Chen, H. Y., & Peng, H. H. (1997). Antioxidant and pro-oxidant effects of various
tea extracts. Journal of Agricultural and Food Chemistry, 45, 30-34.
Yue, X., & Xu, Z. (2008). Changes of anthocyanins, anthocyanidins, and antioxidant activity in
bilberry extract during dry heating. Journal of Food Science, 73, C494-C499.
Yun-Zhong, F., Sheng, Y., & Guoyao, W. (2002). Free radicals, antioxidants, and nutrition.
Nutrition (Burbank, Los Angeles County, Calif.), 18, 872-879.
Zabetakis, I., Koulentianos, A., Orruño, E., & Boyes, I. (2000). The effect of high hydrostatic
pressure on strawberry flavour compounds. Food Chemistry, 71, 51-55.
Zabetakis, I., Leclerc, N., & Kajda, P. (2000). The effect of high hydrostatic pressure on the
strawberry anthocyanins. Journal of Agricultural and Food Chemistry, 48, 2749-2754.
Zadernowski, R., Nowak-Polakowska, H., & Rashed, A. A. (1999). The influence of heat
treatment on the activity of lipo- and hydrophilic components of oat grain. Journal of Food
Processing and Preservation, 23, 177-191.
Zadernowskl, R., Nowak-Polakowska, H., & Rashed, A. A. (1999). The influence of heat
treatment on the activity of lipo-and hydrophilic components of oat grain. Journal of Food
Processing and Preservation, 23, 177-191.
Zern, T. L., Wood, R. J., Greene, C., West, K. L., Liu, Y. Z., Aggarwal, D., Shachter, N. S., &
Fernandez, M. L. (2005). Grape polyphenols exert a cardioprotective effect in pre- and
115
postmenopausal women by lowering plasma lipids and reducing oxidative stress. Journal of
Nutrition, 135, 1911-1917.
Zhang, D. L., & Hamauzu, Y. (2004). Phenolics, ascorbic acid, carotenoids and antioxidant
activity of broccoli and their changes during conventional and microwave cooking. Food
Chemistry, 88, 503-509.
Zhang, D. L., & Quantick, P. C. (1997). Effects of chitosan coating on enzymatic browning and
decay during postharvest storage of litchi (Litchi chinensis Sonn.) fruit. Postharvest Biology
and Technology, 12, 195-202.
Zhang, Y., Liao, X. J., Ni, Y. Y., Wu, J. H., Hu, X. S., Wang, Z. F., & Chen, F. (2007). Kinetic
analysis of the degradation and its color change of cyanidin-3-glucoside exposed to pulsed
electric field. European Food Research and Technology, 224, 597-603.
Zhou, K. Q., & Yu, L. L. (2004). Effects of extraction solvent on wheat bran antioxidant activity
estimation. Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology, 37,
717-721.
Zielinski, H., & Kozlowska, H. (2000). Antioxidant activity and total phenolics in selected cereal
grains and their different morphological fractions. Journal of Agricultural and Food
Chemistry, 48, 2008-2016.
Zielinski, H., Kozlowska, H., & Lewczuk, B. (2001). Bioactive compounds in the cereal grains
before and after hydrothermal processing. Innovative Food Science & Emerging
Technologies, 2, 159-169.
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CHAPTER THREE
COLORED POTATOES (SOLANUM TUBEROSUM L.) DRIED FOR
ANTIOXIDANT-RICH VALUE-ADDED FOODS
ABSTRACT
Colored potatoes (Solanum tuberosum L.) are a significant source of antioxidants from
polyphenols, carotenoids, and ascorbic acid. In this study, retention of total antioxidants in fresh
colored potatoes and processed potato flakes prepared as potential ingredients for snack foods
was studied. Total antioxidant capacity, total phenolics and total anthocyanins were higher in
purple potato flesh compared to those from red, yellow and white potato cultivars. Peeled purple
potatoes were blanched and dehydrated by freeze-drying, drum-drying and refractance window-
drying to prepare potato flakes. Results showed no significant losses (P > 0.05) in total
antioxidant capacity and total phenolic content in flakes in all drying methods obtained under
study. However, 45, 41 and 23 % losses in total anthocyanins content were observed in potato
flakes after freeze-drying, drum-drying and refractive window-drying, respectively. Colored
potatoes could provide an excellent source of antioxidant-rich ingredient for the production of
nutritionally enhanced food products.
1. INTRODUCTION
Antioxidant phytochemicals in plants have recently attracted great attention from the
research community, food industry, and consumers. A large number of scientific papers report
the important role of phytochemicals in preventing many chronic diseases that are related to
oxidative stress caused by free radicals (van den Berg et al., 1999). Free radicals are associated
117
with cancer, inflammation, atherosclerosis and ageing (Halliwell et al., 1992). Phytochemicals,
such as polyphenols in fruits and vegetables, possess high antioxidant activities that control the
generative oxidative reaction caused by reactive oxygen in living tissues (Kaur and Kapoor,
2001; Lachman et al., 2005). It has been reported that phenolic compounds including
anthocyanins (Figure 3.1) have potential to scavenge free radicals (Kalt et al., 1999).
Antioxidant capacity has been highly correlated to the amount of phenolic compounds and
anthocyanins present in dark colored fruits and vegetables (Brown et al., 2003; Prior et al.,
1998). Among common commercial fruits, blueberries have the largest antioxidant capacity
(Wang et al., 1996). It has also been reported that Andean purple corn and red-fleshed sweet
potatoes have even higher antioxidant capacity and antiradical activity than blueberries and
higher or similar content of phenolic compounds and anthocyanins (Cevallos-Casals and
Cisneros-Zevallos, 2003).
Potatoes (Solanum tuberosum L.) have traditionally been perceived by consumers as a
starchy food. Lewis, Walker, Lancaster & Sutton (1998) and more recently Jansen & Flamme
(2006) have reported phenolic, anthocyanin, and flavonoid contents of different varieties of
colored potato cultivars and breeding clones. Colored potatoes have attracted the attention of
investigators as well as consumers due to their antioxidant activities, taste, and appearance. The
antioxidant activity in colored potatoes are associated with the presence of polyphenols
anthocyanins, flavonoids, carotenoids, ascorbic acid, tocopherols, alpha-lipoic acid and selenium
(Lachman et al., 2005). Therefore, colored potatoes have the potential to be one of the richest
sources of antioxidants in the human diet. Brown (2005) reported 0.5 to 1 µg of carotenoids per
gram fresh weight (FW) in white, up to 20 µg per gram FW in deeply yellow to orange and 0.09
to 0.38 mg of total anthocyanins per gram FW in purple and red potato cultivars. The
118
investigator also reported that an average potato contains 0.20 mg of vitamin C per gram FW that
contributes 13 % of total antioxidant activity in the tuber. Han et al., (2006a) reported that
purple potato extract prevented liver injury induced by D-galactosamine in rats. They also
reported that purple potato flakes have radical scavenging activities inhibiting linoleic acid
oxidation and improved antioxidant potential in rats by enhancing hepatic Mn-superoxide
dismutase (SOD), Cu/Zn-SOD and glutathione peroxidase (GSH-Px) mRNA expression.
Important food processing operations such as drying, cooking, and extrusion may affect the
retention of antioxidants in food matrices (Nicoli et al., 1999). However, other than vitamin C,
there is limited literature available on the effects of drying/thermal treatments on antioxidant
activities of potatoes.
Blanching and drying are the two most important unit operations in preparing shelf-stable
potato flakes as ingredients for commercial production of a wide range of foods products,
O+
OH
OH
OH
O G
Pelargonidin-3-glucoside
O+
OH
OH
OH
O G
OH
Cyanidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
OCH3
Malvidin-3-glucoside
O+
OH
OH
OH
O G
OH
OH
Delphidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
Petunidin-3-glucoside
O+
OH
OH
OH
O G
OCH3
Peonidin-3-glucoside
OH
Figure 3. 1. Structure of anthocyanins; G – Glucosides.
119
including mashed potato and extruded snack foods. The objectives of this study were (i) to
quantify the total antioxidant capacity (TAC), total phenolics (TP), and total anthocyanins (TA)
in selected potato cultivars and (ii) to study the effect of blanching and consequent drying on the
retention of TAC, TP, and TA in purple potato cultivars.
2. MATERIALS AND METHODS
2.1. Raw Materials
Red (‗Red Rodeo‘) potatoes were purchased from an Oregon State (USA) potato grower.
Yellow (‗Yukon Gold‘) and white (‗Russet‘) potatoes were purchased from a local store in
Pullman, WA (USA). ‗Purple Majesty‘ potatoes were purchased in Fall of 2007 from Kiska
Farms, Burbank, WA (USA) and SLV Research Center, Colorado State University. The potatoes
were placed in a storage room at 4°C and 80 % relative humidity. Fresh purple potatoes procured
from Kiska Farms, were used for production of flakes by refractance window-drying comparison
with fresh red, yellow and white potatoes. Purple potatoes procured from Colorado State
University were used for the production of drum dried and freeze dried flakes.
2.2. Production of potato flakes
Based on the screening of potato varieties, flakes were prepared only from purple potatoes
for further investigation of the effects of blanching and dehydration on TAC, TP and TA. Stored
purple potatoes were peeled with an abrasive peeler for about 75 seconds. Peeled potatoes were
sliced with a mechanical slicer (Machine type RG-7, Ab Hallade Maskiner, Spanga, Sweden) to
6 mm thick before blanching in a steam blancher for 8 min to inactivate polyphenolic oxidase
(PPO) similar to the procedures reported for peroxidase inactivation (Reyes and Cisneros-
Zevallos, 2007). Blanched potato slices were cooled in ice water for 8 min and then pureed
120
using a mixer (The Hobart Mfg Company, Troy, OH, USA). Additional water was added to the
puree to make it of uniform consistency, before placing the puree into the dryers.
2.2.1. Freeze-drying
Freeze-drying was selected as the reference method for drying. The potato puree was poured
on a tray to approximately 2 mm thick, frozen at -35°C for 1 h, placed into a freeze dryer and
dehydrated for 24 h at 3.33 Pa. The upper plate of the dryer was maintained at 20°C while the
condenser temperature was set at -64 °C. The dried samples were packed in polyethylene bags,
flushed with nitrogen, wrapped in aluminum sheets and stored at -30 °C for further analysis and
use.
2.2.2. Drum-drying
A 15.24cm × 20.32cm drum pilot scale counter rotating twin drum dryer (Blaw Knox Food
& Chemical Equipment Co., Buffalo, NY, USA) was used to dehydrate the puree. Pressurized
steam to the drums was maintained at 413 kPa corresponding to a saturation temperature of
water at 145 °C. The surface temperature of the drums was 135 - 138 °C. The gap between the
drums, rotating at 1.13 rpm, was set to 0.3 mm. The dried samples, packed in polyethylene bags,
were flushed with nitrogen wrapped in aluminum sheet and stored at -30 °C for further analysis
and use.
2.2.3. Refractance window-drying (RW)
A pilot scale refractance window dryer of an effective length and width of 1.83 × 0.60 m
developed by MCD Technologies, Inc. (Tacoma, Washington, USA) was used to dehydrate the
potato puree. The dryer consisted of a plastic conveyer belt rotating at speed of 1.04 m/min in
121
contact with hot water circulated at 95 °C. Potato puree prepared using a Hobert mixer was
poured on a roller feeder, which deposited a thin layer (1 mm) of puree on the conveyor belt.
The conveyer belt transported the puree over a heating section for drying, then through a cooling
section, before the dried flakes were scraped from the end. Average air velocity over the
conveyer was 0.7 m/s and residence time of the puree on the belt was 1min 55 sec. Dried flakes
were packed in polyethylene bags, flushed with nitrogen, kept in aluminum bags, heat sealed and
stored at -30 °C for further analysis and use.
2.3. Chemical Analysis
Moisture content of the raw cultivars and dried flakes was determined by the vacuum oven
method (AOAC, 1995).
Folin-Ciocalteu reagent, potassium chloride, sodium acetate, Trolox (6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid), DPPH (2, 2-diphenyl-1-picrylhydrazyl) and gallic acid
were purchased from Sigma-Aldrich. Laboratory grade methanol and ethanol were used in
extraction and preparation of samples.
2.3.1. Total antioxidant capacity (TAC)
Total antioxidant capacities of raw potatoes were quantified using the DPPH (2, 2-diphenyl-
1-picrylhydrazyl) assay (Brandwilliams et al., 1995). DPPH, a stable radical deep purple in
color, is reduced in the presence of antioxidants decolorizing the solution. The loss of color
results in a decrease in the absorbance intensity, thus providing a basis for measurement of
antioxidant activities in the extracts. Thirty gram of potatoes peeled by an abrasive peeler
(Model 15A, MJM Mfg Co., Culver city, CA, USA) were chopped and homogenized with 100
ml of HPLC grade methanol to a uniform consistency by a homogenizer (Omni Mix
122
Homogenizer, Omni International, Waterbury, CT, USA). The samples were centrifuged
(Beckman J2-HS Centrifuge, USA) at 30000g at 4 °C for 20 min and the supernatants stored at -
20 °C for further analysis. A 6 × 10-5
M DPPH solution was prepared in methanol and stored at -
20 °C for analysis.
Stored supernatants were diluted two fold with methanol and 0.05 ml of diluted supernatants
was added to 1.95 ml of DPPH solution in a cuvette. Aliquots in cuvettes were covered with
Parafilm and vortexed with a mini vortexer (MV1, Wilmington, NC, USA) before taking
absorbance readings at selected times with a Ultraspec 4000 UV/visible spectrophotometer
(Pharmacia Biotech, Cambridge, England) until absorbance values reached a plateau. The
spectrophotometer was blanked with methanol. DPPH without sample was taken as control. For
each sample measured, the percentage of DPPH remaining was calculated as follows:
100)(0@
@
Tabsorbance
tTabsorbance
remainingDPPH
DPPHDPPH
where DPPH absorbance@T=t is the absorbance of DPPH at time t min and DPPH absorbance@T=0 is the
absorbance of DPPH at zero min. Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid) was used as a standard. The absorbance readings at 2h after which there was no additional
change were used to calculate TAC from the trolox standard curve. All the values of TAC were
expressed as micrograms of trolox equivalent per gram of dry weight sample (µg TE/g DW) ±
SD for three replications.
Total antioxidant capacity in purple dried flakes extracts were prepared from 10 g of dried
flakes initially rehydrated with 40 ml water and blended with 40 ml HPLC grade methanol. The
final volume of the mixture was made to 100 ml with aqueous methanol (50:50 v/v). The
mixture was centrifuged at 30,000g at 4 °C for 20 min and supernatants stored at -20 °C for
further analysis.
123
2.3.2. Total phenolics (TP)
Total phenolics were determined from extracts prepared for TAC using an Folin-Ciocalteau
colorimetric method as described by Swain & Hillis (1959) and Singleton & Rossi (1965).
Supernatants were diluted four-fold with methanol. To 0.5 ml aliquots 8 ml of deionized water
was added followed by 0.5 ml of 0.25 N Folin-Ciocalteu (FC) reagent, the samples were
vortexed, kept for 3 min and 1 ml of 1 N sodium carbonate was added and the mixture vortexed.
The samples were kept at room temperature for 2 hours before taking readings at 725 nm in the
UV Spectrophotometer. One-half milliliter methanol was treated in the same way as the diluted
samples and used as a blank. The TP analyses were triplicated and means (± SD) reported as
micrograms gallic acid equivalent per gram of dry weight sample (µg GAE/g DW) from a
standard curve developed for gallic acid.
2.3.3. Total anthocyanins
Potatoes were extracted for anthocyanin analysis using the procedure of Fuleki & Francis
(1968) with modifications. Raw stored potatoes were peeled with an abrasive peeler for 75 sec
and the surface moisture removed with tissue paper. Peeled potatoes were chopped manually to
small pieces and 30 g homogenized with 150 ml of acidified ethanol (95% ethanol/1.5 N HCl,
85:15 v/v) to a uniform consistency. The samples were kept for 10 min at room temperature and
extracts were decanted into a beaker. The residues were washed with 100 ml of acidified ethanol
and extracts from first and second washings and residues were mixed, covered with Parafilm,
and stored for 90 min at 4 °C. The samples were centrifuged at 23000 x g at 4 °C for 15 min
and supernatants stored at -20 °C for further analysis.
Quantification of anthocyanins was carried out using the pH differential method described
124
by Giusti & Wrolstad (2001). Diluted aliquots containing 0.2 ml of anthocyanin extract and 1.8
ml buffer were prepared in 2 ml cuvettes with KCl buffer (pH 1.0) or sodium acetate buffer (pH
4.5), respectively. The diluted aliquots were vortexed with a mini vortexer and equilibrated for
15 min at room temperature. Absorbance readings were taken at maximum wavelength (λ-max)
of 535nm for purple cultivars, 515nm for red and yellow cultivars and 700 nm for correcting for
turbidity (Reyes and Cisneros-Zevallos, 2003) in a UV/visible spectrophotometer, previously
blanked with distilled water. Total anthocyanins in purple cultivars were quantified by
considering malvidin-3-glucoside (Han et al., 2006b; Jansen and Flamme, 2006; Lewis et al.,
1998) as the major anthocyanin with MW =718.5 g/mol and molar extinction coefficient of
30200 l/cm per mol. Pelargonidin-3-glucosides having e = 27300 l/cm per mol and MW = 486.5
g/mol (Fuleki and Francis, 1968) were considered as the major anthocyanins in red cultivars,
whereas, cyanidin-3-glucosides were considered as the major anthocyanin in yellow cultivars.
Total anthocyanins content in potato extracts were calculated according to the formula (Giusti
and Wrolstad, 2001):
de
DFMWAlmgC
*
**)/(
where A = absorbance of the sample, MW = molecular weight of major anthocyanin, DF =
dilution factor, e = molar extinction coefficient of major anthocyanin and d = path length of the
cuvette (1 cm). The absorbances of the samples were calculated as
5.4700max0.1700max )()( pHpH AAAAA . Total anthocyanins were expressed as mg of major
anthocyanin per gram of DW sample mean ± SD for three replications.
For total anthocyanins determination in dehydrated purple potato flakes, 5 g of flakes were
rehydrated with 40 ml water and blended with 40 ml of acidified ethanol (95 % ethanol/1.5 N
125
HCl (85:15 v/v)). The samples were thoroughly mixed by stirring manually and decanted to a
separate beaker. The residues were washed again, the extracts decanted and mixed with the first
decanted extracts. The total extracts were made to a total volume of 100 ml with 50:50 (v/v)
water: acidified ethanol and centrifuged at 23000g at 4 °C for 15 min. The supernatants were
stored at -20 °C for further analysis.
2.4. Color determination
Color of raw cultivars and dehydrated flakes was determined using a color meter (Minolta
Chroma CR200, Minolta Co., Osaka, Japan) with L*a*b* values depicting brightness,
greenness/redness and blue/yellowness, respectively. The color meter was calibrated with white
and black standards provided by the manufacturer. The hue angle h° [h°=arctan(b*/a*)] and
Chromaticity C* [C*=(a*2+b*
2)1/2
] were computed from a* and b*. Color differences between
dehydrated samples and raw samples were expressed as ∆E* where ∆E*= [(∆L*) 2
+ (∆a*) 2
+
(∆b*) 2]
1/2. Peeled potatoes were sliced to measure the color attributes in raw cultivars.
Dehydrated flakes were ground with a mortar and pestle and covered with an ultra-thin
transparent polyethylene sheet before measurements were taken.
2.5. Statistical analysis
Color, TAC, TP and TA data for raw and dehydrated potato samples were analyzed with
SAS software (version 9.1). Significant differences among treatments were determined using
ANOVA followed by Tukey‘s pair-wise comparisons at 95 % confidence level (P≤0.05).
Triplicate (n=3) data obtained from the different analyses were reported as arithmetic mean and
standard deviation.
126
3. RESULTS AND DISCUSSION
3.1. Moisture content, total antioxidant capacity (TAC), total phenolics (TP) and total
anthocyanins (TA) in raw potatoes
Average moisture contents of the selected raw potato tubers were 77% (wet basis). There was
no significant difference (P > 0.05) in moisture contents between cultivars. Also, no significant
differences (P > 0.05) were observed in TAC, TP, and TA contents between purple potatoes
procured from Washington and Colorado state locations. However, it was observed that purple
potatoes contained more TAC, TP, and TA than red, yellow and white potatoes cultivars (Table
3.1). The TAC among the different sample cultivars ranged from 2403 to 9605 µg TE/g DW.
Samples from purple cultivars without skin had the highest TAC with 9605 µg TE/g DW.
White, red and yellow had similar TAC of ~2400–2500 µg TE/g DW. Reddivari, Hale & Miller
(2007) reported that antioxidant activity of whole potato cultivars varied from 157 to 832 µg
TE/g of FW using DPPH assay and 810 to 1622 µg TE/g of FW using ABTS assay. A higher
antioxidant capacity of whole purple and red-fleshed potatoes ranging from 513 to 1426 µg
TE/g of FW has also been reported (Reyes et al., 2005).
Table 3. 1. Total antioxidant capacity by DPPH assay, total phenolics content and total
anthocyanins of selected raw potato cultivars expressed as quantity per gram of dry weight
sample (n=3).
Cultivars Dry matter
(g/g of sample)
Total Antioxidant
Capacity
(µg Trolox/g DW)
Total Phenolics
(µg GAE /g DW)
Total
Anthocyanins
(mg/g DW)
Purple 0.23 9605 ± 404a 3347 ± 198
a 1.08 ± 0.09
a
Red 0.24 2542 ± 120 b 1457 ± 240
c 0.014 ± 0.004
b
Yellow 0.23 2403 ± 90 b 1425 ± 200
c 0.014 ± 0.004
b
White 0.23 2518 ± 471 b 2096 ± 482
b -
Significant differences within the values in the same column are indicated by different
superscript letters (P < 0.05).
127
The amount of TP varied from 1425 to 3347 µg GAE/g DW among the cultivars under study.
The TP content in the flesh of purple potato cultivars was significantly higher (3347 µg GAE/g
DW) than those obtained in white (2096 µg GAE/g DW), red (1457 µg GAE/g DW), and yellow
(1425 µg GAE/g DW) cultivars. Andre et al.(2007) have previously reported TP content in white
and colored potato cultivars in the range of 2360 to 12370 μg GAE/g FW.
Total anthocyanins content in the flesh of purple potato cultivars (from Washington state)
was 1.08 mg MV-3-GLU /g DW, while the red and yellow cultivars contained smaller amount of
TA (Table 3.1). Similarly, Reyes et al. (2005) reported that the TA content in the flesh of purple
potatoes ranged from 0.11 to 1.74 mg CY-3-GLU / g FW and from 0.21 to 0.55 mg CY-3-GLU /
g FW in the flesh of red potatoes. Dark purple-black tubers having 2 to 5 mg of MV-3-GLU /g
FW and red-fleshed potato cultivars with 0.02 to 0.40 mg pelargonidin-3- glucoside/g FW was
reported by Rodriguez-Saona, Giusti & Wrolstad (1998). The differences in TAC, TP and TA
content for the studied cultivars could be due to environmental conditions, growing location,
harvesting date, maturity, sample preparation, and extraction methods used for their evaluation.
3.2. Effects of blanching on TAC, TP and TA
Preliminary refractance window-drying (RWD) of potato puree from purple cultivars without
blanching caused complete loss of purple color in the flakes (Figure 3.2). This loss in color may
be due to PPO activity (Reyes and Cisneros-Zevallos, 2007). Color loss in purple potato puree
was observed, once tubers were cut into slices and ground. The color of the puree turned brown
and became colorless after RW dehydration. Therefore, in an effort to prevent enzymatic
browning to occur, we steam blanched purple potato slices for 8 minutes prior to processing
them into puree for subsequent drying.
128
Purple potato product obtained from steam blanched puree, prior to drum-drying and freeze-
drying, retained about 90 % of TA compared to raw puree (Table 3.2). Reyes et al. (2007)
reported that red and purple fleshed potatoes of ~5 mm thickness steam blanched for 3 min had
98 % reduction in peroxidase activity. Retention of 23 % of TA in blueberries was reported by
Rossi, Giussani, Morelli, Lo Scalzo, Nani & Torreggiani (2003) after blanching compared to
12% without blanching, before processing into juice. Using a reverse-phase HPLC separation
system for separating individual anthocyanins, Rossi et al. (2003) observed that recovery of
anthocyanin from blueberries after blanching was maximum for delphinidin-3-arabinoside
(1936%) followed by petunidin (586 %) and cyanidin (191 %) glucosides, whereas malvidin
glucosides showed the least recovery (143 %). The investigators also reported that the minor
recovery of malvidin glucosides could be due to the structure of the anthocyanin having a single
hydroxyl group on its phenolic ring that was least affected by PPO compared to the other
anthocyanins. In the present study, puree from purple potatoes without blanching prior to RWD
did not retain any color. The complete loss of color in the puree could be due to the activity of
PPO. On the other hand, retention of anthocyanins and color in potato puree after blanching
Figure 3. 2. Color of potato flakes; (A) without blanching; (B) with blanching
129
could be due to inactivation of PPO and subsequent reduction in enzymatic anthocyanin
degradation.
Blanching of sliced potatoes also showed a significant increase (P < 0.05) in TAC (75 %)
and TP (108 %) in the puree compared to unblanched puree (Table 3.2). Wang (2002) reported
an increase in total antioxidant activities of 7 and 26 % after water and microwave blanching of
asparagus spears, respectively. The increases in the TAC and TP content after blanching could
be due to the greater extraction yield by opening up the cell and increasing cell permeability to
bound phenolics (Kalt et al., 2000). Release of lycopene in tomatoes (Dewanto et al., 2002a),
phenolics in apple peels (Wolfe and Liu, 2003) and ferulic acid in corn (Dewanto et al., 2002b)
have also been reported during heat processing. These reports agreed with the results of the
present study with regard to the beneficial effect of blanching of retention of TA activity in the
final product.
Table 3. 2. Total antioxidant capacity (TAC) by DPPH assay, total phenolics (TP) and total
anthocyanins (TA) of raw, blanched and dried potato flakes from purple cultivars. TAC, TP &
TA of blanched and dried flakes were compared with raw cultivars and expressed in dry weight
basis (n=3).
Total Antioxidant Capacity
(µg Trolox/g DW)
Total Phenolics
(µg GAE /g DW)
Total Anthocyanins
(mg/g DW)
Raw (CO) 8787 ± 630b 3835 ± 296
c 1.74 ± 0.16
a
Blanched 15358 ± 2948a 7993 ± 488
a 1.58 ± 0.08
a
Drum dried 7021 ± 911 b 4021 ± 136
c 1.03 ± 0.02
b
Freeze dried 8151 ± 37 b 3950 ± 124
c 0.96 ± 0.17
b
Raw* (WA) 9605 ± 405b* 3347 ± 198
c* 1.08 ± 0.09
a*
Refractance
window dried 7331 ± 246
b* 4680 ± 120
b* 0.83 ± 0.01
b*
* Cultivar from a different location and storage time was used for Refractance Window-drying,
CO: Colorado, WA: Washington. Significant differences within the values in the same column
are indicated by different superscript letters (P < 0.05).
130
3.3 Total antioxidant capacity (TAC), total phenolics (TP) and total anthocyanins (TA) in
dehydrated potato flakes
All of the dehydrated flakes had similar moisture contents: 6.0 % (wet basis) for freeze dried
flakes; 5.9 % (wet basis) for RW dried flakes; and 5.2 % (wet basis) for drum dried flakes.
There was no significant difference (P > 0.05) among the moisture contents of the flakes. The
TAC content in freeze dried, refractance window dried, and drum dried flakes were 8151, 7331,
and 7021 µg TE/g DW, respectively. There was no significant change (P > 0.05) in TAC content
of dry flakes compared to raw sample for all drying methods used in this study. The TP content
in flakes prepared by drum-drying and freeze-drying were 4021 and 3950 µg GAE/g DW,
respectively. Also, significant changes were observed in TP, DD, and FD. However, there was a
significant increase (P < 0.05) of these components observed in the flakes prepared by
refractance window-drying (4680 µg GAE/g DW). Flakes prepared by drum-drying, freeze-
drying and refractance window-drying had TA contents of 1.03, 0.96 and 0.83 mg MV-3-GLY/g
DW, respectively. There were significant losses of 23-45 % TA contents observed in potato
flakes compared to raw samples (Table 3.2).
Many researchers have reported that processing causes major losses in the antioxidant
concentration of fruits and vegetables, mainly due to enzymatic and chemical oxidation of
antioxidants. However, most of the literature focuses on the degradation of vitamin C in fruits
and vegetables during blanching, dehydration or heating (Van den Broeck et al., 1998) and very
few data are available on polyphenols and other compounds that contribute to the antioxidant
activities in fruits and vegetables. In the present study, no significant change (P > 0.05) in TAC
was observed in the dehydrated potato flakes compared to raw samples, regardless of drying
method used. Blessington et al. (2007) reported an increase in the content of carotenoids and
131
DPPH antioxidant activities during frying and microwave-drying of potatoes. Dewanto et al.
(2002b) also reported an increase of 44 % in TA activities in heat processed sweet corn (115 °C
for 25 min) despite a 25 % loss in vitamin C. There is no significant change (P > 0.05) observed
in antioxidant activities during freeze-drying of apple peels (Wolfe and Liu, 2003), baking (at
177 °C for 20 min) of wheat bran (Singh et al., 2007) and canning of chick pea proteins
processed at 121 °C for 20 min (Arcan and Yemenicioglu, 2007). Retention of antioxidant
activities in processed fruits and vegetables have been attributed to protein hydrolysis, Maillard
reaction and fermentation process (Nicoli et al., 1999). In the present study, the observed
retention of TAC might be due to a combination of the natural phytochemicals present in the
potato and Maillard Reaction Products (MRPs) that contribute to the overall antioxidant
activities of the potato flake. Reports by Nicoli et al., (1999) also supports that Maillard
products formed during processing contributed to the formation of antioxidants in roasted coffee
enhancing total antioxidant capacity of the product.
Phenolics were also retained after dehydration of purple potatoes by DD and FD and a
significant increase was observed due to RWD when compared to raw tubers. There is limited
literature available to compare the effect of drying and other thermal treatments on total
phenolics in potatoes. In a study on antioxidant values of phenolic acids in potatoes, Friedman
(1997) reported complete loss of phenolic acids by cooking. In contrast, Blessington (2005)
observed higher TP content in potatoes processed by microwaving, frying and baking than by
boiling. Wolfe et al., (2003) reported a significant increase of phenolic contents in freeze dried
apple peels compared to fresh peels. The retention and increase in TP during blanching and
dehydration may be attributed to opening of the cell matrix and release of bound phenolics.
Blanching followed by drying of peeled purple potatoes reduced the content of total
132
anthocyanins by 20 to 40 % in the flakes. Singh et al., (2007) reported complete loss of
anthocyanins in purple wheat bran during baking. Other researchers reported either an increase
or no change in TA content during drying of carrots (Uyan et al., 2004). It has been well studied
that temperature, pH, oxygen and light affect the stability of anthocyanins. Reyes et al., (2007)
reported a first order thermal degradation of anthocyanin extracts from colored potatoes at
different pH. Anthocyanins lose the glucosides by deglycosylation to form chalcones that further
degrades to acids and aldehydes (Sadilova et al., 2006).
3.4. Correlation between TP and TAC
A small negative linear correlation was found between TP and TAC (r2= -0.119) in dried
potato flakes indicating little relationship between these parameters. Therefore, these data do not
suggest that TP are primarily responsible for the total antioxidant capacity in potato flakes
dehydrated by DD, FD and RW. However, in colored (purple, red, yellow) and white potato
cultivars a large positive correlation between TP and TAC (r2 = 0.886) was observed (Figure
3.3). Reyes et al. (2003) reported that wound-induced phenolic compounds in potatoes were
positively correlated to antioxidant activities. Many researchers also reported a high positive
acid equivalent) in whole raw potatoes (Reddivari et al., 2007; Reyes et al., 2005). In contrast,
Hale (2003) observed that concentration of phenolic acid contributed very little to antioxidant
correlation between antioxidant capacity and total phenolics content (in terms of chlorogenic
activities in raw potatoes (r2=0.18). There has been little information reported on the correlation
between TP and TAC in processed potatoes. The observed difference in correlation between raw
tubers and dehydrated potato flakes suggests that MRPs formed during processing may
contribute significantly to the total antioxidant capacity in the extracts from dehydrated flakes.
133
3.5. Color in raw cultivars and dehydrated flakes
Visual color attributes of the raw cultivars expressed in terms of lightness, hue, and
chromaticity are shown in Table 3.3. Lightness (L*) of white potato was significantly higher (P
< 0.05) among the cultivars tested and purple potato had the smallest L* value (P < 0.05), while
the red and yellow cultivars had similar L* values. Chromaticity values followed the same
pattern observed for lightness that is white potatoes showed the highest and purple potatoes the
smallest color saturation. Red and yellow cultivars were not different in lightness or
Table 3. 3. Color attributes of raw potato cultivars (n=3)
Cultivars Lightness
(L*)
Chromaticity
(C*)
Hue angle
(h°)
Purple 19.3 ± 2.4c 8.2 ± 0.5
c 320.2 ± 0.9
a
Red 65.6 ± 2.1b 24.3 ± 1.7
a 271.7 ± 0.2
b
Yellow 63.8 ± 2.2 b
25.0 ± 0.5 a 89.6 ± 0.3
c
White 69.4 ± 1.6a 15.6 ± 0.6
b 271.6 ± 0.1
b
Significant differences within the values in the same column are indicated by different
superscript letters (P < 0.05)
R2 = 0.8858
1000
3000
5000
7000
9000
1000 2000 3000 4000 5000
TP (µgGAE/g DW)
TA
C (
µg
TE
/g D
W)
Figure 3. 3 Correlations between total antioxidant capacity by DPPH assay and total
phenolics in colored (purple, red, yellow) and white potato cultivars.
134
chromaticity, but were significantly different in hue angle (P < 0.05). The purple potato cultivar
was significantly (P < 0.05) different in all color attributes from other cultivars.
Upon dehydration of white and colored potato samples, a significant increase in lightness
was observed for all samples under different dehydration processes (Table 3.4). Freeze dried
and RW dried flakes had similar L* values and were significantly brighter (P < 0.05) than drum
dried flakes. Similar results were reported on heating of black carrot, strawberry and elderberry
extracts (Sadilova et al., 2006). Color purity of the freeze dried flakes expressed as chroma was
similar to the raw potatoes whereas significantly higher values (P < 0.05) were observed for DD
and RW dried flakes. The higher saturation of color observed in the DD and RW dried samples
could be due to exposure of potato puree to air and light for shorter times during these drying
processes than samples processed by freeze-drying technology. Since, the residence time of
potato puree on DD and RW driers was around 53 sec and 115 sec, respectively. Whereas, puree
samples were kept for 24 h in the freeze dryer; this provided a higher exposure time under this
drying condition. Similarly, Sadilova et al. (2006) observed a significant decrease in chroma of
black carrot, strawberry and elderberry extracts after heating 7 hours depicting dullness/less
saturated color in the samples.
Hue values of all the potato flakes dehydrated under the different drying processes were
significantly different (P < 0.05) from each other (Table 3.4). Flakes from the DD were higher
in hue value depicting more reddish than RW and freeze dried flakes. Also, the color of raw
purple cultivars was shifted towards red with higher hue value than that of dehydrated flakes.
Reyes et al., (2007) reported an increase in hue-value in red and purple-fleshed potato extracts
when exposed to 98 °C. On the other hand, Sadilova et al. (2006) reported an initial decrease in
hue-values after 3h of heating of black carrot, strawberry and elderberry extracts at 98 °C,
135
Table 3. 4. Color attributes of purple dehydrated flakes (n=3)
Drying process Lightness
(L*)
Chromaticity
(C*)
Hue angle
(h°)
Color difference
(∆E*)
Raw 19.3 ± 2.4c 8.2 ± 0.5
b 320.2 ± 0.9
a 0.0
Freeze dried 54.0 ± 2.a 7.2 ± 0.6
b 270.5 ± 0.1
d 35.3 ± 0.99
a
Drum dried 45.7 ± 1.2 b
12.2 ± 1.0 a 296.2 ± 0.3
b 27.0 ± 3.37
b
RW dried 55.3 ± 0.9a 12.2 ± 0.7
a 293.6 ± 0.2
c 36.5 ± 2.01
a
Significant differences within the values in the same column are indicated by different
superscript letters (P < 0.05).
followed by an increase in the hue value more than that of unheated extracts. The overall color
attributes of the dehydrated potato flakes were determined by color differences compared to the
raw potato sample. Lesser differences in color indicate better stability of pigments in the
samples. Color differences (∆E) in DD flakes were significantly less (P < 0.05) that those
obtained from FD and RW dried flakes, indicating more preservation of the original color in
flakes dried by DD technology. Despite the observed variation in color difference between DD
and FD flakes, the concentration of anthocyanins in those flakes was not significantly different
(P > 0.05).
4. CONCLUSION
Evaluation of antioxidant compounds present in purple, red, yellow, and white potatoes
showed that purple potato cultivars contain significantly higher total antioxidants, total phenolic
and total anthocyanins than other potato cultivars. Dry flakes prepared from steam-blanched
purple potatoes retained the purple color. However, those flakes obtained from purple potatoes
without previous blanching lost the purple color. Therefore, blanching was demonstrated to be an
important operation for treating purple potatoes before dehydration. Different drying
technologies (drum-drying, freeze-drying and refractance window-drying) used to prepare
dehydrated purple potato flakes did not significantly change (P > 0.05) total antioxidant content.
136
Similar results were also observed in total phenolics content in the dehydrated purple potato
flakes prepared by freeze-drying and drum-drying. Conversely, a significantly higher (P < 0.05)
total phenolic content was obtained by refractance window-drying technology of potato flakes.
Losses of 23 to 45 % in total antioxidant content were observed in dehydrated potato flakes
processed under all drying methods. The results suggest that processing colored potatoes into
value-added antioxidant-rich ingredients may contribute to the production of healthy snacks and
other foods. A detailed kinetic study of antioxidants during processing of potatoes is needed to
better understand the behavior of antioxidants in food systems.
ACKNOWLEDGEMENTS
We acknowledge the financial support from the Washington State Potato Commission,
Moses Lake, WA, USA and partial support from the Washington State University Agricultural
Research Center.
137
REFERENCES
Andre, C.M., Ghislain, M., Bertin, P., Oufir, M., Herrera, M.D., Hoffmann, L., Hausman, J.F.,
Larondelle, Y., Evers, D. 2007. Andean potato cultivars (Solanum tuberosum L.) as a source
of antioxidant and mineral micronutrients. Journal of Agricultural and Food Chemistry. 55,
366-378.
AOAC. 1995. Official methods of analysis (16th. ed.). Association of Official Analytical
Chemistry, Washington DC.
Arcan, I., Yemenicioglu, A. 2007. Antioxidant activity of protein extracts from heat-treated or
thermally processed chickpeas and white beans. Food Chem. 103, 301-312.
Blessington, A., Miller, J.C., Nzaramba, M.N., Hale, A.L., Redivari, L., Scheuring, D.C.,
Hallman, G.J. 2007. The effects of low-dose gamma irradiation and storage time on
carotenoids, antioxidant activity, and phenolics in the potato cultivar Atlantic. American
Journal of Potato Research. 84, 125-131.
Blessington, T. 2005. The effects of cooking, storage and ionizing irradiation on carotenoids,
antioxidant activity and phenolics in potato. Texas A&M, College station.
Brandwilliams, W., Cuvelier, M.E., Berset, C. 1995. Use of a Free-Radical Method to Evaluate
Antioxidant Activity. Food Science and Technology-Lebensmittel-Wissenschaft &
Technologie. 28, 25-30.
Brown, C.R. 2005. Antioxidants in potato. American Journal of Potato Research. 82, 163-172.
Brown, C.R., Wrolstad, R., Durst, R., Yang, C.P., Clevidence, B. 2003. Breeding studies in
potatoes containing high concentrations of anthocyanins. American Journal of Potato
Research. 80, 241-249.
138
Cevallos-Casals, B.A., Cisneros-Zevallos, L. 2003. Stoichiometric and kinetic studies of
phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato. Journal of
Agricultural and Food Chemistry. 51, 3313-3319.
Dewanto, V., Wu, X.Z., Adom, K.K., Liu, R.H. 2002a. Thermal processing enhances the
nutritional value of tomatoes by increasing total antioxidant activity. Journal of Agricultural
and Food Chemistry. 50, 3010-3014.
Dewanto, V., Wu, X.Z., Liu, R.H. 2002b. Processed sweet corn has higher antioxidant activity.
Journal of Agricultural and Food Chemistry. 50, 4959-4964.
Friedman, M. 1997. Chemistry, biochemistry, and dietary role of potato polyphenols. A review.
Journal of Agricultural and Food Chemistry. 45, 1523-1540.
Fuleki, T., Francis, F.J. 1968. Quantitative methods for anthocyanins-I. Extraction and
determination of total anthocyanin in cranberries. Journal of Food Science. 33, 72-77.
Giusti, M.M., Wrolstad, R. 2001. Characterization and measurement of anthocyanin by UV-
visible spectroscopy In: Current Protocols in Food Analytical Chemistry., (R.E. Wrolstad,
ed.) pp. pp F1.2.1-F1.2.13, John Wiley & Sons, New York.
Hale, A.L. 2003. Screening potato genotypes for antioxidant activity, identification of the
responsible compounds and differentiating Russet Norkotah strains using AFLP and
microsatellite marker analysis., Texas A&M University, College Station, Texas.
Halliwell, B., Gutteridge, J.M.C., Cross, C.E. 1992. Free-Radicals, Antioxidants, and Human-
Disease - Where Are We Now. Journal of Laboratory and Clinical Medicine. 119, 598-620.
Han, K.H., Hashimoto, N., Shimada, K., Sekikawa, M., Noda, T., Yamauchi, H., Hashimoto, M.,
Chiji, H., Topping, D.L., Fukushima, M. 2006a. Hepatoprotective effects of purple potato
139
extract against D-galactosamine-induced liver injury in rats. Bioscience Biotechnology and
Biochemistry. 70, 1432-1437.
Han, K.H., Sekikawa, M., Shimada, K., Hashimoto, M., Hashimoto, N., Noda, T., Tanaka, H.,
Fukushima, M. 2006b. Anthocyanin-rich purple potato flake extract has antioxidant capacity
and improves antioxidant potential in rats. British Journal of Nutrition. 96, 1125-1133.
Jansen, G., Flamme, W. 2006. Coloured potatoes (Solanum tuberosum L.) - anthocyanin content
and tuber quality. Genetic Resources and Crop Evolution. 53, 1321-1331.
Kalt, W., Forney, C.F., Martin, A., Prior, R.L. 1999. Antioxidant capacity, vitamin C, phenolics,
and anthocyanins after fresh storage of small fruits. Journal of Agricultural and Food
Chemistry. 47, 4638-4644.
Kalt, W., McDonald, J.E., Donner, H. 2000. Anthocyanins, phenolics, and antioxidant capacity
of processed lowbush blueberry products. Journal of Food Science. 65, 390-393.
Kaur, C., Kapoor, H.C. 2001. Antioxidants in fruits and vegetables - the millennium's health.
International Journal of Food Science and Technology. 36, 703-725.
Lachman, J., Hamouz, K., Orsak, M. 2005. Red and purple potatoes - A significant antioxidant
source in human nutrition. Chemicke Listy. 99, 474-482.
Lewis, C.E., Walker, J.R.L., Lancaster, J.E., Sutton, K.H. 1998. Determination of anthocyanins,
flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum tuberosum L.
Journal of the Science of Food and Agriculture. 77, 45-57.
Nicoli, M.C., Anese, M., Parpinel, M. 1999. Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology. 10, 94-100.
Prior, R.L., Cao, G.H., Martin, A., Sofic, E., McEwen, J., O'Brien, C., Lischner, N., Ehlenfeldt,
M., Kalt, W., Krewer, G., Mainland, C.M. 1998. Antioxidant capacity as influenced by total
140
phenolic and anthocyanin content, maturity, and variety of Vaccinium species. Journal of
Agricultural and Food Chemistry. 46, 2686-2693.
Reddivari, L., Hale, A.L., Miller, J.C. 2007. Determination of phenolic content, composition and
their contribution to antioxidant activity in specialty potato selections. American Journal of
Potato Research. 84, 275-282.
Reyes, L.F., Cisneros-Zevallos, L. 2003. Wounding stress increases the phenolic content and
antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of
Agricultural and Food Chemistry. 51, 5296-5300.
Reyes, L.F., Cisneros-Zevallos, L. 2007. Degradation kinetics and colour of anthocyanins in
aqueous extracts of purple- and red-flesh potatoes (Solanum tuberosum L.). Food Chemistry.
100, 885-894.
Reyes, L.F., Miller, J.C., Cisneros-Zevallos, L. 2005. Antioxidant capacity, anthocyanins and
total phenolics in purple- and red-fleshed potato (Solanum tuberosum L.) genotypes.
American Journal of Potato Research. 82, 271-277.
Rodriguez-Saona, L.E., Giusti, M.M., Wrolstad, R.E. 1998. Anthocyanin pigment composition
of red-fleshed potatoes. Journal of Food Science. 63, 458-465.
Rossi, M., Giussani, E., Morelli, R., Lo Scalzo, R., Nani, R.C., Torreggiani, D. 2003. Effect of
fruit blanching on phenolics and radical scavenging activity of highbush blueberry juice.
Food Research International. 36, 999-1005.
Sadilova, E., Stintzing, F.C., Carle, R. 2006. Thermal degradation of acylated and nonacylated
anthocyanins. Journal of Food Science. 71, C504-C512.
Singh, S., Gamlath, S., Wakeling, L. 2007. Nutritional aspects of food extrusion: a review.
International Journal of Food Science & Technology. 42, 916-929.
141
Singleton, V.L., Rossi, J.A., Jr. 1965. Colorimetry of Total Phenolics with Phosphomolybdic-
Phosphotungstic Acid Reagents. Am. J. Enol. Vitic. 16, 144-158.
Swain, T., Hillis, W.E. 1959. The phenolic constituents of Prunus domestica-I. The quantitative
abalysis of phenolic constituents. Journal of Food Science and Agriculture. 10, 63-68.
Uyan, S.E., Baysal, T., Yurdagel, O., El, S.N. 2004. Effects of drying process on antioxidant
activity of purple carrots. Nahrung-Food. 48, 57-60.
van den Berg, R., Haenen, G.R.M.M., van den Berg, H., Bast, A. 1999. Applicability of an
improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant
capacity measurements of mixtures. Food Chemistry. 66, 511-517.
Van den Broeck, I., Ludikhuyze, L., Weemaes, C., Van Loey, A., Hendrickx, M. 1998. Kinetics
for Isobaric-Isothermal Degradation of L-Ascorbic Acid. J. Agric. Food Chem. 46, 2001-
2006.
Wang, H., Cao, G.H., Prior, R.L. 1996. Total antioxidant capacity of fruits. Journal of
Agricultural and Food Chemistry. 44, 701-705.
Wang, S.W. 2002. The influence of drying and thermal treatments on antioxidant activity in
asparagus. In: Department of Food Science and Human Nutrition, Washington State
University, Pullman.
Wolfe, K.L., Liu, R.H. 2003. Apple peels as a value-added food ingredient. Journal of
Agricultural and Food Chemistry. 51, 1676-1683.
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CHAPTER FOUR
EFFECT OF EXTRUSION ON THE ANTIOXIDANT CAPACITY AND
COLOR ATTRIBUTES OF EXPANDED EXTRUDATES PREPARED
FROM PURPLE POTATO AND YELLOW PEA FLOUR MIXES
ABSTRACT
Foods with antioxidant capacity contribute health benefits and provide protection against
certain cancers, Alzheimer‘s dementia, and cardio-vascular diseases caused by oxidative
damage. The effect of extrusion cooking on the antioxidant capacity and color attributes of
extruded products prepared from three selected formulations of purple potato and yellow pea
flours using a co-rotating twin screw extruder were studied. Expansion ratios of the extruded
products prepared varied from 3.93 to 4.75. The total antioxidant capacities (TAC) of the
extruded products, using DPPH assay, were 3769 – 4116 µg trolox equivalent/g dry weight
sample and were not significantly different (p > 0.05) from their respective raw formulations.
The total phenolic contents (TP) of the extruded products varied from 2088 to 3766 µg of gallic
acid equivalent/g dry weight sample and retained 73 – 83 % of TP from the raw formulations
after extrusion. The total anthocyanins contents (TA) in the extrudates were 0.116 – 0.228 mg of
malvidin-3-glucosides/g dry weight sample. Compared with their raw formulations, significant
losses (60–70%) of TA in the extruded products occurred during extrusion cooking. Browning
indices and color attributes such as brightness, chroma and hue angles agreed with degradation
of anthocyanins in the extruded products. However, extrusion cooking retained antioxidant
capacities of the raw formulations in the extruded products either in their natural forms or
143
degraded products with radical scavenging activity. This study demonstrated the potential for the
production of puffed extruded food products with improved antioxidant content from colored
potatoes and pulse formulations.
1. INTRODUCTION
Colored potatoes are rich in anthocyanins, which are known for providing natural color to
fruits and vegetables and for exhibiting antioxidant properties (Cevallos-Casals & Cisneros-
Zevallos, 2003; Nayak, Berrios, Powers, Tang & Ji, 2010). Antioxidants play an important role
to protect against diseases by reacting with and quenching oxidative free radicals, reducing
peroxides, chelating transition metals, and stimulating anti-oxidative defense enzyme activities
(Velioglu, Mazza, Gao & Oomah, 1998). Rice-Evans, Miller, Bolwell, Bramley, & Pridham
(1995) reported that anthocyanins are more effective antioxidants in vitro than ascorbic acid and
vitamin E. Pulses (such as yellow peas) are packed with a high content of nutritional ingredients
such as protein, dietary fibers, complex carbohydrates and folate, and are low in fat and sodium
(Madar & Stark, 2002). Most of these nutritional ingredients are also associated with health
benefits including hypochlesterolemic effects (Pusztai, Grant, Buchan, Bardocz, de Carvalho &
Ewen, 1998), prevention of osteoporosis (Messina, 1999), and certain cancers (Lamartiniere,
2000). Therefore, it would be desirable to make commodities such as colored potatoes and
yellow peas into functional foods in the form of extruded snacks and breakfast cereal-type food
products.
Extrusion cooking is a high temperature, short time process in which food materials are
plasticized and cooked by the combination of moisture, pressure, temperature and mechanical
shear, resulting in molecular transformation and chemical reactions. This technology is
preferable to other processing technologies because of it being a continuous process with high
144
productivity, significant retention of nutritional quality (Singh, Gamlath & Wakeling, 2007),
natural color and flavor of foods (Bhandari, D'Arcy & Young, 2001).
Expansion ratio is one of the important characteristics of puffed extruded products. Extrusion
cooking of legumes restricts the expansion ratio of the extruded product, but addition of starch-
containing ingredients, such as potato flours, could improve puffing of the extrudates for the
development of expanded-type foods.
Some studies have demonstrated that it is possible to puff pulse flours and pulse-based
formulations into potentially commercial nutritious snack and breakfast cereal-type products
(Berrios, Camara, Torija & Alonso, 2002; Berrios, Morales, Camara & Sanchez-Mata, 2010;
Berrios, Wood, Whitehand & Pan, 2004; Patil, Berrios, Tang & Swanson, 2007). The goals of
this study were to (i) produce puffed extrudates from mixes of colored potatoes and yellow pea
flours, and (ii) evaluate the effect of extrusion conditions on antioxidant capacities, color
attributes, and some physical characteristics of the extrudates.
2. MATERIALS AND METHODS
2.1. Materials
Fresh ‗Purple Majesty‘ cultivar potatoes were purchased from SLV Research Center,
Colorado State University, Colorado, USA. White (‗Russet‘ cultivar) potatoes were purchased
from a local store. The potatoes were stored at 4 °C and 80 % relative humidity for a couple of
weeks, before processing. Split yellow peas were purchased from Giusto‘s specialty food, South
San Francisco, CA. The peas were pin milled into fine flours and stored at room temperature
until use.
145
2.1.1. Production of potato flours
Purple and white potatoes were peeled using an abrasive peeler and sliced to 6 mm thickness.
Potato slices were blanched in a steam blancher for 8 min to ensure peroxidase inactivation,
since 98 % reduction in peroxidase activity was previously achieved in ~5 mm thickness red-
and purple-fleshed potatoes slices blanched for 3 min. (Reyes and Cisneros-Zevallos, 2007).
Blanched potato slices were immediately cooled in ice water for 8 min, to reduce thermal shock,
drained, and then pureed using a mixer (The Hobart Mfg Company, Troy, OH, USA). Water
(half of the weight of the blanched potatoes) was added to the puree to make it of uniform
consistency, before applying it to a drum dryer. A 15.24 cm × 20.32 cm pilot-scale, counter-
rotating twin-drum dryer (Blaw Knox Food & Chemical Equipment Co., Buffalo, NY, USA) was
used to dehydrate the puree. The surface temperature of the drums was maintained at 135 – 138
°C. The gap between the drums, rotating at 1.13 rpm, was maintained at 0.3 mm. The dehydrated
flakes prepared by drum-drying were pin milled into flour and stored at -30 °C until further use.
The mean particle sizes of pin milled purple potato flour (PPF) and white potato flour (WPF)
were 225 and 220 μm, respectively, as analyzed on a laser scattering particle size distribution
analyzer (Horiba LA-900, Horiba Instruments Incorporated, Irvine, CA).
2.1.2. Sample preparation
A mix of 35/65 (w/w) WPF and split yellow pea flours (SYPF) were prepared in a mixing
bowl under continuous mixing for 10 min. Three formulations with ratios of 35/65, 50/50, 65/35
(w/w), purple potato flour (PPF) and SYPF, respectively, were prepared in the same way. These
formulations will henceforth be referred to as 35, 50 and 65% PPF. All the formulations were
kept in polyethylene bags and stored at room temperature overnight before extrusion processing.
146
2.1.3. Extrusion conditions
A co-rotating twin-screw extruder (Micro 18, American Leistritz Extruder Corp., NJ, USA)
was used to process the different extrudates under study. The extruder was equipped with five
independently-controlled heating zones that were electrically heated and water cooled by means
of a water cooling system. The temperature of the feeding zone was maintained constant at 80
°C. The detailed temperature profile in the heating zones, feed moisture and screw speeds is
given in Table 4.1. Barrel wall and die temperatures were monitored by respective
thermocouples attached to the top and bottom of the five heating/cooling zones and the die. A
Table 4. 1. Extrusion parameters for preparing extruded products using split yellow pea flours
and white potato flours.
Parameters
Feed rate (g/min) : 45
Feed moisture (% wb) : 17, 21, 25
Screw speed (rpm) : 200, 250, 300
Temperature profiles (°C)
(i) die temperature at 120 °C
(ii) die temperature at 130 °C
(iii) die temperature at 140 °C
: 80, 90, 100, 110, 120
: 80, 100, 110, 120, 130
: 80, 100, 115, 130, 140
pressure transducer was also attached at the die to monitor the operating pressure. Raw
formulations were fed at a constant rate of 45 g/min using a volumetric twin-screw feeder (K-
Tron Process Group, Pitman, NJ, USA). A Bran Luebee metering pump (Pumps & Process
Equipment, Inc., IL, USA) connected to the feeding zone was used to add water to the feed
during processing. The feed moisture content was maintained by changing the water flow rate
while keeping the feed rate constant. Extrusion parameters were displayed on an in-built monitor
to the extruder and the data were auto-saved on a personal computer. Extruded samples were
collected once the operation reached a steady state condition, i.e., the drift in torque was minimal
147
for at least 5 min. The samples were collected for 3 min, cooled at room temperature under
natural convection conditions, double packed in polyethylene bags, flushed with nitrogen, and
stored at -30 °C for further analysis.
2.1.4. Experimental design
Extrusion cooking of the WPF and SYPF formulations was designed following the
procedures of Box and Behnken (1960). Three independent extrusion parameters, namely feed
moisture content (% wb), screw speed (rpm), and die temperature (°C) were considered for a
three level (+1, 0, -1) design. The effects of these parameters on the expansion ratio of the
extrudates were investigated using response surface methodology (RSM). A total 15 experiments
were designed according to N = k2 + k + cp, where N is the total number of experiments, k is the
factor number, and cp is the number of replicates at the central point. Three factors with three
levels and three replicates at the central point were considered for the experimental design. The
optimum extrusion conditions to obtain acceptable expansion ratios were considered for
producing extruded products from PPF and SYPF.
2.1.5. Determination of Expansion ratio
Five randomly chosen extruded rods per extrusion run were considered for measurement.
Five readings at the nodes and space between the nodes of the rods were taken with a caliper for
calculating the mean diameter of the extrudates. The expansion ratio was calculated as the ratio
of mean cross-sectional diameter of an extrudate to diameter of the die (Camire, Dougherty &
Briggs, 2007).
148
2.1.6. Moisture content determination
Moisture contents of the raw PPF, SYPF and extruded samples were determined using the
standard procedures of AACC moisture-air-oven method number 44-45A (AACC, 2000).
2.1.7. Pasting profile of potato flours
Pasting profile of WPF and PPF was evaluated using a Rapid Visco Analyzer (RVA)
following the procedures of Batey, Cutin & Moore (1997). Three grams (dry basis) of pin milled
WPF or PPF was put into an RVA canister followed by adding deionized water to a final net
weight of 28.0 g and analyzed with continuous stirring at 160 rpm. The mixture was initially held
at 60 °C for 2 min, followed by 4 minutes of heating to 95 °C at 5.83 °C/min and held there for 4
min. The mixture was then cooled to 50 °C in four minutes at 11.25 °C/min and held for 4 min
for a total run time of 20 minutes The parameters such as time-to-peak viscosity, peak viscosity
(the maximum hot paste viscosity), trough viscosity (the trough at the minimum hot paste
viscosity), and final viscosity (the viscosity at the end of the test) were measured. Breakdown
and total set back associated with the degree of collapse of swollen starch granules
corresponding to release of solubilized starch capable of re-association during cooling were
calculated as following:
Breakdown = Peak viscosity – Trough viscosity
Total setback = Final viscosity – Trough viscosity
2.1.8. Color evaluation
Extruded samples were ground into flour using a food processor to pass through a US # 35
sieve (0.5 mm), whereas raw flour formulations required no further preparation for evaluation of
color attributes. Color attributes were determined using a computer vision system (CVS)
149
following the procedures of Pandit, Tang, Liu & Mikhaylenko (2007). Briefly, the CVS included
a digital camera (Nikon D70 model) with 18 to 70 mm zoom lens providing 6.1 megapixel
resolutions, a lighting system, and a personal computer. Flour samples were kept in a cylindrical
container and placed on a white plate inside a shooting tent. Images of the samples were taken
with the digital camera mounted downwards on the top of the tent at 50 cm above the sample
plate. The images were downloaded to a PC and analyzed using Adobe Photoshop CS2 software
(version 8.0, Adobe Systems Inc, San Jose, CA, USA) to determine the color parameters (CIE L,
a and b). Since color values in Photoshop software are encoded from 0 to 255, standard scaling
values were determined using the method of Briones & Aguilera (2005):
5.2*
LL (1)
120255
240*
aa (2)
120255
240*
bb (3)
where L, a and b values are from Photoshop and L*,a* and b* values are standardized values
depicting brightness, greenness/redness and blue/yellowness, respectively. The hue angle h° and
Chromaticity C* were computed from a* and b* using the following equations:
)*
*(tan 1
abh (4)
22 *)(*)(* baC (5)
Hue angle (h°), the angular representation of color, is often described as "red," "blue," etc.,
whereas chromaticity describes the purity (saturation) of color. The reference values for h° at
0/360°, 90°, 180°, 270° are magenta red, yellow, bluish-green, and blue, respectively. Color
150
differences between extruded samples and their respective raw formulations were expressed as
∆E* where,
222 *)(*)(*)(* baLE (6)
2.2. Chemical Analyses
Folin-Ciocalteu reagents, potassium chloride, sodium acetate, trolox (6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid), DPPH (2, 2-diphenyl-1-picrylhydrazyl) and gallic acid
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Laboratory grade methanol and
ethanol were used in extraction and preparation of samples.
2.2.1. Total antioxidant capacity (TAC)
Ten grams of flour from the raw or extruded samples were blended with aqueous methanol
(50:50 v/v) under constant stirring for 2 min at room temperature. The final volume of the
mixture was brought to 100 mL and kept for 90 min at 4 °C, before centrifuged at 30,000 g at 4
°C for 20 min. The supernatants were collected and stored at -20 °C for further analysis.
TAC on the raw and extruded samples was determined using DPPH assay following the
procedures of Brandwilliams, Cuvelier, & Berset (1995). DPPH (a stable, deep purple color
radical) is reduced in the presence of antioxidants decolorizing the solution. Loss of color results
in a decrease in the absorbance intensity, which can be monitored spectrophotometrically at 515
nm, provides the basis for measurement of the antioxidant capacity of the extracts. Stored
supernatants were brought to room temperature and DPPH assay was prepared by adding 0.05
mL of supernatants to 1.95 mL of freshly prepared DPPH solution (6 × 10-5
M) in a cuvette. The
cuvettes were covered with parafilms and thoroughly mixed before taking readings at 515 nm
using a UV/visible spectrophotometer (Ultraspec 4000, Pharmacia Biotech, Cambridge,
151
England) until a plateau was reached (Brandwilliams et al., 1995). The absorbance at 2 h was
considered optimum for determining the TAC of samples as there was little change in their
absorbance reading toward the end of time. Methanol was used as a blank, whereas DPPH
without sample was taken as control. For each measured sample, the percentage of DPPH
remaining was calculated as:
100)(0@
@
Tabsorbance
tTabsorbance
remainingDPPH
DPPHDPPH (7)
where DPPH absorbance@T=t is the absorbance of DPPH at time t min and DPPH absorbance@T=0 is the
absorbance of DPPH measured at zero min. The TAC was quantified from a trolox standard
curve and expressed as micrograms of trolox equivalent per gram of dry weight sample (µg TE/g
DW). Three replicates were considered for determination of TAC.
2.2.2. Total phenolics (TP)
Extracts from the total antioxidant assay were used for determining TP on the raw and
extruded samples using Folin-Ciocalteu colorimetric method, following the procedures of Swain
and Hillis (1959) and Singleton and Rossi (1965). Briefly, in presence of phenolates, the Folin-
Ciocalteu reagents reduce and produce molybdenum-tungsten blue, which can be measured with
a spectrophotometer. Extracted supernatants were brought to room temperature before diluting
them four times with 50% aqueous methanol. To 0.5 mL supernatants, 8 mL of deionized water
was added followed by 0.5 mL of 0.25 N FCR. The samples were mixed thoroughly and
equilibrated for 3 min at room temperature. After 3 to 4 min, 1 mL of 1 N sodium carbonate was
added to the mixture and mixed. Sodium carbonate raises the pH of the phenols to be oxidized
rapidly in an alkaline medium to form phenolates. The aliquots were kept at room temperature
for 2 h before taking readings 725 nm. 0.5 mL methanol was treated in the same way as the
152
diluted samples and used as blank. TP was quantified from a gallic acid standard curve and
expressed as micrograms gallic acid equivalent per gram of dry weight sample (µg GAE/g DW).
Three replicates were considered for determination of the TP.
2.2.3. Total anthocyanins (TA)
Determination of TA on the raw and extruded samples was carried out following the
procedures of Fuleki & Francis (1968) with modifications. Five grams of flour from the raw or
extruded samples were blended with aqueous acidified ethanol (50/50 water/acidified ethanol,
v/v) and thoroughly stirred for 2 min. Acidified ethanol was prepared from 85:15 95%
ethanol/1.5 N HCl. The final volume of the mixture was brought to 100 mL with aqueous
acidified ethanol. The mixture was covered with Parafilm and kept for 90 min at 4 °C to
equilibrate, before centrifuging at 23,000 g at 4 °C for 15 min. The supernatants were collected
and stored at -20 °C for further analysis.
TA contents were quantified using the pH differential method, following the procedure of
Giusti & Wrolstad (2001). Anthocyanin assays were prepared by adding 0.2 mL of supernatant
to 1.8 mL of KCl buffer (pH 1.0) or sodium acetate buffer (pH 4.5). The cuvettes containing
aliquots were covered with Parafilm, thoroughly mixed and equilibrated for 15 min at room
temperature, before reading absorbances. Malvidin-3-glucoside was considered the major
anthocyanin in purple potato flour (Han et al., 2006; Jansen & Flamme, 2006; Lewis, Walker,
Lancaster & Sutton, 1998) at the maximum wavelength (λmax) of 535 nm with molecular weight
(MW) of 718.5 g/mol and molar extinction coefficient of 30200 L-1
cm-1
mol-1
. Absorbance
readings at 535 nm (λmax) and 700 nm (for correcting turbidity) (Reyes & Cisneros-Zevallos,
2003) were taken in a UV/visible spectrophotometer, previously blanked with distilled water.
153
The TA of the raw formulations and the extrudates were calculated according to the following
formula (Giusti et al., 2001):
de
DFMWAlmgC
*
**)/( (8)
where A = Absorbance of the sample given by 5.4700max0.1700max )()( pHpH AAAAA MW =
molecular weight of malvidin-3-glucoside, DF = dilution factor, e = molar extinction coefficient
and d= path length of the cuvette (1 cm). TA contents were expressed as mg of malvidin-3-
glucosides per gram of DW sample (mg mv-3-glu/g DW). Individual anthocyanin were not
identified or calculated.
2.2.4. Browning Index (BI)
Degradation of anthocyanins and formation of brown Maillard Reactions Products (MRP)
was assessed based on the BI of the products. Extracts from the total anthocyanins were used to
determine BI in the raw and the extruded samples, following the procedures of Jackman, Yada &
Tung (1987). Absorbance readings of 2 mL of supernatants were taken at 535 (λmax), 420, and
700 nm for calculating Browning Index of the samples as:
700535
700420
AA
AABI
(9)
where A420, A535 and A700 were absorbances at 420, 535, and 700 nm, respectively.
2.3. Statistical analysis
All physical and chemical data obtained from the raw formulations and extruded samples
were collected and analyzed with SAS (version 9.1, SAS Institute Inc, Cary, NC, USA) using
analysis of variance (ANOVA). Tukey‘s pair-wise comparison at 95 % confidence level was
154
used to identify statistical significant differences (p < 0.05). All the data were expressed as mean
± standard deviation.
3. RESULTS AND DISCUSSION
3.1. Expansion ratio (ER)
The use of RSM allowed determining how the ER of the extrudates produced from WPF and
SYPF formulations varied under the influence of the selected extrusion parameters of screw
speed (200, 250, and 300 rpm) and die temperature (120, 130, and 140 oC). At the selected
extrusion parameters, the ER of the extrudates produced from the WPF and SYPF formulation
ranged from 1.78 to 5.18 (Figure. 4.1). The amount of PPF was limited compared to that of
WPF. Therefore, experimental designs for the extrusion of the PPF and SYPF formulations were
based on the optimized conditions for ER of the extrudates produced using WPF and SYPF
formulations. Expansion ratio of about 5.0 was selected from the optimized extrusion conditions
with 17% (wb) feed moisture, 250 rpm screw speed and 140 °C temperature. However, actual
extrusion processing of PPF and SYPF formulations were conducted at a screw speed of 300 rpm
and temperature at 130 °C in addition to 140 °C, to reduce the residence time and exposure to
heat of the mix in the extruder barrel, to achieve maximum color retention in the final extrudates.
The expansion ratios of the extrudates prepared from the PPF and SYPF formulations varied
from 3.93 to 4.75 (Table 4.2). Camire et al., (2007) reported diametric expansions of 1.90 to
1.93 in extruded products, processed at extrusion conditions of 175 rpm screw speed, 163 °C die
temperature, and a feed rate of 255 g/min. The formulation was prepared from 84.3% cornmeal
and other food ingredients (sucrose and dehydrated blueberry, cranberry, raspberry, and concord
grape powders). The ER of the extrudates produced from formulations containing 50 and 65%
155
PPF were not significantly different (p > 0.05) at 130 and 140 °C. This tended to indicate that a
difference in processing temperatures of 10 °C might not be sufficient to promote a significant
increase (p < 0.05) in ER of the extrudates. However, the ER of extrudates produced from
formulations containing 35% PPF were significantly smaller (p < 0.05) than those produced from
formulations containing 50 and 65% PPF, under the two die temperatures under study of 130 and
140 oC. It is known that starch has a positive effect on increasing expansion, while fiber and/or
protein have a negative and lowering effect on expansion of extrudates (Conway, 1971a;
Conway, 1971b; Guy & Horn, 1988; Kim & Maga, 1987). These findings support the results on
ER reported in this study, as high potato flour (source of starch) and low dry pea flour content
(source of protein and fiber) in the extrudates produced from formulations containing 50 and
3002
250
3
4
5
18 20 20022 24
Expansion
Ratio
% Feed Moisture
Screw Speed
Figure. 4. 1. Effects of screw speed and feed moisture on the different expansion ratio of
extrudates prepared from white potato and yellow pea flours at 140 °C die temperature
156
65% PPF, presented extrudates with the highest values of ER. Conversely, low concentration of
potato flour and high concentration of dry pea flour, in the extrudates produced from
formulations containing 35% PPF, resulted in extrudates with significantly (p < 0.05) lower
values of ER. Additionally, extrudates produced from formulations containing 35% PPF showed
large variability of diameter (used for calculation of ER) among the different extrudates.
Therefore, the difference (p < 0.05) of ER observed between extrudates produced at 130 and 140
oC is attributed to this indicated variability.
Table 4. 2. Moisture content and expansion ratios of the extrudates prepared from yellow pea
and purple potato flours (n=3); Feed moisture content: 17% (wet basis).
Formulations
(Potato/Pea)
Treatment Moisture content
(% wet basis)
Expansion Ratio
35/65 w/w
Raw Formulation 9.32 0
Extruded @130 °C 7.03 4.28 ± 0.11b
Extruded @140 °C 6.97 3.93 ± 0.22c
50/50 w/w
Raw Formulation 9.01 0
Extruded @130 °C 7.26 4.74 ± 0.10a
Extruded @140 °C 7.39 4.48 ± 0.12a
65/35 w/w
Raw Formulation 8.69 0
Extruded @130 °C 7.43 4.75 ± 0.27a
Extruded @140 °C 7.19 4.53 ± 0.26a
Significant differences within the values in the same column are indicated by different
superscript letters (p < 0.05, Tukey‘s pairwise comparison test)
3.2. Pasting behavior of potato flours
Differences and/or similarities in the pasting behavior of white and purple potato flours can
be explained based on their RVA‘s pasting curves. The pasting behaviors of both the WPF and
PPF followed similar patterns with regard to times to attain their peak, trough and final
viscosities (Figure. 4.2). Breakdown and total setback were 433 and 601 cP, for WPF, and 314
and 629 cP for PPF (Table 4.3). The similar proximate composition presented by the white and
157
Table 4. 3. Pasting behaviors of white and purple potato flours.
Flours Peak
viscosity (cP)
Trough
viscosity (cP)
Final
viscosity (cP)
Breakdown*
(cP)
Total
Setback# (cP)
White potato 1380 947 1548 433 601
Purple potato 1285 971 1600 314 629
*Breakdown = Peak viscosity – Trough viscosity;
#Total setback = Final viscosity – Trough viscosity.
purple potato flours may be responsible for the similar pasting profile patterns displayed by the
two flours. Similar observations were previously reported for pasting profiles of white and
colored potato flours (Hoover, 2001). Use of PPF was justified and used with SYPF for further
analyses with the optimized extrusion process parameters.
3.3. Color attributes
The brightness (L*) or color lightness among the different raw formulations prepared from
PPF and SYPF varied significantly (p < 0.05) from 77 to 88 on a scale of 0–100. The raw
formulation containing 35% PPF was significantly (p < 0.05) brighter than formulation
containing 50% PPF and this one was significantly (p < 0.05) brighter than formulation
containing the brightness color parameter. That is, a decrease in chroma and and hue values with
an increase of PPF in the formulations (Table 4.4). The raw SYPF flours had a whitish color
while potato flour has a purplish color, due to their anthocyanins content. Therefore, as the
amount of PPF increased in the formulations, the color parameters of brightness, chroma and hue
significantly (p < 0.05) decreased.
When comparing the brightness values (L*) of the different raw formulations with those of
their extrudates it was observed that extrusion processing, at die temperatures at 130 and 140 °C,
caused a significant (p < 0.05) decrease in brightness, chroma and hue, at all levels of PPF
addition. It is known that reducing sugars and proteins (amino acids) in foods can react under
158
hue values with and 65% PPF. The values of chroma and hue followed the same trend as
high processing temperatures to promote nonenzymatic browning (Maillard reaction), which
result in darkening of the final product. Potatoes are high in sugars and dry peas are high in
protein (amino acids). Therefore, the observed decrease in brightness is attributed to the Maillard
reaction, as a consequence of extrusion processing. Similarly, previous researchers have
indicated that extrusion of the whey protein concentrate and corn starch gave higher color
differences with increasing amylose content (Matthey & Hanna, 1997). Additionally, the
degradation of purple anthocyanins due to extrusion temperatures could have generated Maillard
reaction products that promoted the changes in color parameters of brightness, chroma and hue
values, observed between the unprocessed (raw) and extruded products.
Color difference (∆E*) was used to represent the color change between the unprocessed and
Time (Sec)
0 200 400 600 800 1000 1200 1400
Vis
cosi
ty (
cP)
0
200
400
600
800
1000
1200
1400
1600
1800
Tem
per
atu
re (
°C)
40
50
60
70
80
90
100
Purple flour
White flour
Temp (°C)
Figure. 4. 2. RVA profile of white and purple potato flours.
159
processed flours (effect of processing). In this study, the values of ∆E* for all flours increased
significantly (p < 0.05) as the processing temperature increased from 130 to 140 oC (Table 4.4).
The values of ∆E* for the extruded flours containing 35 and 50% PPF were similar, at both
indicated processing temperatures. However, those from extruded flours containing 65% PPF
were significantly (p < 0.05) greater. These results indicate that the color in the PPF had, along
with processing temperatures, a direct effect on the values of ∆E*. Additionally, they indicated
that the results obtained for ∆E* support those obtained for the color parameters of brightness,
chroma and hue values. Berrios et al., (2004) reported that there are no established threshold or
cut-off values for color development of an acceptable legume-based snack, due to the lack of this
type of product in the market place. Therefore, the color data in this study may have important
value for future product development of legume pulse-based snack type products.
Table 4. 4. Color attributes of the extruded products prepared from yellow pea and purple potato
flours (n = 3).
Formulations
(Potato/Pea) Treatment
Brightness
(L*)
Chroma
(C*)
Hue
(h°)
Color difference
(∆E*)
35/65 w/w
Raw Formulation 88.10 ± 0.4a 8.60 ± 0.3
f 89.31 ± 0.4
a 0.00
Extruded @130 °C 73.00 ± 1.6d 26.48 ± 0.8
d 87.85 ± 1.2
a 23.41 ± 0.8
c
Extruded @140 °C 73.82 ± 1.5d 29.87 ± 0.4
b 87.61 ± 1.5
a 25.62 ± 0.7
b
50/50 w/w
Raw Formulation 82.10 ± 1.3b 5.20 ± 0.3
g 67.83 ± 0.6
c 0.00
Extruded @130 °C 70.57 ± 0.7e 22.73 ± 0.8
e 87.71 ± 1.4
a 21.31 ± 1.3
c
Extruded @140 °C 72.72 ± 1.4d 28.33 ± 0.4
c 87.47 ± 1.3
a 25.29 ± 0.5
b
65/35 w/w
Raw Formulation 77.03 ± 1.0c 4.04 ± 0.2
h 21.35 ± 2.4
d 0.00
Extruded @130 °C 69.46 ± 1.7e 27.28 ± 0.7
d 83.64 ± 1.6
b 26.75 ± 0.6
b
Extruded @140 °C 73.80 ± 0.4d 33.19 ± 0.8
a 84.61 ± 0.3
b 31.74 ± 0.8
a
a Significant differences within the values in the same column are indicated by different
superscript letters (p < 0.05, Tukey‘s pairwise comparison test).
3.4. Total antioxidant capacity
The TAC of the raw formulations and extrudates were determined using DPPH assay and
expressed as µg TE/g DW (Figure. 4.3). The TAC of the raw formulations with 35, 50 and 65%
160
PPF were 3936 ± 71, 4025 ± 35 and 4083 ± 37 µg TE/g DW, respectively. These values were not
significantly different (p > 0.05). However, data clearly showed that TAC increased with
increasing PPF in the formulations. Those increases represented 2.21 and 1.42%, from 35 to 50
and from 50 to 65% PPF in the formulations, respectively. On the other hand, 0 – 35% PPF
addition resulted in a 60.5% increase in TAC (data not shown in Figure. 4.3). This significant (p
< 0.05) increase in TAC can be attributed to the purple color in the PPF. Since, studies on the
TAC of colorful fruits and vegetables, at the Jean Mayer USDA Human Nutrition Research
Center on Aging at Tuffs University, revealed that a large group of color compounds are
flavonoids (including anthocyanins) with potent antioxidant protection against peroxyl radicals
(McBride, 1996; Wang, Cao & Prior, 1997). When comparing the TAC values of the different
raw formulations with those of their extrudates it was observed that, even though the TAC values
of the extrudates were lower, they were not significantly (p > 0.05) different. Similar TAC
content in the raw formulations and extruded products could be attributable to the effect of
extrusion on (i) breaking complex polyphenols into low molecular weight phenolic compounds
with scavenging activity, (ii) interaction of the phenolics with protein under heat treatment, and
(iii) formation of Maillard Reaction Products. On the other hand, with the exception of
extrudates formulated with 50% PPF, the TAC values of those formulations extruded at die
temperatures of 140 °C showed significantly (p < 0.05) lower TAC values than their raw
counterparts. High temperature extrusion promotes the Maillard reaction and the formation of
brown compounds that may have had an effect on the TAC values of the extrudates (Anese,
Manzocco, Nicoli & Lerici, 1999). Additionally, the potential binding of phenolic compounds to
the protein matrix may account for the decrease in TAC values observed in those formulations
extruded at die temperatures at 140 °C compared to the raw samples. At high protein
161
concentration complex interactions and cross-linking of different protein molecules with
phenolic compounds forms a hydrophobic surface (Mcmanus, Davis, Beart, Gaffney, Lilley &
Haslam, 1985). No significant effect (p > 0.05) as a result of extrusion temperatures of 130 and
140 °C was observed on the TAC of extruded products formulated with 35, 50, and 65% PPF,
respectively. This result may indicate that an in order to see an effect on TAC, an increase in die
temperature greater than 10 oC needs to be used. Camire et al., (2007) reported no significant
change (p > 0.05) in antioxidant activity in control and cranberry extruded products prepared
with corn at a substitution level of 1% and extrusion temperature of 165 °C.
3500
3600
3700
3800
3900
4000
4100
4200
4300
35% PPF 50% PPF 65% PPF
Formulations
To
tal
an
tio
xid
an
t ca
pa
city
(μg
TE
/g D
W)
Non-extruded
Extruded @130 °C
Extruded @140 °C
ab
bc
c
aa
a
a
ab
b
Figure. 4. 3. Total antioxidant capacities by DPPH assay of raw formulations and extruded
products. The formulations contained x% of purple potato flour and (100-x)% of dry pea
flour.aValues in each bar with no letters in common are significantly different (p < 0.05).
162
3.5. Total phenolics
The TP of the raw formulations and extruded products were determined using the FC reagent
method and expressed as µg of GAE/g DW (Figure. 4.4). Comparing the TP content of different
raw formulations it was determined that the TP content of the 65% PPF formulation (4548 ± 117
µg of GAE/g DW) was significantly higher (p < 0.05) than that of 50% PPF (3838 ± 286 µg of
GAE/g DW) formulation; and this one was significantly higher (p < 0.05) than the TP of the
35% PPF (2818 ± 46 µg of GAE/g DW) formulation. Previous researchers have demonstrated
that purple-fleshed potato cultivars had higher phenolic contents than white-fleshed cultivars
(Nayak et al., 2010; Stushnoff et al., 2008) and yellow peas (Xu & Chang, 2008). These reports
support the results obtained in the present study that purple-fleshed potatoes had higher content
of TP than yellow peas. Therefore, those raw formulations containing the highest proportion of
PPF had also the highest content of TP. A similar pattern on TP content was observed in the
extruded products. However, significant losses (p < 0.05) in TP were determined in products
processed at die temperature of 130 °C prepared from formulations containing 50 and 65% PPF,
when compared to TP of their raw formulations. One exception was observed for extruded
products prepared from formulations containing 35% PPF, whose TP contents were not
significantly different (p > 0.05) from the raw formulations. This could be attributed to high
standard deviation values determined for those extruded samples. Similarly, products processed
at a die temperature of 140 °C prepared from formulations containing 35, 50 and 65% PPF had
significantly (p < 0.05) less TP content than their respective raw formulations. Viscidi,
Dougherty, Briggs & Camire (2004) reported significant loss of total phenolics during extrusion
of oat cereals. Additionally, Zadernowski, Nowak-Polakowska & Rashed (1999) reported losses
of up to ~60% of phenolic compounds in extruded oat samples, compared to their respective raw
163
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
35% PPF 50% PPF 65% PPF
Formulations
To
tal
ph
eno
lics
(μ
g G
AE
/g D
W)
Non-extruded
Extruded @130 °C
Extruded @140 °C
c
cd
d
b
c
c
a
b b
Figure. 4. 4. Total phenolic contents of raw formulations and extruded products. The
formulations contained x% of purple potato flour and (100-x)% of dry pea flour. aValues in each
bar with no letters in common are significantly different (p < 0.05).
samples. These reports corroborate well with the findings of the present study. Contrary to the
previous reports, Camire et al.(2007) reported a higher content of soluble phenolics, as ferulic
acid equivalents, in Concord grape and raspberry extrudates compared to their control samples.
Phenolic compounds are heat-liable and can break upon exposure to high temperatures.
Therefore, losses in the TP content of formulations under extrusion are expected to occur, due to
break down of complex polyphenols into other phenolic or non-phenolic compounds, as a
consequence of high temperatures conditions. However, extrusion die temperatures of 130 and
140 °C had no significant effect (p > 0.05) on the TP content of the extrudates (Figure. 4.4).
This indicated that, under the extrusion possessing conditions of the study, a die temperature
differential of 10 °C had not detrimental effect on TP.
164
3.6. Total anthocyanins
The TA content in the raw formulations and extrudates was determined using the pH
differential method and expressed as mg of mal-3-glu/g DW (Figure. 4.5). Results obtained from
the different raw formulations demonstrated that the TA content in the 65% PPF formulation
(0.729 ± 0.04 mg of mal-3-glu/g DW) was significantly higher (p < 0.05) than TA in formulation
containing 50% PPF (0.584 ± 0.05 mg of mal-3-glu/g DW) followed by formulation with 35%
PPF (0.363 ± 0.01 mg of mal-3-glu/g DW). In general, the TA content in the extruded products
prepared from formulations containing 65, 50 and 35% PPF and processed at die temperatures of
130 and 140°C followed same trend observed for the raw formulations. This is a progressive and
significant decrease in TA content as the percentage of PPF in the formulations decreased from
65 to 35%. Higher concentrations of PPF in the formulations contributed to higher content of
TA. Since the purple color in potato flour is mainly due to the presence of the anthocyanins
petunidin and malvidin glucosides present in the potato flesh and skin (Stushnoff et al., 2008);
whereas, preliminary TA results in yellow pea flour showed negligible values (data not shown).
Compared to their raw counter parts, extruded samples showed a significant loss in TA at all the
different levels of PPF in the formulations. The losses in TA were more evident in extrudates
processed at the highest die temperature of 140 °C. Different from the results obtained
previously, where a die temperature differential of 10 °C had no detrimental effect on TP, the
extrudates containing 35 and 50% PPF processed at a die temperature of 140 °C reflected a
significant loss in TA compared to those processed at 130 °C. Extrudates containing the highest
percentage of PPF (65%) processed at die temperatures of 130 and 140 °C presented similar TA
losses. Stability of anthocyanins is affected by a number of factors such as temperature, pH,
light, oxygen, enzymes, ascorbic acid, sulfur dioxide, sugars, metal ions, etc., (Francis, 1989).
165
Degradation of anthocyanins in the extruded products could be attributed to the breaking of
anthocyanin structures at high temperatures. High temperature at the initial step could open
either the pyrylium ring of the anthocyanins and form chalcone (Sadilova, Carle & Stintzing,
2007), or hydrolyze the glycosidic moiety and form aglycon (Sadilova, Stintzing & Carle, 2006),
0
0.2
0.4
0.6
0.8
1
35% PPF 50% PPF 65% PPF
Formulations
To
tal
an
tho
cya
nin
s
(mg
MV
-3-G
ly/g
DW
)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Bro
wn
ing
in
dex
Non-extruded Extruded @130 °C Extruded @140 °C
Non-extruded Extruded @130 °C Extruded @140 °C
c
fg
b
d
e
a
dd
Total anthocyanins
Browning index
Figure. 4. 5. Total anthocyanins contents and browning indices of raw formulations and
extruded products. The formulations contained x% of purple potato flour and (100-x)% of dry
pea flour. aValues in each bar with no letters in common are significantly different (p < 0.05).
providing degradation products as quercetin, phloroglucinaldehyde and protocatechuic acid.
Degradation of anthocyanins in the extruded products might be also due to the formation of
browning compounds caused by the Maillard reaction at high temperatures (Nicoli, Anese &
Parpinel, 1999). A study on the extrusion cooking of blueberry and grape anthocyanins, used as
breakfast cereal colorants, by Camire, Chaovanalikit, Dougherty & Briggs (2002) reported losses
of 90% of blueberry anthocyanins and 74% of grape anthocyanins, induced by extrusion
166
processing. Similarly, Camire et al., (2007) reported almost 90% loss in anthocyanins content in
extruded corn products containing fruit powders. These reports support the results of the present
study and indicate that when processing food materials that are a good source of anthocyanins,
special attention should be giving to the processing conditions to avoid significant losses.
3.7. Browning Index (BI)
Results of BI determined in raw formulations (Figure. 4.5) showed that the BI of
formulations with 35 and 50% PPF (0.19 ± 0.0, 0.20 ± 0.0, respectively) were not different from
(p > 0.05) each other. However, the BI of formulation with 65% PPF (0.15 ± 0.0) was lower (p <
0.05) than the former formulations. Since BI values are calculated based on the ratio of
absorbances at 420, 535 nm subtracting haziness at 700 nm, a higher absorbance value of the
65% PPF formulation at 535 nm (because of higher concentration of PPF than other
formulations) contributed to the observed lower BI values. The BI of the extrudates processed at
130 and 140 °C were significantly higher (p < 0.05) than their respective raw formulations.
Additionally, the BI of extrudates prepared at 140 °C (0.34 ± 0.04, 0.34 ± 0.00 and 0.49 ± 0.04
for 35, 50 and 65% PPF, respectively) were significantly higher (p < 0.05) than those processed
at 130 °C (0.29 ± 0.01, 0.29 ± 0.01 and 0.33 ± 0.03 for 35, 50 and 65% PPF, respectively).
Higher BI in the extrudates might be due to the formation of browning compounds by the
Maillard reaction of amino acids present in peas and reducing sugar in potato flours at high
temperature. Degradation of anthocyanins in the extrudates with change in color attributes also
agrees with higher BI in the extrudates. Karel and Labuza (1968) reported hydrolysis of sucrose
giving reducing sugars, which has potential for browning in a model system containing sucrose.
Browning of extruded products could have related to the feed moisture content in the
formulation, concentration of ingredients and other extrusion parameters. Dominance influence
167
of water on the rate of browning in systems containing carbonyl compounds have been reported
in the literature (Erlandson & Wrolstad, 1972).
3.8. Correlation analyses
Correlations (r2) among the TP, TAC, TA, BI and color attributes were analyzed using
Pearson‘s correlation coefficients method. The TAC was not strongly correlated with the TP
(r2 = 0.53, p > 0.05) or TA (r
2 = 0.46, p > 0.05). The correlation coefficients showed that the
phenolic compounds including anthocyanins were not solely responsible for antioxidant capacity
in the formulations and extruded products. Presence of other secondary metabolites such as
volatile oils, carotenoids and vitamins also might have contributed to the total antioxidant
capacity in the raw formulations and extrudates. Strong correlation of the TA with the TP (r2 =
0.76, p < 0.05) agrees the contribution of anthocyanins to the phenolic compounds. Contents of
the TA in the formulations and extruded products were negatively correlated with the BI (r2 = -
0.71, p < 0.05), chroma (r2 = -0.89, p < 0.05), and hue (r
2 = -0.86, p < 0.05) but positively
correlated with brightness (r2 = 0.52, p < 0.05). Anthocyanins are responsible for the purple color
of PPF. The negative correlations of TA with the BI, chroma and hue were attributable to the
concentrations of PPF in the formulations and degradation of anthocyanins in the extruded
products.
4. CONCLUSIONS
Naturally colored extruded puffed food products rich in antioxidants can be produced from
yellow pea and purple potato flours using extrusion cooking technology. Although degradation
in total anthocyanins content was determined, some purple color was retained in the final
extruded products. This indicated that high temperature-short time extrusion processing is a
168
suitable process for the fabrication of products from antioxidant-rich colored ingredients. The
total antioxidant capacities in the extruded products were retained due to the preservation of
phenolics during processing. Addition of the purple potato flour to yellow pea flour provided an
acceptable expansion ratio to the extruded products. Presence of natural color in the final
extrudates could play a major role in consumer attraction and acceptability, as well as
marketability of the developed extruded food products. More research on the use of extrusion
parameters and their effect on the kinetics of anthocyanins are necessary to study the stability of
natural color in the final extrudates. Naturally colored food ingredients, such as purple potato
flour, has potential for substituting in place of artificial colors, which are generally added as
coatings during the downstream processes.
ACKNOWLEDGEMENTS
We gratefully acknowledge the assistance of James Pan and Matthew Tom, Processed Foods
Research Unit, WRRC, USDA-ARS, Albany, CA for their assistance and feedback during the
extrusion experiment. We also acknowledge the US Dry Pea and Lentil Council for their
financial support for this project.
169
REFERENCES
AACC (2000). Approved methods of the American Association of Cereal Chemists (10th ed.). St.
Paul, MN, USA: American Association of Cereal Chemists.
Anese, M., Manzocco, L., Nicoli, M. C., & Lerici, C. R. (1999). Antioxidant properties of
tomato juice as affected by heating. Journal of the Science of Food and Agriculture, 79(5),
750-754.
Batey, I. L., Curtin, B. M., & Moore, S. A. (1997). Optimization of rapid-visco analyser test
conditions for predicting Asian noodle quality. Cereal Chemistry, 74(4), 497-501.
Berrios, J. D., Camara, M., Torija, M. E., & Alonso, M. (2002). Effect of extrusion cooking and
sodium bicarbonate addition on the carbohydrate composition of black bean flours. Journal
of Food Processing and Preservation, 26(2), 113-128.
Berrios, J. D., Morales, P., Camara, M., & Sanchez-Mata, M. C. (2010). Carbohydrate
composition of raw and extruded pulse flours. Food Research International, 43(2), 531-536.
Berrios, J. D., Wood, D. F., Whitehand, L., & Pan, J. (2004). Sodium bicarbonate and the
microstructure, expansion and color of extruded black beans. Journal of Food Processing
and Preservation, 28(5), 321-335.
Bhandari, B., D'Arcy, B., & Young, G. (2001). Flavour retention during high temperature short
time extrusion cooking process: a review. International Journal of Food Science and
Technology, 36(5), 453-461.
Box, G. E. P., Behnken, D.W., (1960). Some new three level designs for the study of quantitative
variables. Technometrics, 2, 455-475.
170
Brandwilliams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to
evaluate antioxidant activity. Food Science and Technology-Lebensmittel-Wissenschaft &
Technologie, 28(1), 25-30.
Briones, V., & Aguilera, J. M. (2005). Image analysis of changes in surface color of chocolate.
Food Research International, 38(1), 87-94.
Camire, M. E., Chaovanalikit, A., Dougherty, M. P., & Briggs, J. (2002). Blueberry and grape
anthocyanins as breakfast cereal colorants. Journal of Food Science, 67(1), 438-441.
Camire, M. E., Dougherty, M. P., & Briggs, J. L. (2007). Functionality of fruit powders in
extruded corn breakfast cereals. Food Chemistry, 101(2), 765-770.
Cevallos-Casals, B. A., & Cisneros-Zevallos, L. (2003). Stoichiometric and kinetic studies of
phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato. Journal of
Agricultural and Food Chemistry, 51(11), 3313-3319.
Conway, H. F. (1971a). Extrusion cooking of cereals and soybeans-I. Food Product
Development, 5(2), 27 - 29.
Conway, H. F. (1971b). Extrusion cooking of cereals and soybeans-II. Food Product
Development, 5(3), 14 - 17.
Erlandson, J. A., & Wrolstad, R. E. (1972). Degradation of anthocyanins at limited water
concentration. Journal of Food Science, 37(4), 592-595.
Francis, F. J. (1989). Food colorants - Anthocyanins. Critical Reviews in Food Science and
Nutrition, 28(4), 273-314.
Fuleki, T., & Francis, F. J. (1968). Quantitative methods for anthocyanins-I. Extraction and
determination of total anthocyanin in cranberries. Journal of Food Science, 33, 72-77.
171
Giusti, M. M., & Wrolstad, R. (2001). Characterization and measurement of anthocyanin by UV-
visible spectroscopy In: R. E. Wrolstad, Current Protocols in Food Analytical Chemistry.,
vol. F 1.2 (pp. pp F1.2.1-F1.2.13): John Wiley & Sons, New York.
Guy, R. C. E., & Horn, A. W. (1988). Extrusion and co-extrusion of cereals. In: J. M. V.
Blanshard, & J. V. Michelle, Food structure - Its creation and evaluation (pp. 331 - 349).
Butterworths, London.
Han, K. H., Sekikawa, M., Shimada, K., Hashimoto, M., Hashimoto, N., Noda, T., Tanaka, H., &
Fukushima, M. (2006). Anthocyanin-rich purple potato flake extract has antioxidant capacity
and improves antioxidant potential in rats. British Journal of Nutrition, 96(6), 1125-1133.
Hoover, R. (2001). Composition, molecular structure, and physicochemical properties of tuber
and root starches: a review. Carbohydrate Polymers, 45(3), 253-267.
Jackman, R. L., Yada, R. Y., & Tung, M. A. (1987). A review - Separation and chemical
properties of anthocyanins used for their qualitative and quantitative analysis. Journal of
Food Biochemistry, 11(4), 279-308.
Jansen, G., & Flamme, W. (2006). Coloured potatoes (Solanum tuberosum L.) - anthocyanin
content and tuber quality. Genetic Resources and Crop Evolution, 53(7), 1321-1331.
Karel, M., & Labuza, T. P. (1968). Nonenzymic browning in model systems containing sucrose.
Journal of Agricultural and Food Chemistry, 16(5), 717-719.
Kim, C. H., & Maga, J. A. (1987). Properties of extruded whey-protein concentrate and cereal
flour blends. Lebensmittel-Wissenschaft & Technologie, 20(6), 311-318.
Lamartiniere, C. A. (2000). Protection against breast cancer with genistein: a component of soy.
American Journal of Clinical Nutrition, 71(6), 1705s-1707s.
172
Lewis, C. E., Walker, J. R. L., Lancaster, J. E., & Sutton, K. H. (1998). Determination of
anthocyanins, flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum
tuberosum L. Journal of the Science of Food and Agriculture, 77(1), 45-57.
Madar, Z., & Stark, A. H. (2002). New legume sources as therapeutic agents. British Journal of
Nutrition, 88, S287-S292.
Matthey, F. P., & Hanna, M. A. (1997). Physical and functional properties of twin-screw
extruded whey protein concentrate corn starch blends. Food Science and Technology-
Lebensmittel-Wissenschaft & Technologie, 30(4), 359-366.
McBride, J. (1996). Plant pigments - Paint a rainbow of antioxidants. Agricultural Research, 44,
4 - 8.
Mcmanus, J. P., Davis, K. G., Beart, J. E., Gaffney, S. H., Lilley, T. H., & Haslam, E. (1985).
Polyphenol interactions .1. Introduction - Some observations on the reversible complexation
of polyphenols with proteins and polysaccharides. Journal of the Chemical Society-Perkin
Transactions 2(9), 1429-1438.
Messina, M. J. (1999). Legumes and soybeans: overview of their nutritional profiles and health
effects. American Journal of Clinical Nutrition, 70(3), 439s-450s.
Nayak, B., Berrios, J. J., Powers, J. R., Tang, J., & Ji, Y. (2010). Colored potatoes (Solanum
tuberosum L.) dried for antioxidant-rich value-added foods. Journal of Food Processing and
Preservation (in Press).
Nicoli, M. C., Anese, M., & Parpinel, M. (1999). Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology, 10(3), 94-100.
173
Pandit, R. B., Tang, J., Liu, F., & Mikhaylenko, G. (2007). A computer vision method to locate
cold spots in foods in microwave sterilization processes. Pattern Recognition, 40(12), 3667-
3676.
Patil, R. T., Berrios, J. D. J., Tang, J., & Swanson, B. G. (2007). Evaluation of methods for
expansion properties of legume extrudates. Applied Engineering in Agriculture, 23(6), 777-
783.
Pusztai, A., Grant, G., Buchan, W. C., Bardocz, S., de Carvalho, A. F. F. U., & Ewen, S. W. B.
(1998). Lipid accumulation in obese Zucker rats is reduced by inclusion of raw kidney bean
(Phaseolus vulgaris) in the diet. British Journal of Nutrition, 79(2), 213-221.
Reyes, L. F., & Cisneros-Zevallos, L. (2003). Wounding stress increases the phenolic content
and antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of
Agricultural and Food Chemistry, 51(18), 5296-5300.
Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M., & Pridham, J. B. (1995). The
relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radical
Research, 22(4), 375-383.
Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyanins and its
impact on color and in vitro antioxidant capacity. Molecular Nutrition & Food Research,
51(12), 1461-1471.
Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Thermal degradation of acylated and
nonacylated anthocyanins. Journal of Food Science, 71(8), C504-C512.
Singh, S., Gamlath, S., & Wakeling, L. (2007). Nutritional aspects of food extrusion: a review.
International Journal of Food Science & Technology, 42(8), 916-929.
174
Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic., 16(3), 144-158.
Stushnoff, C., Holm, D., Thompson, M. D., Jiang, W., Thompson, H. J., Joyce, N. I., & Wilson,
P. (2008). Antioxidant properties of cultivars and selections from the Colorado potato
breeding program. American Journal of Potato Research, 85(4), 267-276.
Swain, T., & Hillis, W. E. (1959). The phenolic constituents of Prunus domestica-I. The
quantitative abalysis of phenolic constituents. Journal of Food Science and Agriculture, 10,
63-68.
Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total
phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food
Chemistry, 46(10), 4113-4117.
Viscidi, K. A., Dougherty, M. P., Briggs, J., & Camire, M. E. (2004). Complex phenolic
compounds reduce lipid oxidation in extruded oat cereals. Lebensmittel-Wissenschaft Und-
Technologie-Food Science and Technology, 37(7), 789-796.
Wang, H., Cao, G. H., & Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins.
Journal of Agricultural and Food Chemistry, 45(2), 304-309.
Xu, B., & Chang, S. K. C. (2008). Effect of soaking, boiling, and steaming on total phenolic
content and antioxidant activities of cool season food legumes. Food Chemistry, 110(1), 1-
13.
Zadernowski, R., Nowak-Polakowska, H., & Rashed, A. A. (1999). The influence of heat
treatment on the activity of lipo- and hydrophilic components of oat grain. Journal of Food
Processing and Preservation, 23(3), 177-191.
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CHAPTER FIVE
BIOAVAILABILITY OF ANTIOXIDANTS IN EXTRUDED PRODUCTS
PREPARED FROM PURPLE POTATO AND DRY PEA FLOURS
ABSTRACT
Measuring antioxidant activity using a biologically relevant assay is more important for
understanding the role of phytochemicals in-vivo than the use of chemical assays. A cellular
antioxidant activity assay using HepG2 liver cancer cells could provide more biologically
relevant information on bioactive compounds in raw as well as processed food products. The
objective of this study was to investigate the complete phytochemical profiles, antioxidant
activity, cellular antioxidant activity, and their contribution to bioactivity in purple potato flour,
dry pea flour, raw formulations and processed products prepared from the above ingredients
using extrusion cooking. Free phenolics in purple potato flour and dry pea flour contributed 68
and 87%, respectively, to the total phenolics and 64 and 86%, respectively, to the total
antioxidant activity (ORAC value). Caffeic, p-coumaric and ferulic acids were mostly observed
in the bound extracts of raw formulations as well as in extrudates whereas chlorogenic acid was
predominant in free extracts. Extruded products prepared from raw formulations using extrusion
cooking of the above ingredients had either retained or increased the total phenolics, ORAC
antioxidant activity and flavonoids compared to the raw formulations. Extrusion processing
increased the cellular antioxidant activity of extrudates prepared from 50:50 (w/w) of ingredients
than control raw formulations in a dose-dependent manner.
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1. INTRODUCTION
Processed fruits and vegetables are generally believed to have fewer naturally occurring
antioxidants than fresh produce resulting in reduced health benefits. One of the major reasons
for measuring vitamin C levels in foods is its perception as an indicator for health benefits of
processed products (1-4). However, Eberhardt et al. (5) observed that vitamin C contributed less
than 0.4% to the total antioxidant activity in apples indicating the importance of phytochemicals
towards total antioxidant activity. Antioxidant compounds derived from fruits and vegetables act
in preventing formation of reactive oxygen, nitrogen, hydroxyl and lipid species either by
scavenging free radicals, or by repairing or removing damaged molecules (6). These bioactive
phytochemicals are proposed to prevent chronic diseases such as cardiovascular, diabetes and
certain forms of cancers (7-10). Bioactive phytochemicals exist in free as well as soluble-
conjugated and bound forms (11). Bound phytochemicals, mostly in cell wall materials, are
difficult to digest in the upper gastrointestine and may be digested by bacteria in the colon to
provide health benefits and reduce the risk of colon cancer (12). Only a small portion of
flavonoids absorbed across the intestinal membrane are partly transformed to glucuronides and
sulfates; however, the majority of the flavonoids are degraded by intestinal microflora. Several
phenolic acids are produced by bacterial enzymes utilizing hydrolysis, dehydroxylation, cleavage
of the heterocyclic oxygen-containing ring and decarboxylation of flavonoids. Reabsorption of
these phenolic acids increases the antioxidant protection with their radical scavenging ability
(13).
Many researchers have reviewed and documented the contributions of phenolic compounds
including anthocyanins, individual phenolic acids and carotenoids present in potato skin and
flesh to antioxidant activity (14-16). Wounding, boiling, baking, drum-drying, freeze-drying and
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microwave cooking of white and colored potatoes had different effects on the total phenolic
content (17-19). Potato protein hydrolysate inhibited lipid oxidation of beef patties by
scavenging free radicals (20) and potato peel extracts retarded oxidation in radiation-processed
lamb meat (21).
Legumes are rich sources of protein and dietary fiber. Consumption of legumes help prevent
osteoporosis (22), certain cancers (23), and reduces body lipid accumulation (24). Various
effects of thermal processed cool season legumes on antioxidant activity, phenolic content and
potential health benefits were reported (25, 26). Combining both purple potatoes and dry peas
may produce healthy snack foods incorporating natural color along with positive effects on
human health. Limited investigations have been done on the effect of processing on formulations
from potatoes and legumes on characterizing the phytochemicals profile and contributions to
antioxidant activity. Metabolism, absorption, and bioavailability of health-beneficial
phytochemicals could be improved by combining protein and individual phytochemicals. Liu (9)
reported the additive and synergistic effects of biologically active compounds from fruits,
vegetables, and whole grains are responsible for their health benefits.
The objective of this study was to investigate the complete phytochemical profiles that exist
in free and bound forms as well as their contribution to the total antioxidant activity in the raw
ingredients (purple potato flour and dry pea flour), raw formulations and processed products
prepared from the above ingredients using extrusion cooking. The bioactivity of phytochemicals
in the raw and processed samples was also determined using the cellular antioxidant activity
assay.
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2. MATERIALS AND METHODS
2.1. Chemicals and Reagents
Folin-Ciocalteu reagent, sodium nitrite, catechin, sodium borohydride, chloranil, and vanillin
and gallic acid were purchased from Sigma (St. Louis, MO). Sodium hydroxide, hexane,
aluminum chloride, and acetonitrile were obtained from Fisher Scientific (Pittsburgh, PA), while
ethyl acetate, trifluoroacetic acid, methanol, hydrochloric acid, acetic acid, acetone and ethanol
were of analytical grade and were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ).
Tetrahydrofuran and aluminum chloride were purchased from Fisher Scientific (Fair Lawn, NJ).
2.2. Preparation of Samples
‗Purple Majesty‘ potatoes were peeled, sliced, blanched and drum dried in the school of Food
Science pilot plant, Washington State University. Split dry peas and drum dried potato flakes
were pin milled to produce flours at the Processed Foods Research Unit, Western Regional
Research Center, USDA, Albany, CA. Raw formulations were prepared from purple potato flour
(PPF) and dry pea flour (DPF) at selected concentrations (35/65, 50/50 and 65/35 PPF/DPF
w/w). Extruded products were prepared from the raw formulations using a Clextral twin screw
extruder at 130 °C die temperature, 300 rpm and 17% feed moisture. Detailed extrusion
conditions were elaborated in chapter 4. The extrudates were milled to flour using a coffee
grinder and stored at -80 °C until further use. The flours for routine analysis were stored at -20
C.
2.3 Extraction of Free Phenolic Compounds
Free phenolic compounds in the ingredients, raw formulations, or extruded flours were
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extracted using the method previously reported in our laboratory (27, 28). Briefly, 1-2 g of flour
sample was homogenized with 50 mL of 80% chilled acetone for 10 min using a high speed
homogenizer. After centrifugation of homogenized flour at 2500g for 5 min, the supernatant was
removed and extraction was repeated one more time. Supernatants were pooled, evaporated at 45
C to dryness and reconstituted with water to a final volume of 10 mL. The extracts were stored
at -75 °C until use.
2.4. Extraction of Bound Phenolic Compounds
Bound phenolics of the ingredients, raw formulations, or extruded flours were extracted
using the method previously reported in our laboratory (11, 28, 29). Briefly, 1-2 grams of flour
samples were extracted twice with 80% chilled acetone with centrifugation at 2500g for 5 min,
and the supernatant was discarded after each extraction. The residues were digested with 20 mL
of 2 M sodium hydroxide at room temperature for 1 h with shaking under nitrogen gas. The
mixture was neutralized with an appropriate amount of hydrochloric acid and extracted with
hexane to remove lipids. The final solution was extracted five times with ethyl acetate. The ethyl
acetate fraction was evaporated to dryness. The resulting residues were reconstituted in 10 mL of
water and stored at -75 °C until use.
2.5. Determination of Total Phenolics
The total phenolics of each extract was determined using methods previously described by
Singleton et al. (30) and modified in our laboratory (31, 32). Briefly, 400 µL of deionized water
and 100 µL of a known dilution of the extract or standard solution were added to a test tube.
Folin-Ciocalteu reagent (100 µL) was added to the solution and allowed to react for 6 min. Then,
1 mL of 7% sodium carbonate solution and 800 µL of deionized water was added into the test
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tubes, and the mixture mixed well. The color developed for 90 min at room temperature, and
absorbance was read at 760 nm using a MRX II DYNEX spectrophotometer (DYNEX
Technologies, Inc., Chantilly, VA). The measurements for free, bound and total phenolics were
compared to a standard curve of prepared gallic acid solutions and expressed as micrograms of
gallic acid equivalents (GAE) per gram dry weight (DW) sample ± SD for triplicate extracts.
2.6. Determination of Individual Phenolic Acids
Chlorogenic, caffeic, p-coumaric and ferulic acids in sample extracts were quantified using a
reverse phase HPLC procedure employing a Supelcosil LC-18-DB, 150 mm x 4.6 mm, 3 mm
column as reported previously (33). Briefly, isocratic elution was conducted with 20%
acetonitrile in water adjusted to pH 2 with trifluoroacetic acid, at a flow rate of 1.0 mL/min,
delivered using a Waters 515 HPLC pump (Waters Corp., Milford, MA). A Waters 2487 dual
wavelength absorbance detector (Waters Corp.) was used for UV detection of analytes at 280
nm. Data signals were acquired and processed on a PC running the Waters Millennium software,
version 3.2 (1999) (Waters Corp.). The retention times of the standards, chlorogenic, caffeic, p-
coumaric and ferulic acids, were 3.3, 4.7, 7.4 and 8.4 min, respectively. The phenolic acid
concentrations of sample extracts were extrapolated from the pure phenolic acid standard curves
(chlorogenic acid, r2=0.99; caffeic acid, r
2=0.99; p-coumaric acid, r
2=0.99; and ferulic acid,
r2=0.99). Twenty microliter injections were made in each run, and areas were used for all
calculations. The selected individual peaks were identified by the retention times and co-
injection of the pure standards. The method was validated by the recovery of chlorogenic acid,
and the percentage recovery for chlorogenic acid was 96.5 ± 6.22 (n=3).
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2.7. Determination of Total Flavonoids
The total flavonoid contents of the samples were determined using the method of Sodium
Borohydride/Chloronil (SBC) total flavonoid assay with modifications (34). Briefly, stored
sample extracts for total phenolic analysis were thawed and added into test tubes (15 × 150 mm),
then dried at 45 °C under nitrogen gas, and reconstituted in 1 mL of THF/EtOH (1:1, v/v).
Catechin standards (0.3-10.0 mM) were prepared fresh each day before use in 1 mL of
THF/EtOH (1:1, v/v). Each test tube with 1 mL of sample solution or 1 mL of catechin standard
solution had 0.5 mL of 50 mM NaBH4 solution and 0.5 mL of 74.56 mM AlCl3 solution added
and was then shaken in an orbital shaker (Laboratory-Line Instruments, Inc., Melrose Park, IL)
for 30 min at room temperature. Then an additional 0.5 mL of NaBH4 solution was added into
each test tube with continual shaking for another 30 min at room temperature. Cold acetic acid
solution (2.0 mL of 0.8 M, 4 °C) was added into each test tube, and the solutions were shaken in
the orbital shaker in the dark for 15 min after thorough mixing. Then, 1 mL of 20 mM chloranil
was added into each tube, which was heated at 95 °C with shaking for 60 min in a reciprocal
shaking bath (Precision Scientific Inc., Chicago, IL). The temperature in the reciprocal shaking
bath was maintained using glycerin. The reaction solutions were cooled using tap water, and the
final volume was brought to 4 mL using methanol. Then, 1 mL of 1052 mM vanillin was added
into each tube, followed by mixing. Concentrated HCl (2 mL of 12 M) was added into each tube,
and the reaction solutions were kept in the dark for 15 min after thorough mixing. Aliquots of the
final reaction solutions (200 μL) were added into each well of a 96-well plate after centrifuging
for 3 min at 2500g, and absorbances were measured at 490 nm using a MRX Microplate Reader
with Revelation work station (Dynex Technologies, Inc., Chantilly, VA). Total flavonoids were
expressed as micrograms of catechin equivalents per gram of dry weight sample. Data were
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reported as mean ±SD for three replicates.
2.8. Measurement of Antioxidant Activity (ORAC)
The antioxidant activity of the samples (ingredients, raw formulations and extruded products)
were determined using the oxygen radical absorbance capacity(ORAC) assay described by Prior
et al. (35) and modified in our laboratory (36). Briefly, 20 µL of blank, trolox standard, or
sample extracts in 75 mM potassium phosphate buffer, pH 7.4 (working buffer), was added to
triplicate wells in a black, clear-bottom, 96-well microplate. The triplicate samples were
distributed throughout the microplate and were not placed side-by-side, to avoid any effect on
readings due to location. In addition, no outside wells were used, as use of those wells results in
greater variation. A volume of 200 µL of 0.96 µM fluorescein in working buffer was added to
each well and incubated at 37 C for 20 min, with intermittent shaking, before the addition of 20
µL of freshly prepared 119 mM ABAP in working buffer using a 12-channel pipetter. The
microplate was immediately inserted into a Fluoroskan Ascent FL plate reader
(ThermoLabsystems) at 37 C. The decay of fluorescence at 538 nm was measured with
excitation at 485 nm every 4.5 min for 2.5 h. The areas under the fluorescence versus time curve
for the samples minus the area under the curve for the blank were calculated and compared to a
standard curve of the areas under the curve for 6.25, 12.5, 25, and 50 µM trolox standards minus
the area under the curve for blank. ORAC values were expressed as mean micromoles of trolox
equivalents (TE) per gram of dry weight sample ± SD for triplicate data.
2.9. Cellular Antioxidant Activity (CAA) assay
The assay for CAA was performed following the procedures of Wolfe and Liu (37). Briefly,
HepG2 liver cancer cells from the passages of 5 and 7 were seeded at a density of 6 x 104 in 100
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µL complete growth medium per well on a 96-well microplate in a humidified 5 % CO2
incubator at 37 °C. The wells on the boundary of the microplate were filled with 200 µL of PBS.
Twenty-four hours after seeding, the growth medium was removed and the wells were washed
with PBS. The wells were treated in triplicate with 100 µL of solutions containing different
concentrations of antioxidant extracts plus 25 µM DCFH-DA dissolved in antioxidant treatment
media for 1 h at 37 °C. Then treatment mediums were removed and wells were washed with 100
µL of PBS to remove extracellular residues. One hundred microliters of 600 µM ABAP in
oxidant treatment medium (HBSS) was applied to all the cells and the microplate was
immediately placed into a Fluoroskan Ascent FL plate reader (ThermoLabsystems, Franklin,
MA) at 37 °C. Emission was measured for 1 h at 538 nm after excitement at 485 nm in every 5
min. The blank wells contained cells treated with DCFH-DA, HBSS and antioxidant extracts
without ABAP whereas the control wells contained cells treated with DCFH-DA, HBSS and
ABAP without antioxidant extracts.
The area under curve for fluorescence (after subtraction of blank and initial fluorescence
values) versus time was integrated to calculate the CAA value at each concentration of the
sample extracts as follows:
CAA unit = )/(1 CASA
where SA is the integrated area under the sample fluorescence vs time curve and CA is the
integrated area from the control curve. The median effective dose (EC50), i.e. dose required to
give a 50% inhibition for sample extract, was determined from the median effective plot of
log(fa/fu) vs log(dose), where fa is the fraction affected (CAA unit) and fu is the fraction
unaffected (1 – CAA unit) by the treatment. In the experiments, quercetin was used as a
standard.
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2.10. Statistical Analyses
All results were reported as mean ± SD for at least three analyses for each type of extraction
and parameter. Results were subjected to ANOVA, and significance of differences between
means were determined using Tukey‘s multiple comparison test run on SAS (version 9.1, SAS
Institute Inc., Cary, NC). Correlations among various parameters were also investigated using
Pearson‘s correlation coefficient.
3. RESULTS
3.1. Phenolic Contents
The phenolic contents of the ingredients, raw formulations, and extruded products were
determined using the Folin-Ciocalteu method and expressed as µg GAE/g DW sample (Figure
5.1). Free, bound, and total phenolics in purple potato flour (PPF) were 2008 ± 118, 932 ± 66,
and 2940 ± 183 µg GAE/g DW sample, respectively. Dry pea flour (DPF) had 366 ± 20, 55 ± 2,
and 421 ± 19 µg GAE/g DW sample of free, bound, and total phenolics, respectively. Extruded
products prepared from 35% PPF had significantly higher (p < 0.05) free phenolic contents
(1947 ± 60 µg GAE/g DW sample) than its raw formulation (919 ± 28 µg GAE/g DW sample).
Similarly, free phenolic contents in the extruded products prepared from 50% PPF (2692 ± 42 µg
GAE/g DW sample) and 65% PPF (3977 ± 36 µg GAE/g DW sample) were significantly
higher(p < 0.05) than their raw formulations (1552 ± 53 and 2290 ± 92 µg GAE/g DW sample,
respectively). The bound phenolic contents in the extruded products prepared from 35, 50 and
65% PPF (175 ± 8, 378 ± 28, and 331 ± 35 µg GAE/g DW sample, respectively) were
significantly lower (p < 0.05) than their raw formulations (415 ± 26, 478 ± 21 and 451 ± 31 µg
GAE/g DW sample, respectively). The total phenolic contents of the extruded products prepared
185
0
1000
2000
3000
4000
5000
PPF DPF Raw Extruded Raw Extruded Raw Extruded
Ingredients 35% Purple potato
flour formulation
50% Purple potato
flour formulation
65% Purple potato
flour formulation
Ph
eno
lic
con
ten
ts
(µg
ga
llic
aci
d e
q./
g s
am
ple
, D
W)
Free Bound Total
a
b
c
d
cd
d
eef ff
f
g
h
ii
jj j j j j j
kl
Figure 5. 1. Phenolic contents of ingredients, raw formulations, and extruded products prepared
from raw formulations. The formulation contains x% of purple potato flour and (100-x)% of dry
pea flour (DPF). Bars with different letters are significantly different (p < 0.05).
from 35, 50 and 65% PPF (2122 ± 53, 3070 ± 62 and 4308 ± 64 µg GAE/g DW sample,
respectively) were significantly higher (p < 0.05) than their raw formulations (1334 ± 51, 2030 ±
39 and 2741 ± 120 µg GAE/g DW sample, respectively). Table 5.1 shows the percentage
contributions of free and bound phenolics to the total phenolic content of raw ingredients and
extruded products.
3.2. Flavonoid Contents
The flavonoid contents of the raw ingredients, formulations and extruded products were
determined using the SBC assay and were expressed as µg catechin eq/g DW sample (Figure
186
5.2). The free, bound and total flavonoid contents of potato flour were 5495 ± 42, 724 ± 23, and
6219 ± 417 µg catechin eq/g DW sample, respectively. The free, bound, and total flavonoid
contents of DPF were 1305 ± 36, 731 ± 71, and 2036 ± 72 µg catechin eq/g DW sample,
respectively. The free flavonoid contents of the extruded products prepared from 35, 50 and 65%
PPF were 2606 ± 220, 2929 ± 153, and 3468 ± 64 µg catechin eq/g DW sample, respectively,
and were significantly higher (p < 0.05) than their raw formulations (1944 ± 100, 2196 ± 68, and
2890 ± 103 µg catechin eq/g DW sample, respectively). Extruded products prepared from 35 and
50% PPF had significantly higher (p < 0.05) bound flavonoid contents (592 ± 52 and 470 ± 32
µg catechin eq/g DW sample, respectively) than their raw samples (273 ± 24 and 289 ± 26 µg
catechin eq/g DW sample, respectively). However, the bound flavonoid content in the extruded
products prepared from 65% PPF (290 ± 27 µg catechin eq/g DW sample) was not significantly
different (p > 0.05) from its raw formulation (230 ± 19 µg catechin eq/g DW sample). The total
flavonoid content of the extruded products prepared from 35% PPF (3198 ± 220 µg catechin eq/g
Table 5. 1. Percentage contributions of phytochemicals in free and bound extract of ingredients
(purple potato flour and dry pea flour), raw formulations, and extruded products to total
phenolics, total antioxidant activity and total flavonoids (n=3). Total Phenolics Total Antioxidant Activity
(ORAC value)
Total Flavonoids
Free
(%)
Bound
(%)
Free (%) Bound (%) Free
(%)
Bound
(%)
Purple potato flour (PPF) 68 32 64 36 88 12
Dry pea flour (DPF) 87 13 86 14 64 36
35% PPF* Raw 69 31 63 37 88 12
Extruded 92 8 88 12 82 18
50% PPF Raw 76 24 74 26 88 12
Extruded 88 12 88 12 86 14
65% PPF Raw 84 16 83 17 93 7
Extruded 92 8 93 7 92 8
* The formulation contains x% of purple potato flour and (100-x)% of dry pea flour. Extruded
products were prepared from the raw formulations using extrusion cooking technology.
187
0
1000
2000
3000
4000
5000
6000
7000
PPF DPF Raw Extruded Raw Extruded Raw Extruded
Ingredients 35% Purple potato
flour formulation
50% Purple potato
flour formulation
65% Purple potato
flour formulation
Fla
vo
no
id c
on
ten
ts
(µ
g c
ate
chin
/g s
am
ple
, D
W)
Free Bound Total
a
b
ccdcd
de dee
efe
ffg
fgfg
g
ji
ii
kkkl
h
Figure 5. 2. Flavonoid contents of ingredients, raw formulations and extruded products prepared
from raw formulations. The formulation contains x% of purple potato flour and (100-x)% of dry
pea flour (DPF). Bars with different letters are significantly different (p < 0.05).
DW sample) was significantly higher (p < 0.05) than its raw formulation (2217 ± 124 µg
catechin eq/g DW sample). Similarly, extruded products prepared from 50 and 65% PPF (3400 ±
149 and 3758 ± 89 µg catechin eq/g DW sample, respectively) were significantly higher (p <
0.05) than their raw formulations (2486 ± 53 and 3120 ± 111 µg catechin eq/g DW sample,
respectively).
3.3. Individual Phenolic Acids
Detected individual phenolic acids present in raw formulations and extruded products are
188
Table 5. 2. Quantity of free, bound, and total individual phenolic acids present in extracts of ingredients, raw formulations and
extruded products (Mean ± SD, n=3)
Free
(µg/g sample, DW)
Bound
(µg/g sample, DW)
Total
(µg/g sample, DW)
Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum Ferul
Purple potato
flour 714 ±55
d 14±1
b 52 ±0.3
b n.d. 202 ±8
c 801±55
a 1019 ±34
a 109 ±11
b 916 ±55
e 815 ±56
a 1071 ± 35
a 109 ±11
b
Dry pea flour 2.0 ±1g n.d. n.d. n.d. 236 ±20
b 15 ±3
f n.d. 22 ±3
d 260 ±25
g 15 ±3
f n.d. 22 ±3
d
35%
PPF*
Raw 298 ± 24f 10±1
c 6±1
d n.d. 253±27
b 306±45
b 334±10
e 59±1
c 551±46
f 316±46
b 340±10
e 59±1
c
Exd 984±10c n.d. n.d. n.d. 581±60
a 77±6
e 362±11
d 188±12
a 1565±66
c 77±7
e 362±11
e 188±12
a
50%
PPF
Raw 540±77e 13±1
b 37±8
c n.d. 253±33
b 339±39
b 387±54
cd 57±7
c 794±107
e 353±39
b 425±47
d 57±7
c
Exd 2011±211b n.d. n.d. n.d. 609±60
a 168±17
c 970±113
ab 179±27
a 2620±170
b 168±17
c 970±113
ab 179±27
a
65%
PPF
Raw 1011±42c 23±4
a 117±18
a n.d. 243±21
b 309±52
b 463±35
c 58±4
c 1254±63
d 333±55
b 581±51
c 58±4
c
Exd 2967±267a n.d. n.d. n.d. 574±81
a 93±7
d 857±73
b 159±21
a 3541±348
a 93±7
d 857±73
b 159±21
a
* The formulation contains x% of purple potato flour and (100-x)% of dry pea flour in the formulation. Extruded products were
prepared from the raw formulations using extrusion cooking technology. aValues in each column with no letters in common are
significantly different (p < 0.05). Chloro – Chlorogenic acid; p-Coum – Coumaric acid; n.d. – not detected
189
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
PPF DPF Raw Extruded Raw Extruded Raw Extruded
Ingredients 35% Purple potato
flour formulation
50% Purple potato
flour formulation
65% Purple potato
flour formulation
An
tio
xid
an
t A
ctiv
ity
(O
RA
C)
(µm
ol
Tro
lox
eq
./g
sa
mp
le, D
W)
Free Bound Total
a
a
b
bcc c
de
dee
ee
f
f f
g hh h
hhi j i
k
Figure 5. 3 Antioxidant activity of ingredients, raw formulations and extruded products prepared
from raw formulations. The formulation contains x% of purple potato flour and (100-x)% of dry
pea flour (DPF) . Bars with different letters are significantly different (p < 0.05).
presented in Table 5.2, expressed as µg/g DW sample. Chlorogenic acid was the prominent
phenolic acid in the free phytochemicals of the samples whereas caffeic and p-coumaric acids
were prominent in the bound phytochemicals of the samples followed by chlorogenic acid.
Quantity of caffeic, p-coumaric and ferulic acids were either less or not detected in the free
phytochemicals of the raw formulations and extruded products. Chlorogenic acids in the free and
bound phytochemicals of the extruded products were 984 – 2967 µg/g DW sample and 574 –
609 µg/g DW sample, respectively, and were significantly higher (p < 0.05) than their raw
formulations (297 – 1011 µg/g DW sample and 243 – 253 µg/g DW sample, respectively).
190
Extrusion of raw formulations significantly lowered (p < 0.05) the caffeic acid content (306 –
339 to 77 – 168 µg/g DW sample) whereas processing increased the quantity of p-coumaric acid
(334 – 463 to 362 – 970 µg/g DW sample) in the bound phytochemicals of all raw formulations
except 35% PPF. Quantities of ferulic acid in the extruded products (159 – 188 µg/g DW
sample) were also significantly higher (p < 0.05) than their raw formulations (57 – 59 µg/g DW
sample). Individual phenolic acids except caffeic acid in the total phytochemicals were
significantly increased (p < 0.05) in the extruded products compared to their raw formulations.
However, no significant difference (p > 0.05) was observed in the content of total p-coumaric
acid in the extruded product and raw formulation.
3.4. Total Antioxidant Activity
The total antioxidant activities of the ingredients, raw formulations and extruded products
were determined using the ORAC assay and expressed as µmol TE/g DW sample (Figure 5.3)
The free, bound, and total ORAC values of PPF were 47.82 ± 1.66, 26.80 ± 0.89, and 74.62 ±
2.53 µmol TE/g DW sample, respectively. The free, bound and total ORAC values of DPF
samples were 8.86 ± 0.88, 1.43 ±0.07, and 10.29 ± 0.92 µmol TE/g DW sample, respectively.
The free ORAC values of extruded products prepared from 35, 50 and 65% PPF (45.48 ± 2.34,
75.29 ± 5.32 and 113.83 ± 6.67 µmol TE/g DW sample, respectively) were significantly higher
(p < 0.05) than these of their raw formulations (17.69 ± 1.56, 33.36 ± 4.27 and 57.80 ± 1.44
µmol TE/g DW sample, respectively). The bound ORAC values of extruded products prepared
from 35 and 65% PPF (6.08 ± 0.23, and 8.60 ± 1.42 µmol TE/g DW sample, respectively) were
significantly lower (p < 0.05) than these of their raw formulations (10.28 ± 0.52, and 12.25 ±
0.64 /g DW sample, respectively). However, there was no significant difference (p > 0.05) in the
191
ORAC values of the bound phytochemicals in the extruded products prepared from 50% PPF
(9.93 ± 0.54 µmol TE /g DW sample) formulation compared to its raw formulation (11.58 ± 1.74
0.000
0.050
0.100
0.150
0.200
PPF DPF Raw Extruded Raw Extruded Raw Extruded
Ingredients 35% Purple potato
flour formulation
50% Purple potato
flour formulation
65% Purple potato
flour formulation
Cel
lula
r A
nti
ox
ida
nt
Act
ivit
y
(µm
ol
qu
erce
tin
eq
./g
sa
mp
le, D
W)
0
100
200
300
400
500
EC
50
va
lue
(mg
/mL
)
CAA
EC50
a
b
d
d
c
c c
D
B C
D
A
CC
Figure 5. 4. Cellular antioxidant activity and EC50 value of ingredients, raw formulations, and
extruded products prepared from raw formulations. The formulation contains x% of purple
potato flour (DPF) and (100-x)% of dry pea flour. Bars with different letters are significantly
different (p < 0.05). Capital and small letters are for EC50 and cellular antioxidant activity,
respectively.
µmol TE /g DW sample). Extruded products prepared from 35, 50 and 65% PPF had
significantly higher (p < 0.05) ORAC values (51.57 ± 2.57, 85.22 ± 5.81 and 122.43 ± 7.52 µmol
TE/g DW sample, respectively) than these of their raw formulations (27.98 ± 1.87, 44.95 ± 5.92
and 70.05 ± 1.00 µmol TE/g DW sample, respectively).
192
3.5. Cellular Antioxidant Activity
The CAA of raw formulations and extruded products were quantified using the protocol of
the CAA method and expressed as EC50 in mg/mL and CAA values in µmol quercetin equivalent
(QE)/g DW sample (Figure 5.4). The EC50 value of CAA of PPF sample was 52.1 ± 4.0 mg/mL
(CAA = 0.084 µmol QE/g DW sample) whereas no CAA activity was detected in the DPF
sample. The EC50 values of CAA of the extruded product prepared from 35% PPF (64.7 ± 9.0
mg/mL) and 50% PPF (38.9 ± 7.4 mg/mL) were significantly lower (p < 0.05) than these of their
raw formulations (424.2 ± 344 and 195.8 ± 10 mg/mL, respectively). However, no significant
difference in the EC50 values of CAA was observed between the raw (77.8 ± 19 mg/mL) and
extruded products (70.3 ± 4.4 mg/mL) prepared from 65% PPF. Similarly, the CAA values of the
35, 50 and 65% PPF raw formulations (0.017 ± 0.018, 0.025 ± 0.001, and 0.06 ± 0.015 µmol
QE/g DW sample, respectively) and extruded products (0.56 ± 0.008, 0.128 ±0.023, and 0.063 ±
0.004 µmol QE/g DW sample, respectively) followed the same trend as their EC50 values. The
EC50 value of quercetin standard in all the replicates were in the range of 3.35 – 4.5 mg/mL,
which were similar as reported by Wolfe & Liu (37).
3.6. Correlation Analyses
Relationships among total phenolics, total ORAC values and CAA with individual phenolic
acids and flavonoids in free and bound forms were determined using Pearson‘s correlation
coefficient (Table 5.3). Free phenolic content was significantly correlated (p < 0.05) to total
ORAC values (r2 = 0.98) and CAA values (r
2 = 0.66). Similar correlation patterns were observed
between total phenolics and total ORAC values and CAA values. Free and total flavonoids were
significantly correlated (p < 0.05) to total phenolics, ORAC and CAA values. Among individual
phenolic acids, free and total chlorogenic acid contents were significantly correlated (p < 0.05) to
193
the total phenolics, ORAC and CAA values. p-Coumaric and ferulic acids in the bound and total
extracts significantly correlated (p < 0.05) to the total phenolics, ORAC and CAA values.
However, caffeic acid did not show any positive correlation with phenolics, ORAC or CAA
values. Total ORAC values were significantly correlated (p < 0.05) with CAA values (r2 = 0.69).
4. DISCUSSION
Phytochemicals in food samples are present in both free and bound form (11, 12). Without
accounting for bound phytochemicals, the total phytochemical content will be underestimated
(11). Therefore, studying free and bound phytochemicals provides accurate information on the
total phytochemicals and the contributions of free, soluble conjugated and bound to the total
phenolics and their total antioxidant activity. The cellular antioxidant activity assay, which is
used in this study, is a biologically relevant way to quantify the bioactivity of antioxidants in
food products as it takes into consideration the cellular uptake, metabolism, and distribution of
bioactive compounds in the cell (38). The values of cellular antioxidant activities of samples
were also compared with a chemical assay, ORAC. This study was designed to determine the
phytochemicals, their relationships and contributions to total antioxidant activity in potato and
dry pea flours, raw formulations, and processed snack foods applying extrusion technology on
the raw formulations.
Our study showed that majority of the phenolics in purple potato and dry pea flours were present
in free rather than in bound form (Table 5.1). The quantity of total phenolics in potato flour
prepared from the flesh was within the range (1120 – 12370 µg GAE/g DW sample with skin) of
potato cultivars grown in the Andes mountains of South America (39). Results from a Colorado
potato breeding program have shown similar total phenolic contents in processed (microwaved
and freeze-dried) ‗Purple Majesty‘ potatoes with skin (19). The percentage contribution of free
194
phenolics to the total was 68% whereas bound phenolics contributed 32% in potato flour (Table
5.1). This is similar to the results of Chu et al., (40) who reported the contributions of 60 and
40% by free and bound phenolics, respectively, to the total phenolics in white potatoes with skin
(40). Therefore, the total phenolic contents reported in the previous literature citations were
underestimated by not including the bound phenolics in potatoes. Bound phenolics might be
associated with the plant cell walls that survive upper gastrointestinal digestion and reach the
colon where most of it is released by intestinal microflora (11, 12). Total phenolic content in dry
pea flour in the present study (Figure 5.1) was 3 – 5 fold lower than in the reported literature
(25, 26). This could be due to (i) use of previously milled flours compared to whole raw legumes
by other researchers; (ii) different solvents for extraction of phenolic compounds (41). Free
phenolics in dry pea flour contributed more (87%) to the total phenolics than the bound ones
(13%). Raw formulations from potato and dry pea flours in selected concentrations had 69 – 84%
free phenolics. Extrusion cooking of raw formulations increased the percentage contributions of
free phenolics to the total and decreased the contributions from bound phenolics (Table 5.1).
Increase in the total phenolicsin the extruded food products (50 – 60%) rather than in raw
formulations could be due to (i) breaking of conjugated phenolics into free phenolics, and (ii)
leaching of soluble fibers, proteins and other non-phenolic soluble components such as mono-,
di- and oligosaccharides (26). Han and Baik (26) reported a similar increase in total phenolics in
cooked and soaked chickpeas, yellow peas, green peas and soybeans. However, in another report,
total phenolics in cooked cool season food legumes were significantly reduced (p < 0.05) when
compared to uncooked samples (25).
Free and bound phytochemicals in potato flour contributed 64 and 36%, respectively, to the
total ORAC antioxidant activity, similar to their contributions to the total phenolics. The total
195
Table 5. 3. Correlation analysis of phenolics, antioxidant activity (ORAC) and cellular antioxidant activity of ingredients, raw
formulations and extruded product.
Free extract Bound extract Total
Phenolics
Total
ORAC
value
CAA Phenolics
Total
ORAC
value
CAA Total
Phenolics
Total
ORAC
value
CAA
Phenolics 0.9780
(<.0001)
0.6565
(0.0005)
0.3295
(0.1159)
0.3936
(0.0570)
0.9847
(<.0001)
0.6988
(0.0001)
Flavonoids 0.6636
(0.0004)
0.6288
(0.0010)
0.6008
(0.0019)
-0.3210
(0.1262)
-0.2752
(0.1931)
0.0677
(0.7534)
0.5814
(0.0029)
0.5555
(0.0048)
0.5832
(0.0028)
Chlorogenic
acid
0.8843
(<.0001)
0.9176
(<.0001)
0.6047
(0.0017)
0.5117
(0.0106)
0.5583
(0.0046)
0.5605
(0.0044)
0.8529
(<.0001)
0.8897
(<.0001)
0.6181
(0.0013)
Caffeic acid 0.0240
(0.9113)
-0.0655
(0.7610)
-0.0708
(0.7423)
0.1912
(0.3707)
0.1121
(0.6019)
0.2377
(0.2634)
0.1878
(0.3796)
0.1073
(0.6178)
0.2299
(0.2799)
p-Coumaric
acid
0.1575
(0.4622)
0.0926
(0.6668)
0.0880
(0.6827)
0.8402
(<.0001)
0.8329
(<.0001)
0.8551
(<.0001)
0.8465
(<.0001)
0.8318
(<.0001)
0.8532
(<.0001)
Ferulic acid n.d. n.d. n.d. 0.6419
(0.0007)
0.6583
(0.0005)
0.7197
(<.0001)
0.6419
(0.0007)
0.6583
(0.0005)
0.7197
(<.0001)
ORAC
value
0.8762
(<.0001)
0.3514
(0.0923)
0.3188
(0.1290)
0.2749
(0.1936)
0.9847
(<.0001)
0.6984
(0.0001)
Correlation coefficients were calculated as r2. Significant levels are given in the parenthesis.
196
ORAC value in potato flour was in the range of hydrophilic ORAC values (28.25 – 250.67 µmol
TE/g DW) of Andean potato cultivars (39). Individual ORAC values of free and bound
phytochemicals could not be compared due to limited information in the literature. The total
ORAC value in dry pea flour was comparable to that reported in the literature (42) and had
similar contributions from free (86%) and bound (14%) phytochemicals as was the case in total
phenolics (Table 5.1). Han and Baik (26) reported free and bound phytochemicals contributed
68 and 32% respectively, to the total TEAC antioxidant activity of yellow peas. Processing of all
raw formulations using extrusion cooking significantly increased (p < 0.05) the total ORAC
values of extruded products. Increase in the total ORAC values followed the same pattern as in
total phenolics in the extruded products. It was also observed that ORAC values of bound
phytochemicals significantly decreased (p < 0.05) and ORAC values of free phytochemicals
increased in the extruded products. This phenomenon could be attributed to (i) breaking of
conjugated phytochemicals to release free phytochemicals (31), (ii) prevention of enzymatic
oxidation, and (iii) darker colors of the extruded products indicating formation of Maillard
reaction products having antioxidant properties (43). ORAC values of processed green peas,
yellow peas and chickpeas were significantly increased (p < 0.05) (27 – 114%, 12 – 67% and 25
– 40%, respectively) after pressure boiling as compared to the raw legume (42). However, the
FRAP values and DPPH antioxidant activity of legumes were decreased significantly (p < 0.05)
by conventional and pressure boiling (25, 42). An increase in antioxidant activities of sweet corn,
teas and tomatoes with thermal processing were also reported (4, 31, 44).
Quantities of free flavonoids in potato and dry pea flours were significantly higher (p < 0.05)
when compared to their bound flavonoids. The total flavonoids of both flours followed similar
pattern as free flavonoids, because of the larger contribution from free flavonoids (Table 5.1).
197
There is limited literature available to compare the free, bound and total flavonoids in colored
potatoes (45). The total flavonoids of dry pea flour in our study was higher when compared to
the reported total flavonoid contents of whole (41, 46) and dehulled yellow peas (47). Quantities
of free flavonoids increased after extrusion cooking of all the raw formulations. Interestingly, the
contents of bound flavonoids were also significantly increased (p < 0.05) in the extruded
products when compared to their raw formulations. Choi et al. (48) reported a significant
increase of free flavonoids in Shiitake mushrooms after heat treatment at 100 and 121 °C when
compare to raw mushrooms. However, the investigators observed decreases in the bound
flavonoids after heat treatment. Increase in flavonoid contents in the extrudates could be
attributed to (i) disruption of plant cell walls providing better extractability, (ii) breaking of
chemical bonds of higher molecular weight polyphenols and forming soluble low molecular
weight polyphenol compounds, and (iii) inter-conversion of flavonoids in different forms (49).
Variyar et al. (50) reported an increase in antioxidant potential of soybeans with the dose of γ-
irradiation due to increased levels of genistin (an isoflavone) and degradation products of
diadzein.
Phenolic acids are sources of dietary phenols, which are linked to cellulose, lignin and
proteins though ester bonds. For example, chlorogenic acid has strong antioxidant activity and is
already demonstrated to have several desirable effects on biochemical processes involved in
carcinogenesis (51). p-Coumaric acid was the major phenolic acid observed in potato flour,
followed by chlorogenic, caffeic and ferulic acid (Table 5.2). Most of the p-coumaric and caffeic
acids were observed in the bound phenolic fraction whereas chlorogenic acid was higher in the
free phenolics. Ferulic acid was observed only in the bound phenolics. Adom and Liu reported
that more than 93% ferulic acids were present in the bound form in corn, wheat, oats and rice
198
(11). The content of free chlorogenic acid we reported in this study was similar to the total
chlorogenic content in ‗Purple Majesty‘ potatoes found in a Colorado Potato Breeding Program
(19). Interestingly, our results demonstrated the bound phenolics contributed additional ~20% of
total chlorogenic acid, which was commonly underestimated in the published literature. Lewis et
al. (52) and Lachman et al. (53) reported in detail the presence of chlorogenic, caffeic, p-
coumaric, ferulic and other phenolic acids in colored potatoes. Chlorogenic followed by ferulic
and caffeic acids were detected mostly in the bound phenolics in dry pea flour whereas p-
coumaric acid was not detected. Dry pea flour contained only chlorogenic acid in the free
phenolic fraction. The content of total chlorogenic acid in this study was higher when compared
to the reported content of whole yellow peas (25). The investigators, however, reported having
p-coumaric acid along with chlorogenic and gallic acids in yellow peas. A multifold increase in
chlorogenic acid in free and bound phenolic fractions of formulations rather than raw ingredients
could be due to the binding of o-hydroxy phenolic group in chlorogenic acid to protein via the
bidentate hydrogen bond (54). The mechanism for the binding of chlorogenic acid to sunflower
proteins involved both hydrogen bonding and covalent linkages between oxidized phenolics and
nucleophilic amino acid side chains, such as lysine and cysteine (55). Behavior of caffeic acid in
the raw formulations could also be due to the same mechanism of protein-bound phenolics
interactions as chlorogenic acid. Quantities of chlorogenic acids in the free and bound phenolics
in all extruded products were significantly higher (p < 0.05) when compared to their raw
formulations (Table 5.2). A similar pattern was observed in total chlorogenic acid contents in the
extruded products. Xu and Chang (25) reported significant increases in gallic, chlorogenic and
total phenolic acids in yellow peas after pressure boiling. Significant decreases in caffeic acid in
bound fraction and total phenolics were observed in extruded products when compared to their
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raw formulations whereas those decreases were not detected in free phenolics. A decrease in
caffeic acid contents with exposure of potato strips to home processing conditions was reported
(56). Total p-coumaric and ferulic acid contents were increased in extruded products compared
to their raw formulations, mostly due to increases in their bound phenolics. However, caffeic, p-
coumaric and ferulic acids were not detected in free phenolic extractions after extrusion of raw
formulations. The undetected phenolic acids in extrudates may be due to alkaline hydrolysis that
partly or completely broke down original phenolic acids. Increases in total chlorogenic, p-
coumaric and ferulic contents in the extruded products could be attributed to the alkaline
hydrolysis of caffeic acid present in conjugated phenolics. The reasons for change in the
individual phenolic acids during extrusion cooking could be explained according to Fleuriet and
Macheix (57) as (i) oxidative degradation of phenolic acids; (ii) break down of conjugated
phenolics and release of free phenolic acids; and (iii) formation of complex phenolic acids.
Increases in the contents of chlorogenic, p-coumaric and ferulic acids and low molecular
phenolic acids from the breakdown of caffeic acid could have increased the total phenolics in the
extruded products. Thermal decomposition of caffeic acid generated compounds such as
tetraoxygenated phenylinadan isomers with higher antioxidant activity than caffeic acid (58).
Analysis of individual phenolic acids suggested that the breakdown of higher polyphenols and
formation of novel compounds after processing could have increased the total phenolics as well
as antioxidant activity. Significant positive correlations among individual phenolic acids except
caffeic acid to the total phenolics and total ORAC antioxidant activity justify these assumptions.
Increases in the total phenolics after processing and a strong correlation (r2 = 0.9847) with
ORAC antioxidant activity showed that phenolics were mostly responsible for the chemical
antioxidant activity in the extruded products.
200
The bioavailability of phytochemicals in free phytochemical extracts in the raw ingredients,
raw formulations and extruded products were studied using the cellular antioxidant activity
assay. Potato flour showed potent cellular antioxidant activity with an EC50 of 52 mg/mL. The
lower EC50 values indicate higher cellular antioxidant activity. Dry pea flour had no
measureable cellular antioxidant activity. Cellular antioxidant activities were observed in all the
raw formulations and their extruded products (Figure 5.4). Extruded products prepared from
50% PPF had higher cellular antioxidant activity and lower EC50 value among the extrudates (p <
0.05), and also had much higher cellular antioxidant activity (p < 0.05) and lower EC50 value (p <
0.05) than that of its raw formulation. The effect of extrusion on the products prepared from 65%
PPF was not significantly different (p > 0.05) in the EC50 or CAA values. Similar results were
observed in the 35% PPF raw formulation; CAA values were increased in the extrudates when
compared to its raw formulation (Figure 5.4). There was no specific pattern observed in EC50
values of extruded products. It could be possible that products extruded from a 50% PPF raw
formulation had a better composition of potato and dry pea flour for possible additive and
synergistic effects of phytochemicals responsible for the higher cellular antioxidant activity as
suggested by Liu (9). The CAA values of extruded product from 50% PPF formulation was
comparable with those of plums and red grapes and higher than those of cherries, kiwifruit,
mangoes, peaches and pears (36). Extruded products had higher cellular antioxidant activity
when compared to the raw materials, which may be due to extrusion processing increasing the
amount of bioaccessible phytochemicals as well as increased cellular uptake of these
phytochemicals.
In summary, a complete study on the contributions of free and bound phytochemicals to the
total phenolics, flavonoids, ORAC antioxidant activity, and cellular antioxidant activity in
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‗Purple Majesty‘ potato and dry pea flours was made. Raw formulations of these ingredients and
extruded products prepared were analyzed in detail with regard to changes in individual phenolic
acids, bound and free phytochemicals, and their contributions to ORAC antioxidant activities
after extrusion. In most of the extruded products, the total phenolics, flavonoids and ORAC
values increased after extrusion processing. Cellular antioxidants were observed in potato flour,
raw formulations, and extruded products. Extrusion processing improved the cellular antioxidant
activity of 50% PPF raw formulation. Measuring the antioxidant activity using the CAA assay in
extruded food products is important for assessing the bioavailability of processed foods because
it is more biologically relevant than chemical antioxidant assays (37). Further studies on the
antioxidant activities of individual phytochemicals and changes during processing are needed to
understand the detailed mechanism of breaking and formation of total phytochemicals and their
contribution to bioavailability.
ABBREVIATIONS USED
PPF, purple potato flour; DPF, dry pea flour; PBS, phosphate-buffer saline; ABAP, 2,2′-
azobis (2-amidinopropane) dihydrochloride; CAA, cellular antioxidant activity; DCFH, 2′,7′-
dichlorofluorescin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; HBBS, Hank‘s Balanced Salt
Solution; FRAP, ferric reducing/antioxidant power; ORAC, oxygen radical absorbance capacity;
GAE, gallic acid equivalent; QE, quercetin equivalents; TE, Trolox equivalents; TEAC, Trolox
equivalent antioxidant capacity; TRAP, total radical-scavenging antioxidant parameter.
ACKNOWLEDGEMENT
We gratefully acknowledge the assistance of Jams Pan and Matt, Processed Foods Research
Unit, WRRC, USDA-ARS, Albany, CA for their assistance and feedback during the extrusion
202
experiment. We also acknowledge the US Dry Pea and Lentil Council for their financial support
for this study.
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REFERENCES
1. Burg, P.; Fraile, P., Vitamin C destruction during the cooking of a potato dish.
Lebensmittel-Wissenschaft und-Technologie 1995, 28, (5), 506-514.
2. Lathrop, P. J.; Leung, H. K., Rates of ascorbic acid degradation during thermal
processing of canned peas. Journal of Food Science 1980, 45, (1), 152-153.
3. Murcia, M A.; López-Ayerra, B.; Martinez-Tomé, M.; Vera, A. M.; García-Carmona, F.,
Evolution of ascorbic acid and peroxidase during industrial processing of broccoli.
Journal of the Science of Food and Agriculture 2000, 80, (13), 1882-1886.
4. Dewanto, V.; Wu, X. Z.; Adom, K. K.; Liu, R. H., Thermal processing enhances the
nutritional value of tomatoes by increasing total antioxidant activity. Journal of
Agricultural and Food Chemistry 2002, 50, (10), 3010-3014.
5. Eberhardt, M. V.; Lee, C. Y.; Liu, R. H., Nutrition - antioxidant activity of fresh apples.
Nature 2000, 405, (6789), 903-904.
6. Liu, R. H.; Hotchkiss, J. H., Potential genotoxicity of chronically elevated nitric oxide - a
review. Mutation Research-Reviews in Genetic Toxicology 1995, 339, (2), 73-89.
7. Cohen, J. H.; Kristal, A. R.; Stanford, J. L., Fruit and vegetable intakes and prostate
cancer risk. Journal of the National Cancer Institute 2000, 92, (1), 61-68.
8. Lunet, N.; Lacerda-Vieira, A.; Barros, H., Fruit and vegetables consumption and gastric
cancer: A systematic review and meta-analysis of cohort studies. Nutrition and Cancer-
an International Journal 2005, 53, (1), 1-10.
9. Liu, R. H., Potential synergy of phytochemicals in cancer prevention: mechanism of
action. J. Nutr. 2004, 134, (12), 3479S-3485.
204
10. Liu, R. H., Health benefits of fruit and vegetables are from additive and synergistic
combinations of phytochemicals. Am J Clin Nutr 2003, 78, (3), 517S-520.
11. Adom, K. K.; Liu, R. H., Antioxidant activity of grains. Journal of Agricultural and Food
Chemistry 2002, 50, (21), 6182-6187.
12. Liu, R. H., Whole grain phytochemicals and health. Journal of Cereal Science 2007, 46,
(3), 207-219.
13. Pietta, P.-G., Flavonoids as antioxidants. Journal of Natural Products 2000, 63, (7),
1035-1042.
14. Friedman, M., Chemistry, biochemistry, and dietary role of potato polyphenols. A
review. Journal of Agricultural and Food Chemistry 1997, 45, (5), 1523-1540.
15. Alsaikhan, M. S.; Howard, L. R.; Miller, J. C., Antioxidant activity and total phenolics in
different genotypes of potato (Solanum tuberosum, L). Journal of Food Science 1995, 60,
(2), 341-347.
16. Brown, C. R.; Wrolstad, R.; Durst, R.; Yang, C. P.; Clevidence, B., Breeding studies in
potatoes containing high concentrations of anthocyanins. American Journal of Potato
Research 2003, 80, (4), 241-249.
17. Nayak, B.; Berrios, J. J.; Powers, J. R.; Tang, J.; Ji, Y., Colored potatoes (Solanum
tuberosum L.) dried for antioxidant-rich value-added foods. Journal of Food Processing
and Preservation (in Press) 2010.
18. Reyes, L. F.; Cisneros-Zevallos, L., Wounding stress increases the phenolic content and
antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of
Agricultural and Food Chemistry 2003, 51, (18), 5296-5300.
205
19. Stushnoff, C.; Holm, D.; Thompson, M. D.; Jiang, W.; Thompson, H. J.; Joyce, N. I.;
Wilson, P., Antioxidant properties of cultivars and selections from the Colorado potato
breeding program. American Journal of Potato Research 2008, 85, (4), 267-276.
20. Wang, L. L.; Xiong, Y. L. L., Inhibition of lipid oxidation in cooked beef patties by
hydrolyzed potato protein is related to its reducing and radical scavenging ability.
Journal of Agricultural and Food Chemistry 2005, 53, (23), 9186-9192.
21. Kanatt, S. R.; Chander, R.; Radhakrishna, P.; Sharma, A., Potato peel extract - a natural
antioxidant for retarding lipid peroxidation in radiation processed lamb meat. Journal of
Agricultural and Food Chemistry 2005, 53, (5), 1499-1504.
22. Messina, M. J., Legumes and soybeans: overview of their nutritional profiles and health
effects. American Journal of Clinical Nutrition 1999, 70, (3), 439s-450s.
23. Lamartiniere, C. A., Protection against breast cancer with genistein: a component of soy.
American Journal of Clinical Nutrition 2000, 71, (6), 1705s-1707s.
24. Pusztai, A.; Grant, G.; Buchan, W. C.; Bardocz, S.; de Carvalho, A. F. F. U.; Ewen, S. W.
B., Lipid accumulation in obese Zucker rats is reduced by inclusion of raw kidney bean
(Phaseolus vulgaris) in the diet. British Journal of Nutrition 1998, 79, (2), 213-221.
25. Xu, B.; Chang, S. K. C., Phytochemical profiles and health-promoting effects of cool-
season food legumes as influenced by thermal processing. Journal of Agricultural and
Food Chemistry 2009, 57, (22), 10718-10731.
26. Han, H.; Baik, B. K., Antioxidant activity and phenolic content of lentils (Lens culinaris),
chickpeas (Cicer arietinum L.), peas (Pisum sativum L.) and soybeans (Glycine max),
and their quantitative changes during processing. International Journal of Food Science
and Technology 2008, 43, (11), 1971-1978.
206
27. Sun, J.; Chu, Y. F.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of
common fruits. Journal of Agricultural and Food Chemistry 2002, 50, (25), 7449-7454.
28. de la Parra, C.; Serna Saldivar, S. O.; Liu, R. H., Effect of processing on the
phytochemical profiles and antioxidant activity of corn for production of masa, tortillas,
and tortilla chips. Journal of Agricultural and Food Chemistry 2007, 55, (10), 4177-
4183.
29. Adom, K. K.; Sorrells, M. E.; Liu, R. H., Phytochemical profiles and antioxidant activity
of wheat varieties. Journal of Agricultural and Food Chemistry 2003, 51, (26), 7825-
7834.
30. Singleton, V. L.; Orthofer, R.; Lamuela-Raventos, R. M., Analysis of total phenols and
other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.
Oxidants and Antioxidants, Pt A 1999, 299, 152-178.
31. Dewanto, V.; Wu, X. Z.; Liu, R. H., Processed sweet corn has higher antioxidant activity.
Journal of Agricultural and Food Chemistry 2002, 50, (17), 4959-4964.
32. Adom, K. K.; Sorrells, M. E.; Liu, R. H., Phytochemicals and antioxidant activity of
milled fractions of different wheat varieties. Journal of Agricultural and Food Chemistry
2005, 53, (6), 2297-2306.
33. Okarter, N.; Liu, R. H., Health benefits of whole grain phytochemicals. Critical Reviews
in Food Science and Nutrition 2010, 50, (3), 193-208.
34. He, X.; Liu, D.; Liu, R. H., Sodium borohydride/chloranil-based assay for quantifying
total flavonoids. Journal of Agricultural and Food Chemistry 2008, 56, (20), 9337-9344.
35. Prior, R. L.; Hoang, H.; Gu, L. W.; Wu, X. L.; Bacchiocca, M.; Howard, L.; Hampsch-
Woodill, M.; Huang, D. J.; Ou, B. X.; Jacob, R., Assays for hydrophilic and lipophilic
207
antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and
other biological and food samples. Journal of Agricultural and Food Chemistry 2003, 51,
(11), 3273-3279.
36. Wolfe, K. L.; Kang, X.; He, X.; Dong, M.; Zhang, Q.; Liu, R. H., Cellular antioxidant
activity of common fruits. Journal of Agricultural and Food Chemistry 2008, 56, (18),
8418-8426.
37. Wolfe, K. L.; Liu, R. H., Cellular antioxidant activity (CAA) assay for assessing
antioxidants, foods, and dietary supplements. Journal of Agricultural and Food
Chemistry 2007, 55, (22), 8896-8907.
38. Spencer, J. P. E.; El Mohsen, M. M. A.; Rice-Evans, C., Cellular uptake and metabolism
of flavonoids and their metabolites: implications for their bioactivity. Archives of
Biochemistry and Biophysics 2004, 423, (1), 148-161.
39. Andre, C. M.; Ghislain, M.; Bertin, P.; Oufir, M.; Herrera, M. D.; Hoffmann, L.;
Hausman, J. F.; Larondelle, Y.; Evers, D., Andean potato cultivars (Solanum tuberosum
L.) as a source of antioxidant and mineral micronutrients. Journal of Agricultural and
Food Chemistry 2007, 55, (2), 366-378.
40. Chu, Y. F.; Sun, J.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of
common vegetables. Journal of Agricultural and Food Chemistry 2002, 50, (23), 6910-
6916.
41. Xu, B. J.; Chang, S. K. C., A comparative study on phenolic profiles and antioxidant
activities of legumes as affected by extraction solvents. Journal of Food Science 2007,
72, (2), S159-S166.
208
42. Xu, B.; Chang, S. K. C., Effect of soaking, boiling, and steaming on total phenolic
contentand antioxidant activities of cool season food legumes. Food Chemistry 2008,
110, (1), 1-13.
43. Nicoli, M. C.; Anese, M.; Parpinel, M., Influence of processing on the antioxidant
properties of fruit and vegetables. Trends in Food Science & Technology 1999, 10, (3),
94-100.
44. Manzocco, L.; Anese, M.; Nicoli, M. C., Antioxidant properties of tea extracts as affected
by processing. Food Science and Technology-Lebensmittel-Wissenschaft & Technologie
1998, 31, (7-8), 694-698.
45. Christine, E. L.; John, R. L. W.; Jane, E. L., Changes in anthocyanin, flavonoid and
phenolic acid concentrations during development and storage of coloured potato
(Solanum tuberosum L) tubers. Journal of the Science of Food and Agriculture 1999, 79,
(2), 311-316.
46. Xu, B. J.; Yuan, S. H.; Chang, S. K. C., Comparative analyses of phenolic composition,
antioxidant capacity, and color of cool season legumes and other selected food legumes.
Journal of Food Science 2007, 72, (2), S167-S177.
47. Aharon, S.; Hana, B.; Yoram, K.; Ilan, S.; Michal, O.-S.; Shmuel, G., Determination of
polyphenols, flavonoids, and antioxidant capacity in colored chickpea (Cicer arietinum
L.). Journal of Food Science 2010, 75, (2), S115-S119.
48. Choi, Y.; Lee, S. M.; Chun, J.; Lee, H. B.; Lee, J., Influence of heat treatment on the
antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes)
mushroom. Food Chemistry 2006, 99, (2), 381-387.
209
49. Coward, L.; Smith, M.; Kirk, M.; Barnes, S., Chemical modification of isoflavones in
soyfoods during cooking and processing. American Journal of Clinical Nutrition 1998,
68, (6), 1486s-1491s.
50. Variyar, P. S.; Limaye, A.; Sharma, A., Radiation-induced enhancement of antioxidant
contents of soybean (Glycine max Merrill). Journal of Agricultural and Food Chemistry
2004, 52, (11), 3385-3388.
51. Feng, R. T.; Lu, Y. J.; Bowman, L. L.; Qian, Y.; Castranova, V.; Ding, M., Inhibition of
activator protein-1, NF-kappa B, and MAPKs and induction of phase 2 detoxifying
enzyme activity by chlorogenic acid. Journal of Biological Chemistry 2005, 280, (30),
27888-27895.
52. Lewis, C. E.; Walker, J. R. L.; Lancaster, J. E.; Sutton, K. H., Determination of
anthocyanins, flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum
tuberosum L. Journal of the Science of Food and Agriculture 1998, 77, (1), 45-57.
53. Lachman, J.; Hamouz, K.; Orsak, M., Red and purple potatoes - A significant antioxidant
source in human nutrition. Chemicke Listy 2005, 99, (7), 474-482.
54. Mcmanus, J. P.; Davis, K. G.; Beart, J. E.; Gaffney, S. H.; Lilley, T. H.; Haslam, E.,
Polyphenol interactions .1. Introduction - Some observations on the reversible
complexation of polyphenols with proteins and polysaccharides. Journal of the Chemical
Society-Perkin Transactions 2 1985, (9), 1429-1438.
55. Sastry, M. C. S.; Rao, M. S. N., Binding of chlorogenic acid by the isolated polyphenol-
free 11s protein of sunflower (Helianthusa annuus) seed. Journal of Agricultural and
Food Chemistry 1990, 38, (12), 2103-2110.
210
56. Im, H. W.; Suh, B.-S.; Lee, S.-U.; Kozukue, N.; Ohnisi-Kameyama, M.; Levin, C. E.;
Friedman, M., Analysis of phenolic compounds by high-performance liquid
chromatography and liquid chromatography/mass spectrometry in potato plant flowers,
leaves, stems, and tubers and in home-processed potatoes. J. Agric. Food Chem. 2008,
56, (9), 3341-3349.
57. Fleuriet, A., Macheix, J. J., Phenolics in fruits and vegetables. In Flavonoids in Health
and Disease, Rice-Evans, C., Packer, L, Ed. CRC Press: Boca Raton, FL, 2003; pp 1 - 42.
58. Chen, J. H.; Ho, C. T., Antioxidant activities of caffeic acid and its related
hydroxycinnamic acid compounds. Journal of Agricultural and Food Chemistry 1997,
45, (7), 2374-2378.
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CHAPTER SIX
THERMAL DEGRADATION OF ANTHOCYANINS FROM PURPLE
POTATOES (‘PURPLE MAJESTY’ CV) AND IMPACT ON
ANTIOXIDANT CAPACITY.
ABSTRACT
Thermal degradation kinetics of purified anthocyanins from purple-fleshed potatoes (‗Purple
Majesty‘ cv.) was studied over a temperature range of 100 and 150 °C. Anthocyanin was
prepared by removing salts, sugars and colorless non-anthocyanin phenolics from the crude
extract were quantified using HPLC and spectrophotometry for heat induced degradation
compounds. Colorless phenolics from the anthocyanins were separated using HPLC and
monitored at two wavelengths, i.e., 280 and 520 nm. The thermal degradation of purified
anthocyanins followed a first order reaction with reaction rate constants (k-values) of 0.0262 –
0.2855 min-1
, activation energy of 72.89 kJ/mol, thermal death times (D-values) of 8.06 – 8789
min and z-value of 47.84 °C. The enthalpy and entropy of activation was 59.97 kJ/mol and –
116.46 J/mol K, respectively. But the total antioxidant capacity in the thermally treated samples,
measured using DPPH and ABTS assay, were retained indicating antioxidant activities of
degradation compounds in the samples.
1. INTRODUCTION
Anthocyanins are one of the most important groups of water soluble plant pigments largely
responsible for the cyanic colors of flowers, fruits, vegetables and grains accumulating in the
vacuoles of epidermal or subepidermal cells (1). Recently, anthocyanins have gained increasing
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attention as functional compounds for natural color as a replacement for synthetic dyes in food
products (2). In addition, these compounds have health related beneficial antioxidative (3-5) and
anticarcinogenic properties (6, 7). Mishra et al. (8) studied the degradation of anthocyanins
above 100 °C and suggested that anthocyanins could be used as food colorants in high
temperature processes such as for extruded snacks or baked cakes. However, successful use of
anthocyanins either as natural colors or as nutraceuticals depends on their physical and chemical
stability during different processing conditions.
Commercial production of colored potatoes such as the ‗Purple Majesty‘ cultivar has
attractive taste and appearance. An added benefit of colored potatoes is their high antioxidant
capacity mainly due to the presence of polyphenols including anthocyanins (9, 10). Stushnoff et
al. (10) reported five petunidin glycosides and single glycoside of each of malvidin, peonidin and
delphidin aglycones in ‗Purple Majesty‘ potatoes. Various mechanisms of stability of
anthocyanins such as association between pigments and cofactors (polyphenols, metal ions, other
anthocyanins) have been proposed in numerous reports (11-13). Acylation of aromatic acids to
the structure (14, 15) and side chain double bond (16) has also been attributed to the stability of
anthocyanins.
Besides the structure, stability of anthocyanins depends on temperature, light, enzymes,
metal ions, sugars, ascorbic acid, oxygen, and presence of other phenolic compounds, (17).
Several studies have reported on the degradation of anthocyanins in fruits and vegetables during
processing and storage (18, 19). The investigators either assumed or reported first order kinetics
for thermal degradation of anthocyanin in selected fruits and vegetables (20-23). Those studies
were carried out either at temperatures below 100 °C or in whole pulp puree/extract. In general,
extracts from whole pulp of fruits and vegetables or fruit and vegetable contain anthocyanins
213
along with other compounds such as salts, sugars and other colorless non-anthocyanin phenolics
that could affect the stability or degradation kinetics and antioxidant capacity of anthocyanins
(12, 24). Little information is available regarding the thermal kinetics of purified anthocyanins
and antioxidant potencies of degradation compounds. Such information is desirable as reference
starting point to study the interaction of anthocyanin with other compounds in more complicated
food matrix. Stintzing et al., (4) reported color, visual detection thresholds, hydration constants
and ORAC antioxidant activities of purified cyanidin-based anthocyanins. Stability and
antioxidant activities of several purified anthocyanins have been affected by the B-ring structure
and glycosylation (14, 19, 25). In our previous study, extrusion cooking of products prepared
from ‗Purple Majesty‘ potatoes and dry peas retained total antioxidant activity brought by the
raw ingredient materials used to produce extrudates in spite of 60 – 70% decrease in anthocyanin
content. We hypothesized that at high temperatures, such as in extrusion cooking; the
degradation compounds from anthocyanin could be responsible for the overall antioxidant
activity in the extrudates. In the present study, total anthocyanins purified from ‗Purple Majesty‘
potatoes was used to evaluate the thermal kinetics parameters over a temperature range of 100 –
150 °C and antioxidant potencies of degradation compounds from the anthocyanins.
2. MATERIALS AND METHODS
2.1 Chemicals
Folin-Ciocalteu reagents, potassium chloride, sodium acetate, gallic acid, trolox (6-Hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl),
acetonitrile, ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), CHCA (α-Cyano-4-
hydroxycinnamic acid), ethyl acetate, and potassium persulfate were purchased from Sigma-
214
Aldrich (St. Louis, MO, USA). Laboratory grade methanol was used in extraction and
preparation of samples.
2.2. Materials
Fresh purple potatoes (‗Purple Majesty‘ cv) were purchased from Colorado State University
in Fall 2008. Potatoes were washed with tap water and stored at 4 °C in the Washington State
University Food Processing Pilot Plant. To prepare flakes, potatoes were peeled using a
mechanical abrasive peeler for about 75 seconds, sliced to 6mm thickness pieces and steam
blanched for 8 min to inactivate polyphenolic oxidase (PPO) similar to the procedures reported
for peroxidase inactivation (22). The puree was then dehydrated to approximately 5.2% (wet
basis) using a 15.24cm × 20.32 cm pilot scale counter rotating twin drum dryer (Blaw Knox
Food & Chemical Equipment Co., Buffalo, NY, USA). Pressurized steam to the drums was
maintained at 413 kPa corresponding to a saturation temperature of water at 145 °C. The surface
temperature of the drums was 135 - 138 °C. The gap between the drums, rotating at 1.13 rpm,
was set to 0.3 mm. Potato flakes were cooled at room temperature and stored at -20 °C for
further analysis. Moisture content of the flakes were determined using the standard procedures of
AACC moisture-air-oven method number 44-45A (26) and the samples were expressed in dry
weight (DW) basis.
2.3. Extraction of anthocyanins
Purple potato flakes were ground using a food processer and passed through a sieve US # 35
(0.5 mm). Fifty g of flakes were homogenized in 500 ml of extraction solvent (1.5 N HCl:
Methanol: water; 10/70/20 v/v/v) using a high speed homogenizer (Omni Mix Homogenizer,
Omni International, Waterbury, CT, USA). The homogenate was filtered through a double layer
215
of cheese cloth after keeping the mixture at 4 °C for 90 min. The residue was again homogenized
twice in 250 ml of extraction solvent at room temperature and filtered. All filtrates were pooled
and stored at -20 °C for further analyses.
2.4. Purification of anthocyanins
Potato extracts were centrifuged at 23000g at 4 °C for 15 min using a centrifuge (Beckman
J2-HS Centrifuge, USA) and 10 ml of clear supernatants were loaded to a Sep-Pak C18 column
(part no: WAT023635, Waters, Milford, MA) previously activated with HPLC grade methanol
followed by 0.01% aqueous HCl. Anthocyanins and polyphenolics were adsorbed onto the
column (silica-based bonded phase with strong hydrophobicity) while sugars, acids, and other
water-soluble compounds were removed by washing the column with 20 ml 0.01% aqueous HCl.
The column was further washed with 20 ml ethyl acetate to remove colorless non-anthocyanin
phenolics. Anthocyanins from the extract were collected by washing the column with 40 ml
0.01% HCl in methanol. The anthocyanin-rich extract was dried with a rotary evaporator at 30
°C to dryness and re-suspended in 10 ml deionized water. The pH of the aqueous anthocyanin
extracts ranged from of 5.9 – 6.0. These samples were stored at -20 °C for thermal kinetics study.
Purity of anthocyanins was checked with HPLC at a wavelength of 280 nm.
2.5. Heat treatment of anthocyanins
Thermal kinetics test (TKT) cells (Figure 6.1) were used in an oil bath containing silicon oil
for heat treatment. The test cell was custom designed to reduce the come-up time (CUT). Stored
sample was thawed to room temperature before transferring 1 ml of sample into the TKT cells.
Based on preliminary results of total anthocyanin contents during heat treatment, duplicate
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Figure 6. 1. (A) Specially designed thermal kinetics test (TKT) cells used for heat treatment of
purified anthocyanins (darker area) from ‗Purple Majesty‘ potatoes over a temperature range of
100 -150 °C. (B) TKT cells showing the detail dimensions.
Table 6. 1. Experimental design of heat treatments with selected temperature and time
combinations. Time = 0 min at come-up-time.
Temperature (°C) Heating time (min)
100 0 5 15 30 60
110 0 5 15 30 60
120 0 5 15 30 60
130 0 5 15 30 45
140 0 5 10 15 30
150 0 3 5 10 20
samples were considered. Total anthocyanin content in the purified extracts was measured over a
temperature range of 100 – 150 °C. During the treatment, sample temperatures were measured
using a pre-calibrated type T copper-constantan thermocouple with a diameter of 0.1 mm and
recorded at 0.5 sec intervals with a temperature data logger (TracerDAQ Pro, Measurement
Computing, Norton, MA, USA). The come-up-time (CUT e.g., time to reach 0.5 °C below the
set temperature) was 60 – 135 sec. The treatment time zero was considered to be the end of the
CUT interval. Details for the experimental design of thermal treatment are given in Table 6.1.
The samples in the test cells were cooled immediately in ice water after treatments to reduce the
A B
217
thermal shock. Samples were collected for the cell and stored in 2 ml centrifuge tubes at -20 °C
for further quantification, HPLC and mass spectrometry analyses.
2.6. HPLC Analyses
The analytical reverse phase HPLC technique was used for identification and separation of
anthocyanins. Stored samples (control and heated) were thawed at room temperature, each
sample passed through a Whatman 0.45 μm NYL filter to vials before been applied to HPLC.
The experiments were carried out using a Varian Star HPLC solvent delivery and control system
with automatic sample injector and a variable Varian UV/Vis detector. A Varian Microsorb-MV
100-5 C18 250 x 4.6 mm column fitted with a 10 x 3 mm Varian Chromo guard column was
maintained at room temperature. Twenty μL samples were injected for each test. Two elution
solvents used were 10% (v/v) acetic acid (A) and 50% (v/v) aqueous acetonitrile (B). Flow rate
of the elution solvents was maintained at 1 mL/min with a linear 30 min gradient from 0 to 30%
B followed by 5 min hold at 30% B. The column was then washed with 50% B for 5 min and
then returned to 0% B. The column was re-equilibrated for 10 min before the next analysis. The
eluted compounds were monitored at 280 nm for phenolics and 520 nm for anthocyanins (27).
Standards of gallic, protocatechuic, chlorogenic, caffeic, p-coumaric and ferulic acid with
residence times of 3.336, 4.699, 8.083, 9.664, 15.893 and 20.643 min, respectively, (Figure 6.2)
were used to determine the presence of these colorless phenolic acids in the anthocyanin extracts.
2.6. Matrix-assisted laser desorption ionization (MALDI) mass spectroscopy
MALDI-TOF-MS is a rapid technique to identify a group of anthocyanins with different
masses. The mass spectrometry analyses were performed using a 4800 Plus MALDI TOF/TOF
Analyzer (Applied Biosystems, Framington , MA, USA). The positive ion reflector mode was
used. The instrument was equipped with a pulsed nitrogen laser (337 nm, 3 ns pulse duration,
218
5 10 15 20 25 30 35
Minutes
-5
0
10
20
30
40
Ab
sorb
an
ce (
mA
U)
20.6436. Ferulic acid
15.8935. p-coumaric acid
9.6644. Caffeic acid
8.0833. Chlorogenic acid
4.6992. Protocatechuic acid
3.3361. Gallic acid
Retention
time (min)
Standard
12
3
4
5
6
Figure 6. 2. Chromatogram of standards as detected using HPLC
3Hz frequency). All spectra were obtained by averaging 25 laser shots. The matrix used in this
study was α-cyano-4-hydroxycinnamic acid (CHCA). Five mg of CHCA were solubilized in
1mL acetonitrile/water mixture (1:1 v/v/) for preparing the matrix stock solution. The sample to
be analyzed was prepared by mixing purified anthocyanin extracts (control or heat treated) with
the matrix (1:1 v/v). The sample mixtures were applied to the plate and analyzed.
2.7. Measurement of anthocyanins
Determination of total anthocyanin was based on the pH differential method following the
procedures of Giusti & Wrolstad (27). Absorbance readings were taken at a maximum
wavelength (λ-max) of 535nm and 700 nm for correcting for turbidity (28) in an UV/visible
219
spectrophotometer, previously blanked with distilled water. Total anthocyanins were quantified
and expressed in mg equivalent malvidin-3-glucoside (29-31) according to the following
formula:
de
DFMWALmgC
)/( (1)
where A = absorbance of the sample, MW = molecular weight of malvidin-3-glycoside (718.5
g/mol), DF = dilution factor, d = path length of the cuvette (1 cm), and e = molar extinction
coefficient of malvidin-3-glucoside (30200 l/cm per mol). The absorbances of the samples were
calculated as 5.4700max0.1700max )()( pHpH AAAAA .
2.8. Measurement of Antioxidant Capacity
Antioxidant capacities of the samples were measured using DPPH and ABTS assay.
DPPH assay: DPPH, a stable radical, deep purple in color, is reduced in the presence of
antioxidants. The loss of color results in a decrease in the absorbance intensity, thus providing a
basis for measurement of antioxidant activities in the extracts. The assay was based on the
procedures of Brandwilliams et al. (32). Control or heat treated sample (0.05 ml) was added to
1.95 ml of 6 × 10-5
M DPPH solution in a cuvette and the absorbance readings at 515 nm were
taken after 2 h of equilibration time using an Ultraspec 4000 UV/visible spectrophotometer
(Pharmacia Biotech, Cambridge, England).
ABTS assay: Total antioxidant capacity of the samples was also quantified using ABTS
radical cation decolorization assay (33). Working solution for the assay was prepared by mixing
stock solutions of 7 mM ABTS and 2.4 mM potassium persulfate in equal volumes and allowing
them to react for 12 h at room temperature in the dark. One ml of ABTS.+
solution was diluted
with 60 ml methanol to obtain an absorbance of 0.70 ± 0.01 units at 734 nm. Fresh ABTS.+
220
solution was prepared for each assay. One ml of sample was added to 1 ml of diluted ABTS.+
solution and allowed to equilibrate for 7 min before measuring the absorbance at 734 nm.
In both antioxidant assays, the spectrophotometer was blanked with methanol. Percentage
inhibition of DPPH and ABTS was calculated as:
100)1((%) control
sample
Abs
AbsInhibition (2)
where Abscontrol is the absorbance of DPPH or ABTS radical with methanol; Abssample is the
absorbance of DPPH or ABTS radical with sample extract or standard. The total antioxidant
capacity was quantified from a trolox standard curve and expressed as trolox equivalent per gram
of dry weight sample (µg TE/g DW) ± SD for duplicate samples.
2.9. Determination of thermal kinetics parameters
The order of the reaction in the thermal degradation of anthocyanins was predicted by
differential method using the following model:
nCkdt
dC)( (3)
where k is the rate constant, n is the reaction order, C is the concentration of the total
anthocyanins and t is the reaction time.
Order of reaction was determined by graphical analysis, where exponent n in eq. 3 was set to
zero, half, one and two to compare the coefficients of determination among zero-, half-, 1st and
2nd
order reactions, respectively. The integrated form of zero-, half-, 1st and 2
nd order kinetic
models is listed below:
Zero-order: ktCCt 0 (4)
Half-order: ktCCt 02 (5)
221
First-order: ktC
C
o
t ln (6)
Second-order: ktCCt
0
11 (7)
Using the experimental anthocyanins data, the coefficient of determination was observed to be
minimum for n = 1, predicting a 1st order reaction. According to the activated complex theory for
chemical reaction rates, for 1st order, Arrhenius equation relates the reaction rate constants to the
absolute temperature (34):
RT
EAk a lnln (8)
where Ea is the activation energy (kJmol-1
), A is pre-exponential factor/frequency factor (s-1
), T is
the absolute temperature (K) and R is the universal constant (8.135 Jmol-1
K-1
). The reaction rate
constant k and the activation energy, Ea were determined graphically from a plot of ln (C/Co) vs
time and ln k vs 1/T, respectively.
Thermal death time method (D-z model) was used to estimate the decimal reduction time (D-
value) i.e. heating time required to reduce the anthocyanins concentration by 90% and z-value
i.e. temperature change necessary to alter the thermal death time by one log cycle (34) with the
following relationships:
kD
10ln (9)
zTTDD refref /)()/log( (10)
where Dref is the D-value at temperature Tref. The half-lives (t1/2) of the anthocyanins were
calculated as:
k
t2ln
21 (11)
222
The enthalpy of activation (∆H) and entropy of activation (∆S) was estimated using the
Eyring-Polanyi model based on the transition state theory (35):
RT
STH
b eTh
kk
. (12)
Where T is the absolute temperature (K), kb is the Boltzman constant (1.381 x 10-23 J/K), h is
the Planck constant (6.626x10-34 Js) and R is the gas constant (8.31 J/mol K).
3. RESULTS
3.1. Purification of anthocyanins
Aqueous anthocyanin extracts analyzed using HPLC showed the presence of chlorogenic,
caffeic, p-coumaric, ferulic and protocatechuic acids along with other unknown phenolic
compounds at 280 nm (Figure 6.3). After separation of salts, sugars and colorless phenolics
from the aqueous anthocyanin extracts, purified anthocyanins as detected at 280 nm did not show
any peak coincident with phenolic acid standards other than ferulic acid. When the purified
anthocyanins were detected at 520 nm (Figure 6.4), it was observed that the peak for the major
anthocyanin was eluted at the same time as the ferulic acid peak. Absence of individual ferulic
acid mass (m/z: 176.2) in MALDI data (Figure 6.5A) showed no contamination of phenolic
acids in the purified anthocyanin preparations. Comparison of spectra of the purified
anthocyanins from ‗Purple Majesty‘ potatoes with the pure anthocyanin (36) revealed acylation
of a phenolic acid to the major anthocyanin. The MALDI data (Figure 6.5A) showed the
presence of petunidin glucosides (m/z: 479), petunidin glucosides acylated with ferulic acid
(m/z: 963) and also petunidin glucosides acylated with coumaric acid (m/z: 933) in the purified
anthocyanins (10, 37-39). The latter mass (m/z: 933) agreed with the observations of Stushnoff et
223
5 10 15 20 25 30 35
Minutes
0
25
50
75
100
125
Ab
sorb
an
ce (
mA
U)
Purified Anthocyanins
(acidified methanol portion)
Total Polyphenols
(in crude extract)
Phenolic acids
(ethyl acetate portion)
Wavelength = 280 nm
2
3
4
5
6
Figure 6. 3. HPLC-DAD profile of total polyphenols (in crude extract), phenolic acids (in ethyl
acetate portion) and purified anthocyanins (in HCl/Methanol portion) from ‗Purple Majesty‘
potatoes purified using a Sep-Pak C18 column and as detected at 280 nm in HPLC.
Peak 2: protocatechuic acid; peak 3: chlorogenic acid; peak 4: caffeic acid; peak 5: p-coumaric
acid, and peak 6: ferulic acid. anthocyanin eluted at the same time as the ferulic acid peak.
al. (10) on the presence of petunidin-3-rutinoside-5-glucoside acylated with coumaric acid as the
major anthocyanin in ‗Purple Majesty‘ potatoes. The investigator also reported having petunidin-
3-rutinoside-5-glucoside acylated with ferulic acid in ‗Purple Majesty‘ potatoes along with
delphidin, malvidin and peonidin aglycones with single glucosides. Although 1 – 2 smaller and
broader peaks were also observed in the HPLC chromatogram (Figure 6.4), no other individual
anthocyanin was characterized.
224
5 10 15 20 25 30 35
Minutes
0
25
50
75
Ab
sorb
an
ce (
mA
U)
280 nm
520 nm
Figure 6. 4 HPLC-DAD profile of control unheated purified anthocyanins from ‗Purple
Majesty‘ potatoes as detected at 280 and 520 nm.
99 300 501 702 903 1104
Mass (m/z)
5.0E+4
0
10
20
30
40
50
60
70
80
90
100
933.2020
479.1046 852.4272
317.0674 868.4166
850.4072293.0998
866.3948172.0401 379.0930
190.049
854.4277481.1025
963.1570766.6755318.6223147.6709
464.1113
3
4
2
1
A
% I
nte
nsit
y
99 300 501 702 903 1104
Mass (m/z)
5.0E+4
0
10
20
30
40
50
60
70
80
90
100
933.2020
479.1046 852.4272
317.0674 868.4166
850.4072293.0998
866.3948172.0401 379.0930
190.049
854.4277481.1025
963.1570766.6755318.6223147.6709
464.1113
3
4
2
1
A
99 300 501 702 903 1104
Mass (m/z)
5.0E+4
0
10
20
30
40
50
60
70
80
90
100
933.2020
479.1046 852.4272
317.0674 868.4166
850.4072293.0998
866.3948172.0401 379.0930
190.049
854.4277481.1025
963.1570766.6755318.6223147.6709
464.1113
3
4
2
1
A
% I
nte
nsit
y
225
99 300 501 702 903 1104
Mass (m/z)
7.1E+4
0
10
20
30
40
50
60
70
80
90
100
852.2131
317.0797 868.1868
932.8741379.0927
172.0307479.0848
190.0452
770.9835164.0598
147.0283
303.1453 772.9853 948.8604481.0866130.1362
% I
nte
nsit
y
C
99 300 501 702 903 1104
Mass (m/z)
7.1E+4
0
10
20
30
40
50
60
70
80
90
100
852.2131
317.0797 868.1868
932.8741379.0927
172.0307479.0848
190.0452
770.9835164.0598
147.0283
303.1453 772.9853 948.8604481.0866130.1362
% I
nte
nsit
y
C
Figure 6. 5 MALDI mass spectra of the pigments from purified anthocyanins of ‗Purple
Majesty‘ potatoes; (A) spectra of control purified anthocyanins samples; peak 1: petunidin; peak
2: petunidin monoglucoside; peak 3: petunidin-3-rutinoside-5glucoside acyalated with coumaric
acid; peak 4: petunidin-3-rutinoside-5glucoside acyalated with ferulic acid; (B) spectra of heat
treated samples at 100 °C for 30 min; (C) spectra of heat treated samples at 100 °C for 60 min.
Note: Scales of spectra A, B and C are different.
99 300 501 702 903 1104
Mass (m/z)
2.1E+4
0
10
20
30
40
50
60
70
80
90
100
852.5448
868.5381
317.0477
171.9863332.0712 786.2531
% I
nte
nsit
yB
99 300 501 702 903 1104
Mass (m/z)
2.1E+4
0
10
20
30
40
50
60
70
80
90
100
852.5448
868.5381
317.0477
171.9863332.0712 786.2531
% I
nte
nsit
yB
226
Table 6. 2. Concentrations of total anthocyanins from purple potato extract upon heating at 100 – 150 °C for 0 – 60 min.
(mean ± SD, n = 2; time = 0 min at come-up-time)
Total Anthocyanins (mg mal-3-glu/g dry weight sample)
Heating time (min)
Heating temperature (° C)
100 110 120 130 140 150
Control 0.487 ± 0.030a 0.487 ± 0.030a 0.487 ± 0.030a 0.487 ± 0.030a 0.487 ± 0.030a 0.487 ± 0.030a
0 0.467 ± 0.009a 0.420 ± 0.005b 0.436 ± 0.002b 0.402 ± 0.018b 0.368 ± 0.017b 0.364 ± 0.007b
3 - - - - - 0.098 ± 0.012c
5 0.410 ± 0.013b 0.344 ± 0.017c 0.285 ± 0.020c 0.202 ± 0.004c 0.084 ± 0.008c 0.044 ± 0.005cd
10 - - - - 0.030 ± 0.005cd 0.011 ± 0.008e
15 0.339 ± 0.007c 0.216 ± 0.009d 0.138 ± 0.017d 0.059 ± 0.005d 0.007 ± 0.002e -
20 - - - - - 0.001 ± 0.000f
30 0.186 ± 0.005d 0.082 ± 0.014e 0.042 ± 0.006e 0.010 ± 0.005e 0.001 ± 0.001f -
45 - - - 0.003 ± 0.004f - -
60 0.101 ± 0.003e 0.034 ± 0.007f 0.006 ± 0.003f - - -
Significant differences within the values in the same column are indicated by different superscript letters (p < 0.05, Tukey‘s
pairwise comparison test)
227
3.2. Degradation of anthocyanins
Total anthocyanin contents (TA) in the purified anthocyanin preparation determined before
and after heat treatments are summarized in Table 6.2. The TA of the control unheated extract
was 0.487 ± 0.03 mg mav-3-glu/g of DW sample. Thermal degradation of anthocyanins with
heating at 100 °C is clearly reflected by the reduction in the peaks detected at 520 nm (Figure
6.6A). A similar trend was observed when using 280 nm for detection with a major peak with
elution time of 20.643 min (Figure 6.6B).
The order of thermal degradation was estimated by examining the coefficient of
determination (r2) from plots of TA versus treatment time over the temperature range of
Table 6. 3. Estimation of the order of anthocyanins degradation by examining r2 from plot of
zero-, half-, first and second order reactions. (n = 2) Temperature (°C) Zero order Half-order First-order Second-order
100 0.9316 0.9644 0.9849 0.9792
110 0.8451 0.9207 0.9759 0.8428
120 0.7404 0.8958 0.9978 0.8632
130 0.7041 0.867 0.9918 0.8428
140 0.4993 0.704 0.9655 0.8511
150 0.4886 0.7194 0.9828 0.8428
Mean 0.7015 0.8452 0.9831 0.8703
Table 6. 4. Parameters for first-order kinetics and transition state equations for degradation of
anthocyanins from ‗Purple Majesty‘ potatoes after heat treatment over the temperature range of
100 – 150 °C. (n = 2) Temp
(°C)
k (min-1
)
x 103
r2
t1/2
(min)
D-value
(min)
Z-value
(°C)
Ea
(kJ/mol)
∆H
(kJ/mol)
∆S
(J/mol K)
100 26.2 0.9849 26.456 87.885 47.85 72.89 59.97 – 116.46
110 43.2 0.9759 16.045 53.301
120 71.4 0.9978 9.708 32.249
130 110 0.9918 6.301 20.933
140 194.1 0.9655 3.571 11.863
150 285.5 0.9828 2.428 8.065
228
5 10 15 20 25 30 35
Minutes
0
10
20
30
40A
bso
rba
nce
(m
AU
) 1 min
Wavelength = 520 nm
15 min
30 min
60 min
A
5 10 15 20 25 30 35
Minutes
0
25
50
75
Ab
sorb
an
ce (
mA
U)
1min
15 min
30 min
60 min
Channel 1 = 280 nm
23
5
6
x
y
z
B
Figure 6. 6. Chromatogram of thermal degradation compounds from purified anthocyanins
heated at 100 °C for 1 – 60 min. (A) Peaks detected at 520 nm; (B) Peaks detected at 280 nm.
Peaks 2, 3, 5 and 6 were protocatechuic, caffeic, p-coumaric and ferulic acids, respectively. x, y
and z were unknown peaks.
229
100 – 150 °C (Table 6.3). Based on the mean r2, thermal degradation of anthocyanins appeared
to follow 1st order kinetics (r
2 = 0.98). The temperature dependent rate constants, k-values, from
1st order over 100 – 150 °C as calculated from a plot of ln (C/Co) versus treatment time (Figure
6.7) were 0.0262 – 0.2855 min-1
. As expected, the reaction rate increased almost 10 times and
the D-values decreased 11 times (8.06 – 87.89 min) as the heating temperatures increased from
100 to 150 °C (Table 6.4). The activation energy, Ea and z-value of the degradation reaction
calculated from the Arrhenius plot (Figure 6.8) were 72.89 KJ/mol and 47.84 °C, respectively.
The activation enthalpy (∆H) and entropy (∆S) estimated from transition state theory were 59.97
kJ/mol and –116.46 J/mol K, respectively (Table 6.4).
3.3. Antioxidant activity of the degradation compounds
DPPH and ABTS antioxidant assays were performed to evaluate the total antioxidant
capacity of the anthocyanin preparation before and after heating. The TAC of the unheated
sample using DPPH and ABTS assays were 1237 ± 14 and 1546 ± 5 µg TE/g DW sample,
respectively. For each heating temperature, most of the TAC of heated samples either increased
or remained unchanged (p < 0.05) compared to unheated control samples. However, ABTS
antioxidant assay of degradation compounds for samples treated at 110 and 140 °C showed some
decrease in their TAC values. The TAC of the degradation compounds from anthocyanins ranged
from 1243 ± 97 to 1860 ± 21 µg TE/g DW sample using the DPPH assay (Table 6.5) and 1302 ±
82 to 1715 ± 21 µg TE/g DW sample using the ABTS assay (Table 6.6). Thermally induced
changes for the production of compounds from anthocyanins degradation did not follow a
particular trend. The ratio of TAC and TA in the control unheated purified anthocyanins was
2.54 and 3.17 using DPPH and ABTS assay, respectively, and increased to a maximum of 1316
(using DPPH assay) and 1581 (using ABTS assay) after heating at 150 °C (Table 6.7).
230
Treatment time (min)
0 10 20 30 40 50 60 70
ln (
C/C
o)
-7
-6
-5
-4
-3
-2
-1
0100 °C
110 °C
120 °C
130 °C
140 °C
150 °C
1st order
model
Figure 6. 7. First order plot for the degradation of anthocyanins during heating over a
temperature range of 100 -150 °C. Data were means of duplicate samples.
4. DISCUSSION
Protocatechuic, chlorogenic, caffeic and p-coumaric acids (peaks 2, 3, 4 & 5 in Figure 6.3)
in the crude anthocyanins extract were not observed in the purified anthocyanins when examined
at 280 nm. During purification of anthocyanins, washing of the Sep-Pak C18 column with ethyl
acetate removed the colorless non-anthocyanin phenolics (peaks 2, 3, 4 and 5 in Figure 6.3).
Prior to the removal of colorless phenolics, salts and sugars were also removed from the crude
extract with acidified water. A HPLC chromatogram of the purified anthocyanins showed one
large peak eluting at the same time as ferulic acid and smaller peaks were detected for other
anthocyanins at 520 nm. Purified anthocyanins from elderberries, strawberries and black carrots
have shown similar chromatograms when detected at 280 and 520 nm (14, 19). From the HPLC
231
1000/T in absolute temperature (K)
2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70
ln k
-4
-3
-2
-1
0
Figure 6. 8. Plot of ln (k) versus (1/T) for anthocyanin degradation during heating over the
temperature range of 100 -150 °C.
and MS information, the presence of petunidin glucosides acylated with a phenolic acid in
purified anthocyanins was indicated. Stushnoff et al. (10) reported the presence of petunidin-3-
rutinoside-5-glucoside acylated with coumaric and ferulic acid in addition to other types of
anthocyanins in ‗Purple Majesty‘ potatoes.
Degradation of anthocyanins in the extract is associated with reduction in its color. A similar
observation was noted while heating purple potatoes at high temperature (40). In this study,
anthocyanins in the extracts were reduced to negligible levels after 15 min of heating at 140 °C
and 10 min of heating at 150 °C. Yue & Xu (23) observed undetectable levels of anthocyanins in
the bilberry extracts after 10 min of heating at 150 °C. Reduction to negligible amount of
anthocyanins in heated samples could be due to chalcone formation from anthocyanins in the
232
process of thermal exposure or loss of glycosyl moieties (41). The breakdown of anthocyanin
compounds to other colorless small molecular weight compounds cause the sample to lose its
original color (22). Over the range of 100 – 150 °C, degradation kinetics followed a 1st order
reaction, which is in agreement with previous reports (22, 24, 42-44). The reaction rate constants
and half-lives over the range of 100 – 150 °C confirmed the influence of temperature on
anthocyanins and agreed with a previous report by Yue et al. (23) on dry heating of bilberry
extract. However, the k-value for purified anthocyanins in the present study was 10-fold higher
than the reported values for crude anthocyanins in a blackcurrant juice (pH 3.4) model at 100 °C
(24) and purple-fleshed potatoes (pH 3.0) at 98 °C (22). This shows that purified anthocyanins
degrade at a faster rate compared to anthocyanins in crude or unpurified extracts obtained
directly from food material, which could be due to inter- and intramolecular co-pigmentation
reactions in the pigments and cofactors such as colorless non-anthocyanin phenolic compounds
in the extract (11, 12). The values of half-lives over 100 – 125 °C in the present study at pH 5.95
± 0.05 (Table 6.4) were comparable with the half-lives of bilberry extracts during dry heating
(23) but lower than reported values at 100 °C i.e. 2.18 h in a model blackcurrant juice (24) and
98 °C (1.6, 5.6, 2 and 4.9 h) for purple-flesh potatoes, red-flesh potatoes, grapes and purple
carrots, respectively (22). Sadilova et al. (14, 19) reported that the half-lives of purified
anthocyanins at 95 °C were 1.95, 1.96 and 2.81 h at pH 3.5 and 3.2, 1.9 and 4.1 h at pH 1.0 for
strawberries, elderberries and black carrots, respectively. This indicates that purified
anthocyanins from ‗Purple Majesty‘ potatoes (pH 6.0) are less heat stable than those of
strawberries, elderberries and black carrots. One possible explanation is the difference in the
chemical structure of anthocyanin (45), intramolecular stacking of acylated anthocyanins (46),
types of acylation of anthocyanins (14) and types of sugar moieties (47). For example, cyanidin-
233
3-galactoside-xyloside-glucoside-sinapic acid, cyanidin-3-galactoside-xyloside-glucoside-ferulic
acid, and cyanidin-3-galactoside-xyloside-glucoside-coumaric acid in black carrots have half-
lives of 2.94, 3.43 and 3.1 h, respectively at pH 3.5 and 2.57, 2.39 and 2.16 h at pH 1.0 when
heated at 95 °C (14, 19). The same investigators also reported that different anthocyanins such as
pelargonidin-3-glucoside in strawberries and cyanindin-3-glucoside in elderberries have half-
lives of 2.12 and 1.82 at pH 3.5 and 2.12 and 1.95 at pH 1.0 upon heating at 95 °C. In ‗Purple
Majesty‘ potatoes, anthocyanins such as petunidin/malvidin/delphidin/peonidin-3-rutinoside-5-
glucoside are acylated with coumaric or ferulic acids (10), which might have different stability
when exposed to heat. Matsufuji et al., (48) reported anthocynins in red radish extract acylated
with p-coumaric acid or ferulic acid had a little more stability than anthocyanins acylated with
caffeic acids. The z-value of Purple Majesty potato anthocyanins was higher than the reported
values for all blue and CO94165-3P/P potato varieties (22) but comparable to roselle
anthocyanin extract (35).
Activation energy for the degradation of purified anthocyanins (72.89 KJ/mol ) in the present
study was in agreement with reported values for purple-flesh potatoes (72.49 kJ/mol) over 25 –
98 °C (22), blood orange juice and concentrate (73.2 – 89.5 kJ/mol) over 5 – 90 °C (44), sour
cherry concentrate (73.06 kJ/mol) over –18 to 80 °C (43), and blueberry extract (60 – 80 kJ/mol)
over 80 – 150 °C (23). Higher activation energy implies that a smaller temperature change could
degrade a compound more rapidly. The enthalpy of activation in the degradation of purified
anthocyanins from ‗Purple Majesty‘ potatoes was higher than blackberry (34 kJ/mol) and roselle
extracts (except Thai variety) (44 – 48 kJ/mol) but less than blood orange (63 kJ/mol) over 30 –
100 °C (35). This indicates that the degradation rate of purified anthocyanins from ‗Purple
Majesty‘ potatoes was less affected by temperature, even over a temperature range of 100 – 150
234
Table 6. 5. Antioxidant Activity (µg trolox eq./g DW sample) using DPPH radical scavenging
assay of purified anthocyanins from ‗Purple Majesty‘ potatoes upon heating at 100 – 150 °C for
0 – 60 min. (mean ± SD, n=2; come-up-time = 0 min)
Total Antioxidant Activity (µg trolox eq./g dry weight sample)
Heating
time
(min)
Heating temperature (° C)
100 110 120 130 140 150
Control 1237 ± 14e 1237 ± 14b 1237 ± 14b 1237 ± 14c 1237 ± 14c 1237 ± 14b
0 1352 ±21d 1404 ± 83a 1336 ± 7a 1639 ± 56a 1500 ± 34a 1326 ± 34a
3 - - - - - 1364 ± 89a
5 1511 ± 21b 1355 ± 41b 1341 ± 28a 1525 ± 7b 1500 ± 7a 1355 ± 21a
10 - - - - 1476 ± 41a 1277 ± 117a
15 1860 ±21a 1574 ± 117a 1356 ± 49a 1719 ± 28a 1340 ± 27b -
20 - - - - - 1316 ± 21a
30 1431 ± 7c 1277 ± 124b 1252 ± 14b 1525 ± 63b 1359 ± 14b -
45 - - - 1510 ± 98b - -
60 1431 ± 21c 1389 ± 255a 1243 ± 97a - - -
Significant differences within the values in the same column are indicated by different
superscript letters (p < 0.05, Tukey‘s pairwise comparison test)
°C than those from blood orange juice. Enthalpy of activation measures the energy barrier, which
must be overcome by reacting molecules and is related to the strength of bonds that are broken
and made in the formation of the transition state from the reactants (49). Entropy of activation
relates to the number of molecules with the appropriate energy which actually react and the
values provide an insight into the roles of the reactants in anthocyanins degradation by including
steric and orientation requirements (49). The estimated entropy of activation in ‗Purple Majesty‘
potatoes (–116.46 J/mol K ) was less negative than that of blood orange juice (–149 J/mol.K) ,
blackberry juice (–233 J/mol.K) and roselle extracts (–165 to –205 J/mol.K) (35). The more
negative entropy of activation indicates a smaller number of species in the transition state (49).
235
Antioxidant capacity and color of control unheated purified anthocyanins extracts is due to
high resonance of the fully conjugated 10-electron A-C ring-system in the structure, with some
contribution by the B-ring as well. The resonance of the structure leads to low reactivity and the
groups attached to the structure such as hydroxyl, methoxy, and glycosyl add stability (3, 17). After
thermal treatment of the purified extracts, degradation of original acylated anthocyanins
occurred. The MS data shows spectra of pigments in the samples heat treated at 100 °C for 30
and 60 min and the formation of new compounds at different heating times (Figure 6.5B &
6.5C). Even if the mechanism is not clear, possible explanations include chalcone formation,
deglycosylation and formation of compounds such as coumarin derivatives (50), benzoic
derivatives (51) and trihydroxybenzaldehyde (14). Formation of five different types of
Table 6. 6. Antioxidant Activity (µg trolox eq./g DW sample) using ABTS radical scavenging
assay of purified anthocyanins from ‗Purple Majesty‘ potato upon heating at 100 – 150 °C for 0
– 60 min. (mean ± SD, n=2; come-up-time = 0 min)
Total Antioxidant Activity (µg trolox eq./g dry weight sample)
Heating
time
(min)
Heating temperature (° C)
100 110 120 130 140 150
Control 1546 ± 5c 1546 ± 5a 1546 ± 5a 1546 ± 5b 1546 ± 5a 1546 ± 5b
0 1653 ± 22b 1491 ± 32a 1538 ± 5ab 1663 ± 22a 1539 ± 32a 1715 ± 21a
3 - - - - - 1622 ± 55b
5 1710 ± 19a 1433 ± 34b 1501 ± 21b 1573 ± 39a 1421 ± 14b 1619 ± 64b
10 - - - - 1400 ± 20b 1617 ± 12b
15 1687 ±38a 1433 ± 3b 1488 ± 8b 1576 ± 28a 1386 ± 6c -
20 - - - - - 1581 ± 40b
30 1649 ± 22b 1370 ±
119b 1482 ± 11b 1537 ± 45ab 1365 ± 23c -
45 - - - 1460 ± 64c - -
60 1559 ± 41c 1302 ± 82b 1469 ± 13c - - -
Significant differences within the values in the same column are indicated by different
superscript letters (p < 0.05, Tukey‘s pairwise comparison test)
236
anthocyanidins from heated bilberry extract after cleavage of conjugated sugars from
anthocyanins has been reported (23). The retention or increase in the TAC of heated anthocyanin
preparation compared to unheated purified anthocyanins is emphasized by noting the increasing
ratio of TAC and TA in the samples (Table 6.7) and formation of new compounds as observed in
increasing peak areas detected at 280 nm in chromatograms from the degradation of
anthocyanins when heated at 100 °C for 0 – 60 min (Figure 6.6). The ratio of TAC to TA using
DPPH assay varied from 2.54 – 1359.46, whereas using ABTS assay the ratio was 3.81 – 1581
over the temperature range of 100 – 150 °C at 0 – 60 min. This shows that the anthocyanin
degradation compounds exhibit higher antioxidant activity than unheated anthocyanin, which
agrees with previous reports (14, 23, 48). It could be assumed that acylated anthocyanins are
cleaved into their corresponding acyl-glycosides, then into intermediate chalcones and finally
into colorless phenolics such as phenolic acids and aldehydes that contribute to the TAC in the
heated samples. Sadilova et al., (14) observed retentions of 74.4, 86.5 and 84.6 % antioxidant
activity in the anthocyanin degradation compounds after heating 4 h at 95 °C in strawberry,
black carrot and elderberry anthocyanins isolates, respectively, because of formation of
compounds such as protocatechuic acid, phologlucinaldehyde, and 4-hydroxybenzoic acid.
Sreeram et al. (51) reported that the antioxidant activity of degradation compounds of cyanidin
glycosides from tart cherries were comparable to commercial antioxidants such as butylated
hydroxytoulene (BHT).
5. CONCLUSIONS
The present study evaluated the thermal degradation of purified anthocyanins from ‗Purple
Majesty‘ potatoes after removal of salts, sugars and colorless non-anthocyanin phenolics. The
thermal degradation of purified anthocyanins followed first order kinetics in an Arrhenius type
237
Table 6. 7. Progression of degradation compounds upon thermal exposure as measured by the ratio of total antioxidant capacity
(TAC) and total anthocyanins (TA) in purified anthocyanins samples over 100 – 150 °C. (A) TAC measured using DPPH assay; (B)
TAC measure using ABTS assay.
Ratio of Total Antioxidant Capacity versus Total Anthocyanins
Heating
time
(min)
Heating temperature (° C)
100 110 120 130 140 150
DPPH ABTS DPPH ABTS DPPH ABTS DPPH ABTS DPPH ABTS DPPH ABTS
Control 2.54 3.17 2.54 3.17 2.54 3.17 2.54 3.17 2.54 3.17 2.54 3.17
0 2.89 3.54 3.34 3.55 3.06 3.53 4.08 4.14 4.08 4.18 3.64 4.71
3 - - - - - - - - - - 13.92 16.55
5 3.69 4.17 3.94 4.17 4.71 5.27 7.55 7.79 17.86 16.92 30.79 36.80
10 - - - - - - - - 49.19 46.67 116.10 147
15 5.49 4.98 7.29 6.63 9.82 10.78 29.13 26.71 191.44 198.57 - -
20 - - - - - - - - - - 1315.84 1581
30 7.68 8.85 15.57 16.71 29.82 35.29 152.51 153.70 1359.46 1365 - -
45 - - - - - - 503.40 486.67 - - - -
60 14.14 15.40 40.85 38.29 207.09 244.83 - - - - - -
238
relationship. The total antioxidant capacity of purified anthocyanins after heat treatment was
either increased or retained compared to the unheated samples because of compensation in
antioxidant activity of the degradation compounds. Characterization of degradation compounds
from heat treatments based on their structure and functionality might be needed to understand the
mechanism responsible for the measured antioxidant activity.
239
REFERENCES
1. Wagner, G. J., Cellular and Subcellular Localization in Plant Metabolism. In Recent
Advances in Phytochemistry, Creasy, L. L.; Hrazdina, G., Eds. Plenum Press: New York,
NY, 1982; Vol. 16, pp 1 - 45.
2. Downham, A.; Collins, P., Colouring our foods in the last and next millennium.
International Journal of Food Science and Technology 2000, 35, (1), 5-22.
3. Rice-Evans, C. A.; Miller, N. J.; Paganga, G., Structure-antioxidant activity relationships
of flavonoids and phenolic acids. Free Radical Biology and Medicine 1996, 20, (7), 933-
956.
4. Stintzing, F. C.; Stintzing, A. S.; Carle, R.; Frei, B.; Wrolstad, R. E., Color and
antioxidant properties of cyanidin-based anthocyanin pigments. Journal of Agricultural
and Food Chemistry 2002, 50, (21), 6172-6181.
5. Kahkonen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J. P.; Pihlaja, K.; Kujala, T. S.;
Heinonen, M., Antioxidant Activity of Plant Extracts Containing Phenolic Compounds. J.
Agric. Food Chem. 1999, 47, (10), 3954-3962.
6. Hou, D. X., Potential mechanisms of cancer chemoprevention by anthocyanins. Current
Molecular Medicine 2003, 3, (2), 149-159.
7. Han, K. H.; Hashimoto, N.; Shimada, K.; Sekikawa, M.; Noda, T.; Yamauchi, H.;
Hashimoto, M.; Chiji, H.; Topping, D. L.; Fukushima, M., Hepatoprotective effects of
purple potato extract against D-galactosamine-induced liver injury in rats. Bioscience
Biotechnology and Biochemistry 2006, 70, (6), 1432-1437.
240
8. Mishra, D. K.; Dolan, K. D.; Yang, L., Confidence Intervals for Modeling Anthocyanin
Retention in Grape Pomace during Nonisothermal Heating. Journal of Food Science
2008, 73, (1), E9-E15.
9. Lachman, J.; Hamouz, K.; Orsak, M., Red and purple potatoes - A significant antioxidant
source in human nutrition. Chemicke Listy 2005, 99, (7), 474-482.
10. Stushnoff, C.; Holm, D.; Thompson, M. D.; Jiang, W.; Thompson, H. J.; Joyce, N. I.;
Wilson, P., Antioxidant properties of cultivars and selections from the Colorado potato
breeding program. American Journal of Potato Research 2008, 85, (4), 267-276.
11. Malien-Aubert, C.; Dangles, O.; Amiot, M. J., Color stability of commercial
anthocyanin-based extracts in relation to the phenolic composition. Protective effects by
intra and intermolecular copigmentation. Journal of Agricultural and Food Chemistry
2001, 49, (1), 170-176.
12. Del Pozo-Insfran, D.; Brenes, C. H.; Talcottt, S. T., Phytochemical composition and
pigment stability of acai (Euterpe oleracea Mart.). Journal of Agricultural and Food
Chemistry 2004, 52, (6), 1539-1545.
13. Mazza, G.; Brouillard, R., The Mechanism of Copigmentation of Anthocyanins in
Aqueous-Solutions. Phytochemistry 1990, 29, (4), 1097-1102.
14. Sadilova, E.; Carle, R.; Stintzing, F. C., Thermal degradation of anthocyanins and its
impact on color and in vitro antioxidant capacity. Molecular Nutrition & Food Research
2007, 51, (12), 1461-1471.
15. Giusti, M. M.; Wrolstad, R. E., Acylated anthocyanins from edible sources and their
applications in food systems. Biochemical Engineering Journal 2003, 14, (3), 217-225.
241
16. Redus, M.; Baker, D. C.; Dougall, D. K., Rate and equilibrium constants for the
dehydration and deprotonation reactions of some monoacylated and glycosylated
cyanidin derivatives. Journal of Agricultural and Food Chemistry 1999, 47, (8), 3449-
3454.
17. Delgado-Vargas, F.; Paredes-López, O., Anthocyanins and betalains. In Natural
Colorants for Food and Nutraceutical Uses, CRC Press Boca Raton, 2003; pp 167 - 219
18. Rossi, M.; Giussani, E.; Morelli, R.; Lo Scalzo, R.; Nani, R. C.; Torreggiani, D., Effect of
fruit blanching on phenolics and radical scavenging activity of highbush blueberry juice.
Food Research International 2003, 36, (9-10), 999-1005.
19. Sadilova, E.; Stintzing, F. C.; Carle, R., Thermal degradation of acylated and nonacylated
anthocyanins. Journal of Food Science 2006, 71, (8), C504-C512.
20. Garzon, G. A.; Wrolstad, R. E., The stability of pelargonidin-based anthocyanins at
varying water activity. Food Chemistry 2001, 75, (2), 185-196.
21. Kirca, A.; Ozkan, M.; Cemeroglu, B., Effects of temperature, solid content and pH on the
stability of black carrot anthocyanins. Food Chemistry 2007, 101, (1), 212-218.
22. Reyes, L. F.; Cisneros-Zevallos, L., Degradation kinetics and colour of anthocyanins in
aqueous extracts of purple- and red-flesh potatoes (Solanum tuberosum L.). Food
Chemistry 2007, 100, (3), 885-894.
23. Yue, X.; Xu, Z., Changes of Anthocyanins, Anthocyanidins, and Antioxidant Activity in
Bilberry Extract during Dry Heating. Journal of Food Science 2008, 73, (6), C494-C499.
24. Harbourne, N.; Jacquier, J. C.; Morgan, D. J.; Lyng, J. G., Determination of the
degradation kinetics of anthocyanins in a model juice system using isothermal and non-
isothermal methods. Food Chemistry 2008, 111, (1), 204-208.
242
25. Wang, H.; Cao, G. H.; Prior, R. L., Oxygen radical absorbing capacity of anthocyanins.
Journal of Agricultural and Food Chemistry 1997, 45, (2), 304-309.
26. AACC, Approved methods of the American Association of Cereal Chemists (10th ed.).
American Association of Cereal Chemists: St. Paul, MN, USA, 2000; p Method 44-15A.
27. Giusti, M. M.; Wrolstad, R., Characterization and measurement of anthocyanin by UV-
visible spectroscopy In Current Protocols in Food Analytical Chemistry., John Wiley &
Sons ed.; Wrolstad, R. E., Ed. John Wiley & Sons, New York: 2001; Vol. F 1.2, pp pp
F1.2.1-F1.2.13.
28. Reyes, L. F.; Cisneros-Zevallos, L., Wounding stress increases the phenolic content and
antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of
Agricultural and Food Chemistry 2003, 51, (18), 5296-5300.
29. Han, K. H.; Sekikawa, M.; Shimada, K.; Hashimoto, M.; Hashimoto, N.; Noda, T.;
Tanaka, H.; Fukushima, M., Anthocyanin-rich purple potato flake extract has antioxidant
capacity and improves antioxidant potential in rats. British Journal of Nutrition 2006, 96,
(6), 1125-1133.
30. Jansen, G.; Flamme, W., Coloured potatoes (Solanum tuberosum L.) - anthocyanin
content and tuber quality. Genetic Resources and Crop Evolution 2006, 53, (7), 1321-
1331.
31. Lewis, C. E.; Walker, J. R. L.; Lancaster, J. E.; Sutton, K. H., Determination of
anthocyanins, flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum
tuberosum L. Journal of the Science of Food and Agriculture 1998, 77, (1), 45-57.
243
32. Brandwilliams, W.; Cuvelier, M. E.; Berset, C., Use of a Free-Radical Method to
Evaluate Antioxidant Activity. Food Science and Technology-Lebensmittel-Wissenschaft
& Technologie 1995, 28, (1), 25-30.
33. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C.,
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical Biology and Medicine 1999, 26, (9-10), 1231-1237.
34. Holdsworth, S. D., Thermal Processing of Packaged Foods. Blackie Academic &
Professional: New York, 2000; p 70 - 93.
35. Cisse, M.; Vaillant, F.; Acosta, O.; Dhuique-Mayer, C.; Dornier, M., Thermal
Degradation Kinetics of Anthocyanins from Blood Orange, Blackberry, and Roselle
Using the Arrhenius, Eyring, and Ball Models. Journal of Agricultural and Food
Chemistry 2009, 57, (14), 6285-6291.
36. Hong, V.; Wrolstad, R. E., Use of Hplc Separation Photodiode Array Detection for
Characterization of Anthocyanins. Journal of Agricultural and Food Chemistry 1990, 38,
(3), 708-715.
37. Wang, J.; Kalt, W.; Sporns, P., Comparison between HPLC and MALDI-TOF MS
analysis of anthocyanins in highbush blueberries. Journal of Agricultural and Food
Chemistry 2000, 48, (8), 3330-3335.
38. Giusti, M. M.; Rodriguez-Saona, L. E.; Griffin, D.; Wrolstad, R. E., Electrospray and
tandem mass spectroscopy as tools for anthocyanin characterization. Journal of
Agricultural and Food Chemistry 1999, 47, (11), 4657-4664.
39. Jones, C. M.; Mes, P.; Myers, J. R., Characterization and inheritance of the Anthocyanin
fruit (Aft) tomato. Journal of Heredity 2003, 94, (6), 449-456.
244
40. Nayak, B.; Berrios, J. J.; Powers, J. R.; Tang, J.; Ji, Y., Colored Potatoes (Solanum
Tuberosum L.) Dried for Antioxidant-Rich Value-Added Foods. Journal of Food
Processing and Preservation (in Press, DOI: 10.1111/j.1745-4549.2010.00502.x) 2010.
41. Adams, J. B., Thermal degradation of anthocyanins with particular reference to the 3-
glycosides of cyanidin. I. In acidified aqueous solution at 100 °C. Journal of the Science
of Food and Agriculture 1973, 24, (7), 747-762.
42. Wang, W. D.; Xu, S. Y., Degradation kinetics of anthocyanins in blackberry juice and
concentrate. Journal of Food Engineering 2007, 82, (3), 271-275.
43. Cemeroglu, B.; Velioglu, S.; Isik, S., Degradation Kinetics of Anthocyanins in Sour
Cherry Juice and Concentrate. Journal of Food Science 1994, 59, (6), 1216-1218.
44. Kirca, A.; Cemeroglu, B., Degradation kinetics of anthocyanins in blood orange juice and
concentrate. Food Chemistry 2003, 81, (4), 583-587.
45. Von Elbe, J. H.; Schwartz, S. J., Colorants. In Food Chemistry, Third ed.; Fennema, O.
R., Ed. Marcel Dekker, Inc: New York, NY, 1996; pp 651 - 722.
46. Dangles, O.; Saito, N.; Brouillard, R., Anthocyanin Intramolecular Copigment Effect.
Phytochemistry 1993, 34, (1), 119-124.
47. Attoe, E. L.; Von Elbe, J. H., Photochemial Degradation of Betanine and Selected
Anthocyanins. Journal of Food Science 1981, 46, (6), 1934-1937.
48. Matsufuji, H.; Kido, H.; Misawa, H.; Yaguchi, J.; Otsuki, T.; Chino, M.; Takeda, M.;
Yamagata, K., Stability to light, heat, and hydrogen peroxide at different pH values and
DPPH radical scavenging activity of acylated anthocyanins from red radish extract.
Journal of Agricultural and Food Chemistry 2007, 55, (9), 3692-3701.
245
49. Ariahu, C. C.; Ogunsua, A. O., Thermal degradation kinetics of thiamine in periwinkle
based formulated low acidity foods. International Journal of Food Science & Technology
2000, 35, (3), 315-321.
50. Jackman, R. L.; Smith, J. L., Anthocyanins and betalains. In Natural food colorants,
Hendry, G. A. F.; Houghton, J. D., Eds. Blackie and Son Ltd.: London, 1992; pp 183 -
241.
51. Seeram, N. P.; Bourquin, L. D.; Nair, M. G., Degradation Products of Cyanidin
Glycosides from Tart Cherries and Their Bioactivities. Journal of Agricultural and Food
Chemistry 2001, 49, (10), 4924-4929.
246
CHAPTER SEVEN
CONCLUSIONS AND RECOMMENDATIONS
1. CONCLUSIONS
In this study, the profile of the phenolic antioxidants and their bioactivity in colored potatoes,
value added products prepared from colored potatoes, and thermal kinetics of potato anthocyanin
was investigated. White, red, yellow and purple potato cultivars were investigated for total
phenolics, antioxidant activity and total anthocyanin content. The effects of steam blanching
following drying technologies (freeze-drying, drum-drying and refractance window-drying) on
the phenolic antioxidants were determined. Extruded products prepared from purple potato and
yellow pea flour using extrusion cooking technology were analyzed for their bioavailability as
well as chemical antioxidant activity by determining total phenolics and flavonoids including
existence in free and bound forms. A flow chart showing the detail recommended process for
producing puffed extrudates from purple potato (‗Purple Majesty‘ cv) and yellow pea flours is
given in Appendix 1. The thermal kinetics of purified anthocyanin from ‗Purple Majesty‘
potatoes was studied using specially designed thermal kinetic test cells in an oil bath over a
temperature range of 100 – 150 °C and the degradation products were analyzed for potential
antioxidant activity. From the above studies, it can be concluded that:
Purple potatoes (‗Purple Majesty‘ cv) contains significantly higher total phenolics,
antioxidant activity, and total anthocyanins than red (‗Red Cliff‘ cv), yellow (‗Yukon Gold‘ cv),
and white potato (‗Russet‘ cv) cultivars and can be used as a source for producing antioxidant-
rich value-added products.
247
Blanching is an important step for processing purple potatoes to retain natural color
(anthocyanins) in dehydrated flakes.
Different drying technologies (drum-drying, freeze-drying and refractance window-
drying) used to prepare dehydrated purple potato flakes either retained or increased (P < 0.05)
the total phenolic content and antioxidant capacity.
Losses of 23 to 45 % in total anthocyanin content were observed in dehydrated potato
flakes processed under all drying methods.
Natural colored extruded puffed food products with an acceptable expansion ratio can be
produced from yellow pea and purple potato flours using extrusion cooking technology.
Although major degradation in the quantity of the total anthocyanin content was noticed,
some color in the final product survived high temperature extrusion.
The total antioxidant capacities (DPPH and ORAC) of the extruded products were
retained or increased because of retention of some natural antioxidant compounds and formation
of new compounds with potent antioxidant activity.
The total phenolics and flavonoids in most of the extruded products also increased 33 –
37% and 17 – 30%, respectively, after extrusion cooking.
Positive dose-response inhibitory effects on the HepG2 cells were observed in potato
flour, raw formulations, and extruded products.
Extrusion cooking improved the dose-response inhibitory effect of 50% PPF raw
formulation on the HepG2 cells, thus increasing the CAA value of extruded products.
The thermal degradation of purified anthocyanins followed first order kinetics in an
Arrhenius type relationship.
248
The total antioxidant capacity of purified anthocyanins after heat treatment was either
increased or retained compared to the unheated samples because of compensation in antioxidant
activity of the degradation products.
2. RECOMMENDATIONS
The present study was focused on the phytochemicals present in colored potatoes and their
response to different processing conditions. The following future studies are recommended to
help reveal the effect of processing on phytochemicals in fruits and vegetables more completely:
Although positive results of antioxidant activities of processed products were observed,
the effect of storage on those phytochemicals was not studied.
Colored potatoes contain different types of antioxidant compounds at varying
concentrations, availability and activity depending on their intrinsic properties.
Fundamental research on the effect processing has on individual compounds is needed for
interpreting proper food and health relationships.
Value-added products from potatoes and peas further complicate the existence and
bioavailability of phytochemicals in free and conjugated states. Interactions between
natural and heat-induced antioxidants need to be investigated for possible synergistic
effects using advanced technologies such as Nuclear magnetic resonance (NMR) and
Matrix-assisted laser desorption/ionization (MALDI) is important to help understand the
overall antioxidant activity of the processed products.
In this study, bioavailability of antioxidant compounds was analyzed using HepG2 liver
cancer cells, which should be extended to other types of cells to develop a more complete
understanding of bioavailability.
249
APPENDIX
Flow chart showing recommended processes for producing extrudates from purple potato
(‗Purple Majesty‘ cv) and yellow pea flours
Purple Majesty Potatoes Peeling Slicing
( 6 mm thick)
Blanching
(Steam; 8 min) Pureeing
Dehydration/Drying
(Drum drying; drum surface temp:135 – 138 °C;
speed: 1.13 rpm; gap: 0.3 mm)
Pin Milling
Potato flour
(225 µm) Pea flour
Mixing/Formulations
(8.7 – 9.3% moisture content, wet basis)
Extrusion Cooking
(feed moisture: 17% wb; screw speed:
300 rpm; die temp: 130 & 140 °C)
Extrudates
(Cooling at room temperature)
Cooling
(Ice water; 8 min)
