EFFECT of THERMAL PROCESSING on the PHENOLIC ANTIOXIDANTS of COLORED POTATOES by BALUNKESWAR NAYAK a Dissertation Submitted In

Home , Chlorogenic acid, Cinnamic acid, Quinic acid

EFFECT OF THERMAL PROCESSING ON THE PHENOLIC ANTIOXIDANTS OF

COLORED POTATOES

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

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.

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.

iii

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

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.

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

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

vii

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

viii

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

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

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

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

xii

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

xiii

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

xiv

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

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

xvi

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

xvii

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

xviii

Dedication

This dissertation is dedicated to my parents who provided

both emotional support and encouragement

xix

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

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).

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

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

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

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

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)

 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)

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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

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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

Phytochemicals

Carotenoids Phenolics Alkaloids Nitrogen- Organosulfur containing

Phenolic acids Flavonoids Stilbenes Coumarins Tannins

Hydroxy- Hydroxy- Flavonols Flavones Isoflavonoids Flavanones Flavanols Anthocyanidins benzoic acid cinnamic acid

Apigenin 15 Gallic acid Quercetin Genistein Flavanones Catechin Cyanidin P-Coumaric Chrysin Protocatechuic Kaempferol Daidzein Epicatechin Palargonidin acid Luteolin acid Myricetin Glycitein Epigallocatechin Delphidin Caffeic acid Vannilic acid Galangin formononetin Epicatechin- Peonidin Ferulic acid Syringic acid Fisetin gallate Petunidiin Sinapic acid p-Hydroxy- Epigallocatechin Malvidin benzoic acid -gallate

Figure 2. 1. Classification of Phytochemicals. Adapted and modified from Liu & Felice (2007).

H3CO HO OH H CO H3CO 3 HO O HO CHO HO COOH O HO Vanillin Eugenol Chlorogenic acid

HO OH COOH OH HO O HO O OH OH O O OH O rutinose Rosmaric acid Rutin

OH OH OH HO O HO O HO O OH OH OH

OH OH OH OH OH Luteolin (+)- Catechin (-)- Epicatechin

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).

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

3' 2' 4' 8 B 7 O 2 5' A C 6' 6 3 5 4

Figure 2. 3. Generic structure of Flavonoid

R 1 R1

R COOH 2 R2 CH CH COOH

R 3 R3

Benzoic acid derivatives Cinnamic acid derivatives

Benzoic acid Substitutions Cinnamic acid Substitutions

Derivatives R1 R2 R3 derivatives 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,

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

Food Antioxidants

Primary Secondary /Synergistic Antioxidants Antioxidants

‗Hindred‘ Miscellaneous Oxygen Chelating Secondary Miscellaneous Phenols Phenols Primary Scavengers Agents Antioxidants Antioxidants Antioxidants

Gallates BHA Sulfites Polyphosphates Nitrites Ethoxyquin Thiodipropionic Hydroquinone BHT Ascorbic acid EDTA Amino acids 20 Anoxomer acid Trihydroxy- TBHQ Ascorbyl Tartaric acid Spice extracts Trolox-C Dilauryl, butyrophenone Tocopherols palmitate Citric acid Flavonoids Distearyl esters Nordihydroguairetic Gum Guaiac Erythorbic acid Citrate esters Vitamin A acid Ionox series Phytic acid Β-Carotene Lecithin Tea extracts Zinc Selenium

Figure 2. 5. Classification of food antioxidants. Adapted and modified from Rajalakshmi & Narasimhan (1996)

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

Initiation R H R + H R + O ROO Propagation 2 ROO + R H ROOH + R

Inhibition ROO + A H ROOH + A Termination ROO + A

2 ROO Non-radical ROO +R products R + R

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

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

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 1. ORAC (oxygen radical absorbance capacity) reactions 2. TRAP (total radical-trapping antioxidant . . parameter) ROO + AH  ROOH + A 3. Crocin bleaching assay . . 4. IOU (inhibition oxygen uptake) ROO + LH  ROOH + L 5. Inhibition of linoleic oxidation 6. Inhibition of LDL oxidation Assays by electron transfer reaction 1. TEAC (Trolox Equivalent Antioxidant Capacity) . 2. FRAP (Ferric Reducing Antioxidant Power ) M (n) + e (from AH)  AH + + M (n-1) 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.

Table 2. 3. Comparison of different in vitro total antioxidant capacity assays. Method ORAC TRAP FRAP TEAC TOSC Crocin DPPH Reagents AAPH or AAPH or ABAP, TPTZ, FeCl3, ABTS, K2SO4, KMBA, Crocin, AAPH DPPH ABAP, Trolox, Trolox, sodium acetate Trolox AAPH or or ABAP, Fluorescein R-phycoerythrin ABAP Trolox Instrument Fluorescent Fluorescent plate Spectro- Spectro- Head space Spectro- Spectro- plate reader reader photometer photometer GC photometer photometer spectrometer spectrometer Temperature 37 °C 37 °C 37 °C 37 °C 37 °C 37 °C 37 °C Absorbance Excitation: 485 Excitation: 495 593 415 or 734 Depending on 443 515 (nm) Emission: 520 Emission: 575 detector type pH 7.4 7.4 3.6 7.4 7.4 7.4 Endpoint End of Lag phase Fixed time Fixed time End of Fixed time % remaining fluorescence (4 – 10 min) (4 – 6 min) production of (10 min) DPPH decay ethylene Calibration Trolox solution Trolox solution Fe3+ standard Trolox solution - Trolox Trolox solution solution solution Calculation of Area of the Length of the lag Absorbance of Absorbance Area under the Ka/Kc of Absorbance 25 Results fluorescence phase final reading decrease in kinetic curve antioxidant decrease in curve decay minus blank presence of the divided for presence of the sample Ka/Kc of sample Trolox Dimension of µmol of Trolox µmol of Trolox Absorbance of mM Trolox µmol of % inhibition; 2+ results equivalent equivalent Fe complex equivalent to 1 Trolox EC50; TEC50; produced by mM test equivalent AE = antioxidant substance (1/ EC50)TEC50 reduction of corresponding tripyridyltriazine Fe3+ complex 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).

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

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-

Storage (air, vacuum, inert gas, refrigeration, freezing)

Processed Food

 Boiling  Microwave heating  Blanching Food  Irradiation  Pasteurization Water Electromagnetic  Pulsed electric field Waves  Sterilization  Ultrasound  Evaporation sonication Oil  Extrusion cooking Air

 Roasting Shallow and  Baking Deep Fat Frying  Drying

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,

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,

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

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

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

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

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.

Total antioxidant activity

New antioxidants

Natural antioxidants Antioxidant activity Antioxidant

Heating time

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

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

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

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

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

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

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,

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

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

(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

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

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,

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

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).

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.

OH OCH3 OH OH OH HO + HO O+ O HO O+ OCH3 G O G O O G OH OH OH

Pelargonidin-3-glucoside Cyanidin-3-glucoside Malvidin-3-glucoside

OCH OH OCH3 3 OH OH OH + + HO O HO O+ HO O OH OH

O G O G O G OH OH OH

Petunidin-3-glucoside Peonidin-3-glucoside Delphidin-3-glucoside

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

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)

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; Fan-Chiang & Wrolstad (2005); Cyanidin-3-xyloside Cyanidin-3-malonylglucoside Rommel, Wrolstad, & Heatherbell (1992) Strawberries Pelargonidin-3-glucoside Cyanidin-3-glucoside Jackman et al. (1987) Litchi pericarp Cyanidin-3-rutinoside Malvidin-3-acetylglucoside; Lee & Wicker (1991); Sarni- Quercetin-3-rutinoside; Cyanidin glucoside; Quercetin Manchado, Le Roux, Le Guerneve, glucoside Lozano, & Cheynier (2000) Blood orange Cyanidin-3-glucoside Delphinidin-3,5-diglucoside; Cyanidin-3,5-diglucoside; Krifi et al. (2000) Delphinidin-3-glucoside; Peonidin-3,5-diglucoside; Cyanidin- 3-(acetyl)-glucoside; Cyanidin-3-(feruloyl)-glucoside Acai Cyanidin-3-glucoside Pelargonidin-3-glycoside Del Pozo-Insfran et al. (2004) Bilberry Cyanidin galactoside Delphidin arabinoside; Cyanidin glucoside; Delphidin Yue & Xu (2008) galactoside; Delphidin glucoside; Petunidin glucoside; Malvidin galactoside; Cyanidin arabinoside; Peonidin glucoside; Malvidin arabinoside

52 Elderberry Cyanidin-3-sambubioside Cyanidin-3-glucoside; Stintzing, Stintzing, Carle, Frei, & Cyanidin 3,5-diglucoside; Wrolstad (2002) Cyanidin-3-sambubioside-5-glucoside Red radish Pelargonidin-3-sophoroside-5- Matsufuji et al. (2007) glucoside 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- Kim & Padilla-Zakour (2004) rutinoside Purple carrot Cyanidin 3-(sinapoylxylosyl- Malvidin 3-monoglucoside; Glassgen, Wray, Strack, Metzger, glucosylgalactoside) Peonidin 3-monoglucoside & Seitz (1992) Red cabbage Cyanidin-3-sophoroside-5- Cyanidin-3,5-diglucoside Giusti, Rodriguez-Saona, Griffin, glucoside & Wrolstad (1999) Purple-fleshed Petunidin-3-(p-coumaroyl- Malvidin-3-(p-coumaroyl-rutinoside)-5-glucoside Lewis, Walker, Lancaster, & potato rutinoside)-5-glucoside Sutton (1998) Red-fleshed Pelagonidin-3-(p-coumaroyl- Peonidin-3-(p-coumaroyl- Lewis et al. (1998) potato rutinoside)-5-glucoside rutinoside)-5-glucoside

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

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

Quality of anthocyanin Quantity of  B-ring structrure anthocyanin  Glycosidic substitution pigment  Acylation  Hue  pKA  Rate of reactivity

Reaction with other compounds  Leucoanthocyanins  Flavonols  Acetaldehyde Browning reactions  Acid  Maillard  Polyphenoloxidase  Enzymatic  Ascorbic acid  Ascorbic acid  Proteins  Oxygen  Hydrogen peroxide  Metal ions  Sulfur dioxide

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

OCH3 OCH3 OH OH

+ O O HO O OCH3 + +H OCH3

O G O G OH OH Malvidin-3-glucoside (blue) Malvidin-3-glucoside (red) Quinoidal base or Anhydrobase Deglycosylation

OCH3 OCH3 OH OH HO + HO O HO O OCH 3 OCH3

OH OH OH OH

Malvidin

OCH3 OCH3 OH HO OH OH O OH HO O OCH3 OCH3 O O O HO O HO - Diketone

H2O H2O

OCH HO OH 3 OH CHO +

OCH3 OH HOOC

Protocatechuic acid Phloroglucinaldehyde

Figure 2. 13. Effect of pH on the possible degradation mechanism of anthocyanin (Malvidin-3- glucoside). Adapted from Wong (1989).

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

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

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

HO O OH O O 2 + H2O + Enzyme

O-diphenol O-benzoquinone

O Oxidized Degradation + Anthocyanin + anthocyanin Product O-benzoquinone

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

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

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

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.

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

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,

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

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

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

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).

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

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

OH OH OH B B + + HO O HO O A C A C O glc O glc OH OH Pelargonidin-3-glucoside Cyanidin-3-glucoside

Deglycosylation Deglycosylation OH OH OH B + + B HO O HO O A C A C OH OH OH OH Cyanidin Pelargonidin Clevage

Clevage

Clevage Clevage

OH COOH CHO B COOH A B HO HO OH OH HO Phloroglucinaldehyde Protocatechuic acid OH

4-Hydroxybenzoic acid OH

Clevage OH Clevage + HO O

OH Cyanidin

Clevage OH O OH OH + HO O

Clevage Deglycosylation Coum-gal-glu O gal glc coum OH OH Coumaric acid Cyanidin-3-gal-glc-coum

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).

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):

dC  k(C)n (1) dt 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 2nd order reactions, respectively.

The integrated form of zero-, half-, 1st and 2nd order kinetic models is given in Eq. (2) – (5).

Zero-order: Ct  C0  kt (2)

Half-order: 2 Ct  C0  kt (3)

C First-order: ln t  kt (4) Co

1 1 Second-order:   kt (5) Ct C0

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:

ln 2 t 1  (6) 2 k

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.

ln10 D  (7) k

E  1 1  k  k exp  a    (8) ref R T T    ref 

log( D/ Dref )  (T Tref )/ z (9)

k(T 10) Q10  (10) kT

C C t eq  exp(kt) (11) Co Ceq

-1 where Dref is the D-value at temperature Tref, Ea is the activation energy (kJmol ), T is the

-1 -1 absolute temperature (K), R is the universal constant (8.135 Jmol K ), 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.

C  C exp(k t ) (12) (tij ,Ti ) 0(Ti) (Ti ) ij

  E  C  C exp  k t exp a  (13) (tij ,Ti ) 0(Ti )  o ij     RTi  where C is the concentration of C at time t and temperature T and C is the initial (tij ,Ti ) ij i, 0(Ti ) concentration at time zero for temperature Ti.

Table 2. 5. Degradation kinetic parameters of anthocyanins.(d:days; h:hour) Fruit/vegetable Processing condition Degradation Kinetic parameter Reference kinetics Acai 10 – 30 °C; 1st order k = (7.7 – 13.9) x 103 min-1; Del Pozo-Insfran et al.

H2O2 (0- 30 mmol/L) t1/2 = 90 – 50 min; (2004) Q10 = 1.5 (10 – 20 °C); Q10 = 1.2 (20 – 30 °C) Grapes 25 – 98 °C; 1st order k = 0.0006 – 0.2853 h-1; Reyes & Cisneros-Zevallos pH 3.0 t1/2 = 47 d – 2.4 h; (2007) D-value = 157 d – 8.1 h; z-value = 28 °C; Q10 = 2.28; Ea = 75.03 kJ/mol; Grape skin 25 – 80 °C for 0.25 – 6 h; 1st order k = 3.6 – 15 x 10-3 h-1 (B); Dyrby et al. (2001) (in McIlvaine buffer pH 3.0 k = 2.4 – 320 x 10-3 h-1 (SD); (B) and carbonated Ea = 58 kJ/mol (B); soft drink (SD)) Ea = 77 kJ/mol (SD) -1 Concord grapes Retort (126.7 °C) for >30 min Non-linear regression k110 °C = 0.0607 min ; Mishra et al. (2008) pomace (non-isothermal) Ea = 65.32 kJ/mol Raspberries 4 – 40 °C for 5 months Non-linear regression k = (2.00 – 7.10) x 103 d-1; Ochoa et al. (2001)

76 Ea = 26 kJ/mol 3 -1

Sweet cherries 4 – 40 °C for 5 months Non-linear regression k = (1.28 – 6.95) x 10 d ; Ochoa et al. (2001) Ea = 32.49 kJ/mol Sour cherries 4 – 40 °C for 5 months Non-linear regression k = (1.10 – 5.37) x 103 d-1; Ochoa et al. (2001) Ea = 34.8 kJ/mol st Plum puree 50 – 90 °C for 0 – 20 min 1 order Ea = 37.48 kJ/mol Ahmed et al. (2004) st Blood orange juice 5 – 37 °C & 70 – 90 °C 1 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 103 s; Cisse et al. (2009) Z-value = 36 °C; Ea = 66 kJ/mol; ∆H = 63 kJ/mol; ∆S = -149 J/mol-K Blueberry juice 40 – 80 °C 1st order k = (0.064 – 2.25) x 103 min-1; Kechinski et al. (2010)

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 st Strawberries Stored at 25 °C 1 order t1/2 = 56 – 934 d Garzon & Wrolstad (2001)

-3 -2 -1 Strawberries (fresh- 5 – 20 °C with 80 kPa O2 Non-linear regression kα = 4.4 x 10 – 4.4 x 10 d Odriozola-Serrano et al. cut) flushing (Weibull model) (2009b) st Strawberry 95 °C for 6 – 7 h 1 order t1/2 = 1.95 h (pH 3.5) Sadilova et al. (2007); concentrate t1/2 = 3.2 h (pH 1.0) Sadilova et al. (2006) st Elderberry 95 °C for 6 – 7 h 1 order t1/2 = 1.96 h (pH 3.5) Sadilova et al. (2007); concentrate t1/2 = 1.9 h (pH 1.0) Sadilova et al. (2006) Elderberries 25 – 80 °C for 0.25 – 6 h; 1st order k = 31 – 90 x 10-3 h-1 (B); Dyrby et al. (2001) (in McIlvaine buffer pH 3.0 k = 5.5 – 180 x 10-3 h-1 (SD); (B) and carbonated Ea = 89 kJ/mol (B); soft drink (SD)) Ea = 56 kJ/mol (SD) Blackcurrant juice Heating 1st order k = (0.16 – 310) x 10-3 h-1; Harbourne et al. (2008) (model system) (4 – 100 °C) t1/2 = 180 d – 2.18 h; Ea = 73 kJ/mol (21 – 100 °C) -1 110 °C for 1 – 30 min Non-linear regression k110 °C = 1.02 h ; Harbourne et al. (2008) (non-isothermal) Ea = 81.5 kJ/mol 110 – 140 °C for 1 min Non-linear regression k = 9.954 h-1; Harbourne et al. (2008) (non-isothermal) Ea = 91.09 kJ/mol st -3 -1 77 Blackcurrants 25 – 80 °C for 0.25 – 6 h; 1 order k = 2.4 – 43 x 10 h (B); Dyrby et al. (2001) -3 -1

(in McIlvaine buffer pH 3.0 k = 4.5 – 87 x 10 h (SD); (B) and carbonated Ea = 69 kJ/mol (B); soft drink (SD)) Ea = 50 kJ/mol (SD) Blackberry juice & 60 – 90 °C 1st order k = 0.69 – 3.94 x 103 min-1; Wang & Xu (2007) concentrate (for 8.9 °Brix) t1/2 = 16.7 h – 2.9 h; Ea = 58.95 kJ/mol Blackberry juice (65 5 – 37 °C 1st order k = 2.0 – 59.1 x 103 min-1; Wang & Xu (2007) °Brix) t1/2 = 330.1 h – 11.7 h; Ea = 75.5 kJ/mol Blackberry 5 – 37 °C 1st order k = 5.2 – 89.9 x 103 min-1; Wang & Xu (2007) concentrate t1/2 = 133.3 h – 7.7 h; (65 °Brix) Ea = 65.06 kJ/mol Blackberry juice 100 – 180 °C Non-linear regression Ea = 92 ± 8 kJ/mol (100 – 140 °C); Jimenez et al. (2010) (non-isothermal) Ea = 44 ± 40 kJ/mol (140 – 180 °C); st Blackberry juice 100 – 180 °C 1 order (isothermal) Ea = 74 kJ/mol Jimenez et al. (2010) Blackberry juice 30 – 90 °C 1st order D-value = (30 – 341) x 103 s Cisse et al. (2009) Z-value = 56 – 57 °C Ea = 37 kJ/mol ∆H = 34 kJ/mol; ∆S = -232 to -233 J/mol-K

Roselle extract 30 – 90 °C 1st order D-value = (30 – 2280) x 103 s Cisse et al. (2009) Z-value = 34 – 44 °C Ea = 47 – 61 kJ/mol ∆H = 44 – 58 kJ/mol; ∆S = -205 to -165 J/mol-K st Black Carrot juice 70 – 90 °C; 1 order Ea = 62.5 – 95.1 kJ/mol; Kirca et al. (2007) pH 4.3 and pH 6.0; Q10 = 1.7 – 2.8 (70 – 80 °C); 11 – 64 °Brix Q10 = 2.0 – 2.2 (80 – 90 °C) st 70 – 90 °C; pH 2.5 – 7.0 1 order Ea = 78.1 – 47.4 kJ/mol; Kirca et al. (2007) st Storage at 4 – 37 °C; 1 order Ea = 62.1 – 86.2 kJ/mol; Kirca et al. (2007) pH 4.3 Q10 = 2.3 – 3.1 (4 – 20 °C); Q10 = 2.5 – 3.6 (20 – 37 °C) st Black carrot 95 °C for 6 – 7 h 1 order t1/2 = 2.81 h (pH 3.5) Sadilova et al. (2007); concentrate t1/2 = 4.1 h (pH 3.5) Sadilova et al. (2006) nd Red radish juice Stored (2 or 25 °C for 65 2 order t1/2 = 16 weeks (25 °C) Rodriguez-Saona et al. concentrate weeks) t1/2 > 65 weeks (2 °C) (1999) nd Red-fleshed potato Stored (2 or 25 °C for 65 2 order t1/2 = 10 weeks (25 °C) Rodriguez-Saona et al.

78 juice concentrate weeks) t1/2 = 60 weeks (2 °C) (1999) st -1

Purple-fleshed 25 – 98 °C; 1 order k = 0.0007 – 0.3259 h ; Reyes & Cisneros-Zevallos potatoes pH 3.0 t1/2 = 41 d – 2.1 h; (2007) D-value = 137 d – 7.1 h; z-value = 28.4 °C; Q10 = 2.25; Ea = 72.49 kJ/mol; Red-fleshed potatoes 25 – 98 °C; 1st order k = 0.0003 – 0.0725 h-1; Reyes & Cisneros-Zevallos pH 3.0 t1/2 = 89 d – 9.6 h; (2007) D-value = 297 d – 32 h; z-value = 31.5 °C; Q10 = 2.08; Ea = 66.7 kJ/mol; Purple carrots 25 – 98 °C; 1st order k = 0.0001 – 0.1004 h-1; Reyes & Cisneros-Zevallos pH 3.0 t1/2 = 216 d – 6.9 h; (2007) D-value = 717 d – 23 h; z-value = 26 °C Q10 = 2.44; Ea = 81.34 kJ/mol; Red cabbage 80 °C; 1st order k = 9.0 x 10-3 h-1 (B); Dyrby et al. (2001) (in McIlvaine buffer pH 3.0 k = 3.6 x 10-3 h-1 (SD) (B) and carbonated soft drink (SD))

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.

C t  ekt  (14) Co

t E  1 1    exp  a    (15) 0 R T T    ref 

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

activity (DPPH) of fresh-cut strawberries stored at similar conditions under a high oxygen atmosphere (80 kPa).

 AOX  AOX 0 exp[(t.k ) (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 tm  ( )(1 ) (17) k 

m k  ln[1 exp{c(T Tc )}] (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:

H TS k  k  b Te RT (19) h

-23 where T is the absolute temperature (K), kb is the Boltzman constant (1.381 x 10 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

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.

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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

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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

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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.

OH OCH3 OH OH OH + HO O+ HO O + HO O OCH3 G O G O O G OH OH OH

Pelargonidin-3-glucoside Cyanidin-3-glucoside Malvidin-3-glucoside

OCH OH OCH3 3 OH OH OH + + HO O HO O+ HO O OH OH

O G O G O G OH OH OH Delphidin-3-glucoside Petunidin-3-glucoside Peonidin-3-glucoside

Figure 3. 1. Structure of anthocyanins; G – Glucosides.

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,

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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

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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

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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

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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:

DPPH absorbance@T t (DPPH)remaining  100 DPPH absorbance@T 0 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.

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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

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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):

A*MW *DF C(mg /l)  e*d 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

A  (A max  A700) pH1.0 (A max  A700) pH4.5 . 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

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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.

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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 Total Antioxidant Total Phenolics Total (g/g of sample) Capacity (µg GAE /g DW) Anthocyanins (µg Trolox/g DW) (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).

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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.

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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

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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 Total Phenolics Total Anthocyanins (µg Trolox/g DW) (µg GAE /g DW) (mg/g DW) Raw (CO) 8787 ± 630b 3835 ± 296 c 1.74 ± 0.16 a Blanched 15358 ± 2948a 7993 ± 488a 1.58 ± 0.08a 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 7331 ± 246 b* 4680 ± 120 b* 0.83 ± 0.01 b* window dried * 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).

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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

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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

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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.

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9000

7000 R2 = 0.8858

5000

3000 TAC TAC (µgTE/g DW)

1000 1000 2000 3000 4000 5000 TP (µgGAE/g DW)

Figure 3. 3 Correlations between total antioxidant capacity by DPPH assay and total phenolics in colored (purple, red, yellow) and white potato cultivars.

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 Chromaticity Hue angle (L*) (C*) (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)

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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,

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Table 3. 4. Color attributes of purple dehydrated flakes (n=3) Lightness Chromaticity Hue angle Color difference Drying process (L*) (C*) (h°) (∆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.

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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.

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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

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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

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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.

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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.

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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 : 80, 90, 100, 110, 120 (ii) die temperature at 130 °C : 80, 100, 110, 120, 130 (iii) die temperature at 140 °C : 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

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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).

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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)

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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):

L L*  (1) 2.5

240a a*  120 (2) 255

240b b*  120 (3) 255 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:

h  tan1 (b* ) (4) a*

C*  (a*)2  (b*)2 (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

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differences between extruded samples and their respective raw formulations were expressed as

∆E* where,

E*  (L*)2  (a*)2  (b*)2 (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,

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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:

DPPH absorbance@T t (DPPH)remaining  100 (7) DPPH absorbance@T 0 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

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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-1cm-1mol-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.

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The TA of the raw formulations and the extrudates were calculated according to the following formula (Giusti et al., 2001):

A*MW *DF C(mg /l)  (8) e*d

where A = Absorbance of the sample given by A  (A max  A700) pH1.0 (A max  A700) pH4.5 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:

A  A BI  420 700 (9) A535  A700 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

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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%

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4 Expansion 3 Ratio 2 300 250 18 20 22 24 200 Screw Speed

% Feed Moisture

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

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

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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 Treatment Moisture content Expansion Ratio (Potato/Pea) (% wet basis) Raw Formulation 9.32 0 35/65 w/w Extruded @130 °C 7.03 4.28 ± 0.11b Extruded @140 °C 6.97 3.93 ± 0.22c Raw Formulation 9.01 0 50/50 w/w Extruded @130 °C 7.26 4.74 ± 0.10a Extruded @140 °C 7.39 4.48 ± 0.12a Raw Formulation 8.69 0 65/35 w/w 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

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Table 4. 3. Pasting behaviors of white and purple potato flours. Peak Trough Final Breakdown* Total Flours viscosity (cP) viscosity (cP) viscosity (cP) (cP) 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

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1800 100

1600 90 1400

1200 80

1000 70 800

Viscosity (cP) Viscosity Purple flour

600 60 (°C) Temperature White flour Temp (°C) 400 50 200

0 40 0 200 400 600 800 1000 1200 1400 Time (Sec)

hueFig valuesure. 4. with 2. RVA and 65%profile PPF. of whiteThe values and purple of chroma potato and flours. 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

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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 Brightness Chroma Hue Color difference Treatment (Potato/Pea) (L*) (C*) (h°) (∆E*) Raw Formulation 88.10 ± 0.4a 8.60 ± 0.3f 89.31 ± 0.4a 0.00 35/65 w/w Extruded @130 °C 73.00 ± 1.6d 26.48 ± 0.8d 87.85 ± 1.2a 23.41 ± 0.8c Extruded @140 °C 73.82 ± 1.5d 29.87 ± 0.4b 87.61 ± 1.5a 25.62 ± 0.7 b Raw Formulation 82.10 ± 1.3b 5.20 ± 0.3g 67.83 ± 0.6c 0.00 50/50 w/w Extruded @130 °C 70.57 ± 0.7e 22.73 ± 0.8e 87.71 ± 1.4a 21.31 ± 1.3c Extruded @140 °C 72.72 ± 1.4d 28.33 ± 0.4c 87.47 ± 1.3a 25.29 ± 0.5b Raw Formulation 77.03 ± 1.0c 4.04 ± 0.2h 21.35 ± 2.4d 0.00 65/35 w/w Extruded @130 °C 69.46 ± 1.7e 27.28 ± 0.7d 83.64 ± 1.6b 26.75 ± 0.6b Extruded @140 °C 73.80 ± 0.4d 33.19 ± 0.8a 84.61 ± 0.3b 31.74 ± 0.8a 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%

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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

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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.

4300 a Non-extruded 4200 Extruded @130 °C a Extruded @140 °C ab 4100 a a ab b 4000

bc 3900 c

(μg TE/g (μg DW) 3800 Totalantioxidant capacity 3700

3600

3500 35% PPF 50% PPF 65% PPF Formulations

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).

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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

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5000 a 4500 Non-extruded b 4000 Extruded @130 °C b b Extruded @140 °C 3500 c c 3000 c cd 2500 d

2000

1500 Totalphenolics GAE/g (μg DW) 1000

500

0 35% PPF 50% PPF 65% PPF

Formulations

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.

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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).

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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),

1 0.60

0.50 0.8 a

b 0.40 0.6

0.30

0.4 c Browningindex

Totalanthocyanins 0.20 (mg MV-3-Gly/g (mg DW) d d d e 0.2 f g 0.10

0 0.00 35% PPF 50% PPF 65% PPF

Formulations Total anthocyanins Non-extruded Extruded @130 °C Extruded @140 °C Browning index Non-extruded Extruded @130 °C Extruded @140 °C

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

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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

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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 (r2 = 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 (r2 = -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

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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.

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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, r2=0.99; p-coumaric acid, r2=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 ( SA/ CA) where  SA is the integrated area under the sample fluorescence vs time curve and CAis 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

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5000

Free Bound Total b 4000

cd c d 3000 d e f ef f f 2000 g

h Phenoliccontents i i

(µg gallic (µg acid eq./g DW)sample, 1000 j j j j j j j k l

0 PPF DPF Raw Extruded Raw Extruded Raw Extruded

Ingredients 35% Purple potato 50% Purple potato 65% Purple potato flour formulation flour formulation flour formulation

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

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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 Total Flavonoids (ORAC value) 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 Raw 69 31 63 37 88 12 35% PPF* Extruded 92 8 88 12 82 18 Raw 76 24 74 26 88 12 50% PPF Extruded 88 12 88 12 86 14 Raw 84 16 83 17 93 7 65% PPF 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.

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7000 a

b 6000 Free Bound Total

5000

4000 c cd cd de de e e 3000 ef f fg fg fg

Flavonoidcontents g 2000

(µg catechin/g (µg DW)sample, h

i i 1000 i j k k k l

0 PPF DPF Raw Extruded Raw Extruded Raw Extruded

Ingredients 35% Purple potato 50% Purple potato 65% Purple potato flour formulation flour formulation flour formulation

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

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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 Bound Total

(µg/g sample, DW) (µg/g sample, DW) (µg/g sample, DW) Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum Ferul Purple potato 714 ±55d 14±1b 52 ±0.3b n.d. 202 ±8c 801±55a 1019 ±34a 109 ±11b 916 ±55e 815 ±56a 1071 ± 35a 109 ±11b flour Dry pea flour 2.0 ±1g n.d. n.d. n.d. 236 ±20b 15 ±3 f n.d. 22 ±3d 260 ±25g 15 ±3f n.d. 22 ±3d 35% Raw 298 ± 24f 10±1c 6±1d n.d. 253±27b 306±45b 334±10e 59±1c 551±46f 316±46b 340±10e 59±1c PPF* Exd 984±10c n.d. n.d. n.d. 581±60a 77±6 e 362±11d 188±12a 1565±66c 77±7e 362±11e 188±12a 50% Raw 540±77e 13±1b 37±8c n.d. 253±33b 339±39b 387±54cd 57±7c 794±107e 353±39b 425±47d 57±7c PPF Exd 2011±211b n.d. n.d. n.d. 609±60a 168±17c 970±113ab 179±27a 2620±170b 168±17c 970±113ab 179±27a 65% Raw 1011±42c 23±4a 117±18a n.d. 243±21b 309±52b 463±35c 58±4c 1254±63d 333±55b 581±51c 58±4c PPF Exd 2967±267a n.d. n.d. n.d. 574±81a 93±7d 857±73b 159±21a 3541±348a 93±7d 857±73b 159±21a * 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

188 significantly different (p < 0.05). Chloro – Chlorogenic acid; p-Coum – Coumaric acid; n.d. – not detected

140 a 130 a 120 Free Bound Total 110

100 b

90 bc 80 c c

70 de 60 de e e 50 e f 40

f f AntioxidantActivity (ORAC) (µmol Trolox (µmol eq./g DW)sample, 30 g h 20 h h i h h j i 10 k 0 PPF DPF Raw Extruded Raw Extruded Raw Extruded

Ingredients 35% Purple potato 50% Purple potato 65% Purple potato flour formulation flour formulation flour formulation

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).

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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

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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

0.200 500

D CAA EC50 a 400 0.150

300

b 0.100 D c

c c 200 EC50value (mg/mL)

d CellularAntioxidant Activity 0.050 C (µmol quercetin (µmol eq./g DW)sample, B C d C 100 A

0.000 0 PPF DPF Raw Extruded Raw Extruded Raw Extruded

Ingredients 35% Purple potato 50% Purple potato 65% Purple potato flour formulation flour formulation flour formulation

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).

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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 (r2 = 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

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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

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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

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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 Total Total Total Total Phenolics ORAC CAA Phenolics ORAC CAA ORAC CAA Phenolics value value value Phenolics 0.9780 0.6565 0.3295 0.3936 0.9847 0.6988 (<.0001) (0.0005) (0.1159) (0.0570) (<.0001) (0.0001) Flavonoids 0.6636 0.6288 0.6008 -0.3210 -0.2752 0.0677 0.5814 0.5555 0.5832 (0.0004) (0.0010) (0.0019) (0.1262) (0.1931) (0.7534) (0.0029) (0.0048) (0.0028) Chlorogenic 0.8843 0.9176 0.6047 0.5117 0.5583 0.5605 0.8529 0.8897 0.6181 acid (<.0001) (<.0001) (0.0017) (0.0106) (0.0046) (0.0044) (<.0001) (<.0001) (0.0013) Caffeic acid 0.0240 -0.0655 -0.0708 0.1912 0.1121 0.2377 0.1878 0.1073 0.2299 (0.9113) (0.7610) (0.7423) (0.3707) (0.6019) (0.2634) (0.3796) (0.6178) (0.2799) 195 p-Coumaric 0.1575 0.0926 0.0880 0.8402 0.8329 0.8551 0.8465 0.8318 0.8532

acid (0.4622) (0.6668) (0.6827) (<.0001) (<.0001) (<.0001) (<.0001) (<.0001) (<.0001) Ferulic acid n.d. n.d. n.d. 0.6419 0.6583 0.7197 0.6419 0.6583 0.7197 (0.0007) (0.0005) (<.0001) (0.0007) (0.0005) (<.0001) ORAC 0.8762 0.3514 0.3188 0.2749 0.9847 0.6984 value (<.0001) (0.0923) (0.1290) (0.1936) (<.0001) (0.0001) Correlation coefficients were calculated as r2. Significant levels are given in the parenthesis.

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).

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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

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(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.

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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

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experiment. We also acknowledge the US Dry Pea and Lentil Council for their financial support for this study.

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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

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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-

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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

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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

215

B A

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

216

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,

217

4 Standard Retention time (min) 1. Gallic acid 3.336 40

2. Protocatechuic acid 4.699 ) 3. Chlorogenic acid 8.083 30 mAU 3 4. Caffeic acid 9.664 5. p-coumaric acid 15.893 20 6. Ferulic acid 20.643 5 Absorbance ( 1 10 2 6

0 -5 5 10 15 20 25 30 35 Minutes

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

218

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:

A MW  DF C(mg / L)  (1) e  d 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 A  (A max  A700) pH1.0 (A max  A700) pH4.5 .

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.+

219

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:

Abs Inhibition(%)  (1 sample )100 (2) Abscontrol 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:

dC  k(C)n (3) dt 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 2nd order kinetic models is listed below:

Zero-order: Ct  C0  kt (4)

Half-order: 2 Ct  C0  kt (5)

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C First-order: ln t  kt (6) Co

1 1 Second-order:   kt (7) Ct C0

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):

E ln k  ln A a (8) RT

-1 -1 where Ea is the activation energy (kJmol ), A is pre-exponential factor/frequency factor (s ), T is the absolute temperature (K) and R is the universal constant (8.135 Jmol-1K-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:

ln10 D  (9) k

log( D/ Dref )  (T Tref )/ z (10) where Dref is the D-value at temperature Tref. The half-lives (t1/2) of the anthocyanins were calculated as:

ln 2 t 1  (11) 2 k

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The enthalpy of activation (∆H) and entropy of activation (∆S) was estimated using the

Eyring-Polanyi model based on the transition state theory (35):

H TS k  k  b T.e RT (12) h

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

222

Wavelength = 280 nm

5 Total Polyphenols (in crude extract) 125 Phenolic acids

3 (ethyl acetate portion) ) 100 6 Purified Anthocyanins

mAU (acidified methanol portion)

50 Absorbance ( Absorbance

4 25 2

0 5 10 15 20 25 30 35 Minutes

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.

223

75 280 nm

520 nm )

mAU 50

Absorbance ( Absorbance 25

0 5 10 15 20 25 30 35 Minutes

Figure 6. 4 HPLC-DAD profile of control unheated purified anthocyanins from ‗Purple Majesty‘ potatoes as detected at 280 and 520 nm.

5.0E+4 100 A 933.2020 90 3 2 80 1 70

60 479.1046 852.4272 317.0674 868.4166 50 850.4072

40 293.0998 % Intensity % % Intensity % 866.3948 172.0401 30 379.0930 4 190.049 20 464.1113 854.4277 10 318.6223 481.1025 766.6755 147.6709 963.1570 0 99 300 501 702 903 1104 Mass (m/z)

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100 B 2.1E+4 90 852.5448 80 868.5381 70

% Intensity Intensity % % 40

30 317.0477 20

10 171.9863 332.0712 786.2531 0 99 300 501 702 903 1104 Mass (m/z)

100 7.1E+4 90 C 852.2131 80 317.0797 70 868.1868 932.8741 60 379.0927 172.0307 479.0848 50 190.0452

40 770.9835

164.0598

% Intensity Intensity % % 30 147.0283 20

10 303.1453 481.0866 772.9853 948.8604 130.1362 0 99 300 501 702 903 1104 Mass (m/z)

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.

225

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 temperature (° C) Heating time (min) 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 -

226 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)

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 k (min-1) t D-value Z-value E ∆H ∆S r2 1/2 a (°C) x 103 (min) (min) (°C) (kJ/mol) (kJ/mol) (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

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40 Wavelength = 520 nm

) 1 min 30 15 min

mAU 30 min 60 min

20 Absorbance ( 10

0 5 10 15 20 25 30 35 Minutes

B 6 Channel 1 = 280 nm 75 1min

) 15 min 30 min

mAU 50 60 min z y

25 x Absorbance (

5 3 2 0

5 10 15 20 25 30 35 Minutes

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.

228

100 – 150 °C (Table 6.3). Based on the mean r2, thermal degradation of anthocyanins appeared to follow 1st order kinetics (r2 = 0.98). The temperature dependent rate constants, k-values, from

st 1 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).

229

100 °C 0 110 °C 120 °C -1 130 °C 140 °C -2 150 °C st

) 1 order o model -3

ln (C/C -4

-5

-6

-7 0 10 20 30 40 50 60 70

Treatment time (min)

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

230

-1

k -2

-3

-4 2.30 2.35 2.40 2.45 2.50 2.55 2.60 2.65 2.70

1000/T in absolute temperature (K)

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

231

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-

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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

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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 Heating temperature (° C) time (min) 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).

234

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 Heating temperature (° C) time (min) 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 1370 ± 30 1649 ± 22b 1482 ± 11b 1537 ± 45ab 1365 ± 23c - 119b 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)

235

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

236

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 Heating temperature (° C) time 100 110 120 130 140 150 (min) 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 - -

237 45 ------503.40 486.67 - - - -

60 14.14 15.40 40.85 38.29 207.09 244.83 ------

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.

238

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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.

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 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.

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 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.

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APPENDIX

Flow chart showing recommended processes for producing extrudates from purple potato

(‗Purple Majesty‘ cv) and yellow pea flours

Slicing Purple Majesty Potatoes Peeling ( 6 mm thick)

Cooling Blanching Pureeing (Ice water; 8 min) (Steam; 8 min)

Dehydration/Drying (Drum drying; drum surface temp:135 – 138 °C; Pin Milling speed: 1.13 rpm; gap: 0.3 mm)

Potato flour Pea flour (225 µm)

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)

249