Olive and Olive Oil Bioactive Constituents

Olive and Olive Oil Bioactive Constituents

Dimitrios Boskou

Table of Contents

Cover image

Title page

Copyright

Preface

About the Editor

Contributors

List of Abbreviations

Chapter 1: Olive Fruit, Table Olives, and Olive Oil Bioactive Constituents

Introduction

Olive and Olive Oil Bioactive Ingredients

Phenolic Compounds and Olive Oil Quality and Origin

Analytical Methods for the Rapid Analysis of Total Phenols and Orthodiphenols

Synopsis of Health Properties Attributed to Olive Oil and Its Biophenols

Claims Related to Fatty Acid Composition and Polar Phenols

Recovery of Bioactive Compounds from Olives, Olive Leaves, and Olive Processing Waste Products

Chapter 2: Minor Bioactive Olive Oil Components and Health: Key Data for Their Role in Providing Health Benefits in Humans

Introduction

Bioavailability of Olive Oil Phenolic Compounds in Humans after Dietary Doses of Olive Oils

Lipids, Lipoproteins, and Lipoprotein Oxidation

Conclusion

Acknowledgments

Chapter 3: Cellular and Molecular Effects of Bioactive Phenolic Compounds in Olives and Olive Oil

Introduction

Traditional Medicinal Applications of Olives and Olive Oil

Phenolic Compounds in Olives and Olive Oil

Olive and Olive Oil Products in Cosmetics

Antioxidant and Anti-Inflammatory Effects of Phenolic Compounds in Olives and Olive Oil

Cardiovascular Effects of Hydroxytyrosol and Oleuropein

Anticancer Effects of Phenolic Compounds in Olives and Olive Oil

Acknowledgments

Chapter 4: Olive Oil Phenolic Composition as Affected by Geographic Origin, Olive Cultivar, and Cultivation Systems

Introduction

Phenolic Composition

Factors Affecting Phenolic Composition

Conclusion

Chapter 5: Effect of Fruit Maturity on Olive Oil Phenolic Composition and Antioxidant Capacity

Introduction

VOO Phenolic Compounds

Evaluation of Fruit Maturation

Harvesting Time

Factors Affecting the Concentrations of Phenolic Compounds in VOO

Olive Fruit Ripening

A Comparative Study of the Effect of Maturation in the Phenolic Content of Olive Oils from Tunisia to Greece

Conclusion

Chapter 6: From Drupes to Olive Oil: An Exploration of Olive Key Metabolites

Introduction

General Factors That Influence the Final Concentration and Quality of Biophenols

Quantitative Changes of the Concentrations of Specific Bioactive Components during Olive Oil Production

Waste as a Raw Material for the Isolation of Bioactive Components

Conclusion

Chapter 7: Research and Innovative Approaches to Obtain Virgin Olive Oils with a Higher Level of Bioactive Constituents

Introduction

Virgin Olive Oil Production: Technological Aspects and Minor Components

Storage and Packaging: Bioactive Micro-Constituents and Shelf Life of the Product

Olive Pomace and Olive Pomace Oil as Sources of Bioactive Compounds

Conclusion

Chapter 8: Table Olives as Sources of Bioactive Compounds

Introduction

Antioxidant Activity and Biophenol Levels in Table Olives: Other Bioactives in Table Olives

Table Olive Processing

Influence of Processing on Olive Phenol

Olives Consumed without Any Processing

Innovative Proposals to Retain a Higher Level of Biophenols

Conclusion

Chapter 9: Bioactive Phenolic Compounds from Olea europaea: A Challenge for Analytical Chemistry

Introduction

Extraction of Phenolic Compounds from Olea europaea: Olive Fruit and Olive Oil

Analytical Separation Techniques for the Study of Phenolic Compounds in Olea europaea: Advantages and Disadvantages

Determination of Phenolic Compounds from Olea europaea

Analysis of Polyphenols and Metabolites in Biological Samples

Chapter 10: Analysis of Bioactive Microconstituents in Olives, Olive Oil and Olive Leaves by NMR Spectroscopy: An Overview of the Last Decade

Introduction

Multinuclear and Multidimensional Liquid High-Resolution NMR Spectroscopy

Characterization of the Microconstituents in Olive Fruit (Raw Fruit and Table Olive Fruit)

Characterization of the Microconstituents in Olive Oil

Characterization of the Microconstituents in Olive Leaf

Chapter 11: Recovery of High Added Value Compounds from Olive Tree Products and Olive Processing Byproducts

Introduction

Description and Characterization of the Byproducts of the Olive Processing Industry

Recovery of Bioactive Compounds from Byproducts of Olive Oil and Table Olive Production

Possible Use of Olive Byproducts for the Development of Pharmaceuticals, Nutraceuticals, and Cosmeceuticals

Conclusion

Index

Copyright

AOCS Mission Statement

AOCS advances the science and technology of oils, fats, surfactants and related materials, enriching the lives of people everywhere.

AOCS Books and Special Publications Committee

W. Byrdwell, Chairperson, USDA, ARS, BHNRC, FCMDL, Beltsville, Maryland

N.T. Dunford, Oklahoma State University, Oklahoma

D.G. Hayes, University of Tennessee, Knoxville, Tennessee

V. Huang, Yuanpei University of Science and Technology, Taiwan

G. Knothe, USDA, ARS, NCAUR, Peoria, Illinois

D.R. Kodali, University of Minnesota, Minneapolis, Minnesota

G.R. List, USDA, NCAUR-Retired, Consulting, Peoria, Illinois

R. Moreau, USDA, ARS, ERRC, Wyndmoor, Pennsylvania

W. Warren Schmidt, Surfactant Consultant, Cincinnati, Ohio

P. White, Iowa State University, Ames, Iowa

N. Widlak, ADM Cocoa, Milwaukee, Wisconsin

R. Wilson, Oilseeds & Biosciences Consulting, Raleigh, North Carolina

Copyright © 2015 by AOCS Press, Urbana, IL 61802. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the publisher.

ISBN 978-1-630670-41-2 (print)

ISBN 978-1-630670-42-9 (.epub)

ISBN 978-1-630670-43-7 (.mobi)

Library of Congress Cataloging-in-Publication Data

Olive and olive oil bioactive constituents / Dimitrios Boskou, editor.

pages cm

ISBN 978-1-63067-041-2 (print : alk. paper)—ISBN 978-1-63067-042-9 (epub) — ISBN 978-1-63067-043-6 (mobi) 1. Plant bioactive compounds. 2. Plant polyphenols. 3. Olive oil—Analysis. 4. Olive oil—Therapeutic use. 5. Olive. I. Boskou, Dimitrios, editor.

QK898.B54O55 2015

583′.87—dc23

2015007549

Printed in the United States of America

19 18 17 16 15 5 4 3 2 1

The paper used in this book is acid-free, and falls within the guidelines established to ensure permanence and durability.

www.aocs.org

Preface

Olive oil has a health nimbus as a basic ingredient of the Mediterranean diet. Studies on the favorable effects of biophenols and other olive and olive oil bioactive ingredients on markers of chronic inflammation, platelet aggregation in the blood, oxidized LDL cholesterol, joint health, skin conditions, and neurodegenerative disorders are continuously opening new paths for medical and pharmaceutical research. In addition, the olive oil market is becoming increasingly international because the positive role of virgin olive oil in the diet has become a topic of universal concern. New features of product optimization and development are emerging due to a better understanding of the chemistry of olives and olive oil minor constituents.

The bibliography for the biological activity of some minor compounds of olive and olive oil becomes more extensive every day, and a vast amount of published material has been accumulated. The growth of science aiming at unravelling exactly why olives and olive oil are so healthy co-occurs with experimental approaches for the identification and quantitation of bioactive ingredients in the various matrices (olive fruits, olive paste, virgin and other types of olive oil, intermediate and final products of processing, and biological fluids). Excellent studies do exist, but not all of the publications are equal; there are repetitions, discrepancies, and blanks due to difficulties deriving from the complex nature of compounds present in minute quantities in various matrices. An additional difficulty is the setting up of multidisciplinary studies with the involvement of food chemists, analytical chemists, and toxicologists who may provide guidance for the interpretation of results in relation to the actual chemistry of the compounds or isolates investigated, the expression of results, the presentation of levels and safety, or the efficacy of isolates and formulations. It is obvious that with the myriad of publications scattered in journals, books, and reference proceedings, a comprehensive understanding of olive and olive oil chemistry, biochemistry, and technology becomes difficult but also essential. This makes the systematic search for information necessary to better understand olive and olive bioactives, but this search is a challenge.

This book gathers together specialists who, with their deep reviews, cover the most important aspects of olive and olive oil biologically active constituents. Every effort has been made to provide information from authentic and highly regarded sources and to bring the huge but disconnected bibliography in the area into a single volume resource, as analytically described here:

 The current state of the art in the study of bioactive molecules present in olive fruits, olive oil, olive paste, olive milling waste byproducts, and table olives is analytically presented.

 Olive oil phenol composition as affected by geographic origin, olive cultivar, cultivation systems and maturity, and the development of technology and innovative methods to retain optimum levels of bioactive ingredients in virgin olive oil and table olives are broadly discussed.

 Extended information is embodied for the biological importance of olive products, and individual bioactive compounds present in olive products are considered. The presentations focus on oxidative stress and inflammation, and evidence is provided from randomized, controlled human studies. Topics such as mechanisms of action, including molecular mechanisms emerging from genome-wide transcriptome analyses and findings in wound healing; cardiovascular effects; and anticancer properties of the major phenolics are considered, as well.

 The book contains also overviews of advanced techniques for the isolation, identification, and quantitation of phenolic compounds from various Olea europaea parts including mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The last part of the book describes the main byproducts of olive processing that are rich in bioactives and valorization methods and techniques for their recovery and their possible market utilization.

The subject matter is organized in eleven chapters. Chapter 1 is an introductory one. It provides an outline of the classes of olive and olive oil bioactive constituents, both phenolic and nonphenolic, and emphasizes the differences in the phenol composition between olive fruits and virgin olive oil. It stresses the importance of the level and nature of phenolics for the evaluation of quality characteristics, mainly stability, freshness, and specific sensations (bitterness, pungency). Because the myriad protective benefits of consuming olives and olive oil are supported by a wealth of scientific research and the literature is extremely extended, a synopsis of confirmed and putative health effects attributed to virgin olive oil biophenols is included with a short list of key publications. This chapter is a complement to Chapters 2 and 3 for those researchers who wish to better intergrate the results of past and ongoing experiments in the area of biosciences and relate them to the complexity and diversity of biophenols present in olive and olive oils. The last part of the chapter deals with olive oil substitutes and wellness products based on the addition of olive-derived phenols, derivatization of phenols, functionalization of food, and finally biophenols and culinary applications.

Chapter 2 discusses data related to the health benefits provided by olive oil phenolic compounds, such as the improvement of HDL lipoprotein functionality, reduction of the oxidation of lipids, decrease of inflammation, improvement of endothelial function, and decrease of systolic blood pressure. Postulated mechanisms by which olive oil phenolics can exert their beneficial effects are illustrated. The key work, randomized, controlled human studies that are capable of providing evidence for the benefits of the consumption of olive oils rich in phenolic compounds is summarized. Why this type of evidence is the prerequisite for approving nutritional recommendations at a population level is analyzed.

Chapter 3 explores the traditional medicinal properties and early uses of olives and olive oil. It further discusses the evolution and health benefits of the Mediterranean diet and focuses on cellular and molecular effects of bioactive phenolic compounds in olives and olive oil, mainly hydroxytyrosol and oleuropein. Emphasis is placed on molecular mechanisms of action emerging from genome-wide transcriptome analyses.

Chapter 4 analytically examines the factors affecting the occurrence and abundance of phenolics in olive fruits and the oil produced. These factors—variety, geographical origin, farming, irrigation, fertilization, edaphoclimatic conditions, and processing of olives, isolated or in conjunction, greatly influence olives, table olives, and olive oil phenolic composition. Without proper control of these factors, the availability of phenolic compounds in olive products will be reduced drastically with serious adverse effects on the quality, stability, sensory characteristics, and favorable health properties of the oil.

Chapter 5 focuses mainly on the effect of fruit ripening on the phenolic content and antioxidant potential of the extracted olive oil. It demonstrates the significance of the correct ripeness stage and points out that determining the optimal harvesting period is important to obtain various types of olive oils (fruity, bitter, sweet) to fulfill the required market characteristics and necessary typicality in composition.

Chapter 6 analyzes the benefits of high-resolution mass spectroscopy and the latest generations of analyzers for quantitative and simultaneously qualitative monitoring of small molecules in the complex olive matrices. Experimental work disclosing the transformations and levels of olive key secondary metabolites in the different production steps (from olive drups to olive oil) is extensively and critically discussed in this chapter. The general trends for the formation of bioactives from molecules initially present in the drups during crushing, malaxation, and centrifugation are presented. The generation of important phenols and triterpene acids, such as hydroxytyrosol, tyrosol, oleacein, oleocanthal, and maslinic acid, and reduction of oleuropein and ligstroside oleuropein and ligstroside aglycons is monitored by measuring concentrations in the paste and the final product (olive oil).

Chapter 7 analyzes virgin olive oil production steps in relation to minor bioactive constituents. A lot of information is provided for the preprocessing of olives, crushing, particle size, innovative crushing and malaxation systems, control of oxygen, enzyme activities, separation of phases, filtration, and storage. Theoretical considerations are included for the dissolution of phenols in virgin olive oil during crushing and malaxation, and for the drop diameters and viscosity. Finally, employment of ultrasounds and microwaves and proposed strategies to develop continuous plants are described.

Chapter 8 deals with table olive processing methods in relation to the levels of phenols and other bioactive constituents. Table olives have more or less the same health properties as olive oil, but this aspect was overlooked because, with the exception of Mediterranean countries, this product was seen as a piece of decoration for pizzas and breads. Debittering techniques that are internationally applied are described, as are local practices and popularity of certain preparations that give an image of the rich Mediterranean culture and tradition. Innovative proposals for debittering, targeting of oleuropein hydrolysis, packing under modified atmosphere, improvement of cultivars to obtain larger phenolic levels in fruits, fermentation with the use of probiotic bacteria from olive’s natural flora, and generally the combination of tradition and innovation indicate that this product can now become an important functional food.

Chapter 9 explores advances in analytical science and related methodologies. The determination of minor compounds in olive oil, such as biophenols, is a challenge for today’s analyst, who may combine hyphenation of chromatography and electrophoresis techniques with UV, fluorescence, and mass spectrometry (MS), as well as fluorescence, infrared (IR) and NMR spectroscopies, and ambient MS. High-resolution spectroscopy can be further coupled to the facilities of computerized mathematical and statistical processing. The discussion in this chapter concentrates on the application of these methodologies to determine phenolic compounds in olive fruit and olive oil and their metabolites in biological samples, based mainly on literature published in the last five years.

Chapter 10 covers studies carried out during the last decade focusing mainly on the new developments in multinuclear and multidimensional liquid high-resolution NMR spectroscopy, as well as solid state NMR spectroscopy that made possible the detection and identification of new microconstituents. A number of figures are included with useful spectroscopic parameters and structures or transformations of biophenols and other minor constituents. A lot of references are cited, providing more detailed information about chemical shifts, assignment of signal, and other spectroscopic characteristics of molecules. Examples are given mainly from sources such as olive oil, olive fruit, table olives, and olive leaves.

Chapter 11 features a valuable section on olive bioactive compounds found in olive leaves, pomace, milling waste water, and table olive processing waste water, the main byproducts of the olive processing industry. Among the compounds present in these materials, the phenolic compounds constitute the most interesting group, endowed with a wide array of biological activities. Extraction is described from conventional methods to advanced techniques of fractionation and isolation. Possible use of olive byproducts for the development of pharmaceuticals, nutraceuticals, and cosmeceuticals is discussed.

It is hoped that the book will be of special value to food and health scientists, nutritionists, dieticians, cardiovascular disease epidemiologists, pharmacologists, food technologists, agronomists, analytical chemists, and researchers and professionals in the area of bioscience involved in studies related to natural antioxidants, oxidative stress, inflammation, and chemoprevention.

I thankfully acknowledge the work of the specialists who contributed to this book, and I consider myself fortunate for having the opportunity to work with so many experienced colleagues from universities and research institutes in Spain, Italy, Greece, Portugal, Tunisia, the United States, and Australia. Their reviews may suggest future research not only to understand better the role of phenols in the light of recent chemical knowledge, but also to assess the magnitude of the contribution of each bioactive compound to the overall positive health impact, an issue that has not yet been properly addressed.

Dimitrios Boskou

About the Editor

Dimitrios Boskou,     Professor Emeritus School of Chemistry, Aristotle University, Thessaloniki, Hellas

Dimitrios Boskou received his diploma and doctor’s degree in chemistry from the School of Chemistry, Aristotle University of Thessaloniki, Hellas; his Ph.D. in Food Science from the University of London; and a Doctor of Science degree from the School of Chemistry, Aristotle University. He served as an assistant lecturer, assistant professor, associate professor, professor, and head of the Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University (1970–2006). In the period from 1986 to 1998 he was a member of the IUPAC Oils, Fats, and Derivatives Commission. He served as a member of the Supreme Chemical Council, Athens (1995–2005), and a member of the Scientific Committee for Food of the European Commission and an expert of the Food Additives Panel of the European Food Safety Authority (1995–2012).

Dr. Boskou has published over 90 papers and reviews. He is the editor of 7 books and the author of 20 chapters in books related to the major and minor constituents of fats, natural antioxidants, olive oil, and heated fats, published in the United States, the United Kingdom, France, India, and Croatia. He is also a contributor to international scientific encyclopedias and the Lexicon of Lipid Nutrition, a joint IUPAC/IUNS work.

Contributors

AOCS Press extends gratitude and appreciation to the Olive and Olive Oil Bioactive Constituents authors who helped make this title possible.

Nektarios Aligiannis,     Department of Pharmacognosy and Natural Products Chemistry, National and Kapodistrian University of Athens, Athens, Hellas

Riccardo Amirante,     Department of Mechanics, Mathematics and Management, Polytechnic University of Bari, Bari, Italy

Aikaterini Argyropoulou,     Department of Pharmacognosy and Natural Products Chemistry, National and Kapodistrian University of Athens, Athens, Hellas

Natalie P. Bonvino

Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Victoria, Australia

Department of Pathology, The University of Melbourne, Victoria, Australia

Dimitrios Boskou,     Aristotle University, School of Chemistry, Thessaloniki, Hellas

Salvatore Camposeo,     Department of Agricultural and Environmental Science (DiSAAT), University of Bari, Bari, Italy

A. Segura Carretero

Department of Analytical Chemistry, University of Granada, Spain

Research and Development of Functional Food Centre (CIDAF), PTS Granada, Spain

Nicola Cicero,     Department of Environmental Science, Safety, Territory, of Food and Health, University of Messina, Messina, Italy

Maria Lisa Clodoveo,     Department of Agricultural and Environmental Science (DiSAAT), University of Bari, Bari, Italy

M.d.M. Contreras Gámez

Department of Analytical Chemistry, University of Granada, Spain

Research and Development of Functional Food Centre (CIDAF), PTS Granada, Spain

María-Isabel Covas

Nutritional Projects Assessment, NUPROAS HANDESBOLAG (NUPROAS HB). Nacka. Sweden.

Cardiovascular Risk and Nutrition Research Group, IMIM-Institut Hospital del Mar d’Investigacions Mèdiques

CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) Barcelona, Spain

Photis Dais,     NMR Laboratory, Department of Chemistry, University of Crete, Crete, Hellas

Giacomo Dugo,     Department of Environmental Science, Safety, Territory, of Food and Health, University of Messina, Messina, Italy

Montserrat Fitó,     Cardiovascular Risk and Nutrition Research Group. IMIM-Institut Hospital del Mar d’Investigacions Mèdiques, CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) Barcelona, Spain

A.M. Gómez-Caravaca

Department of Analytical Chemistry, University of Granada, Spain

Research and Development of Functional Food Centre (CIDAF), PTS Granada, Spain

Maria Halabalaki,     Laboratory of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, Hellas

Emmanuel Hatzakis,     Penn State Department of Chemistry, University Park, Pennsy

Andriana C. Kaliora,     Department of Nutrition and Dietetics, Harokopio University, Athens, Hellas

Nick Kalogeropoulos,     Department of Nutrition and Dietetics, Harokopio University, Athens, Hellas

Tom C. Karagiannis

Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, VIC, AUS

Department of Pathology, The University of Melbourne, Victoria, Australia

Nicholas T. Lam

Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Victoria, Australia

Heart Failure Research Group, Baker IDI Heart and Diabetes Institute, Victoria, Australia

J. Lozano-Sánchez

Research and Development of Functional Food Centre (CIDAF), PTS Granada, Spain

Research and Development Functional Olive Oil Department, Granada, Spain

Raymond Luc

Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, VIC, AUS

Department of Pathology, The University of Melbourne, Victoria, Australia

Ricardo Malheiro,     Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Bragança, Portugal

José Alberto Pereira,     Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Bragança, Portugal

Nancy B. Ray,     McCord Research, Coralville, Iowa, USA

Nuno Rodrigues,     Mountain Research Centre (CIMO), School of Agriculture, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Bragança, Portugal

Alexios-Leandros Skaltsounis,     Laboratory of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, Hellas

A. Taamalli,     Laboratoire de Biotechnologie de l’Olivier, Centre de Biotechnologie de Borj Cedria, Tunisia

Aikaterini Termentzi,     Laboratory of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, Hellas

Rafael de la Torre

Human Pharmacology and Clinical Neurosciences Research Group, IMIM-Institut Hospital del Mar d’Investigacions Mèdiques, Pompeu Fabra University

CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) Barcelona, Spain

Nikos Xynos,     Department of Pharmacognosy and Natural Products Chemistry, National and Kapodistrian University of Athens, Athens, Hellas

List of Abbreviations

1-Done-dimensional

3, 4-DHPEAHydroxytyrosol

ABTS2,2-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)

ACAntioxidant capacity

AMFAnhydrous milk fat

APCI-TOF-MSAtmospheric pressure chemical ionization-time-of-flight-mass spectrometry

APIAtmospheric pressure ionization

CADCoronary artery disease

CECapillary electrophoresis

CHDCoronary heart disease

CLCardiolipin

CONICETConsejo Nacional de Investigaciones Científicas y Técnicas

COX2Cyclooxigenase-2

CPCross-polarization

CRAMPSCombined Rotation and Multiple-Pulse Spectroscopy

CSAChemical shift anisotropy

CZECapillary zone electrophoresis

DADDiode array detector

DAGDiacylglycerol

DDDipole-dipole

DFDiafiltration

DHADocosahexaenoic acid

DMEDimethyl ether

DMFDimethylformamide

DOSYDiffusion-ordered spectroscopy

DPGDiphosphatidylglycerol

DPPH1,1-Diphenyl-2-picrylhydrazyl radical

EBVEpstein-Barr virus

EDTAEthylenediaminetetracetic acid

EESIExtractive electrospray ionization

EFSAEuropean Food Safety Authority

EGCGEpigallocatechin-3-gallate

EICExtracted ion chromatogram

ELSDEvaporating light scattering detector

EPCEndolethial progenitor cells

ESIElectrospray ionization

ESI-TOF-MSElectron spray ionization-time-of-flight-mass spectrometry

EVOOExtra virgin olive oil

FAFatty acid

F–CFolin–Ciocalteu assay

FDAFood and Drug Administration

FGFat globule

FIDFree induction decay

FLDFluorescence detector

FRAPFerric ion reducing antioxidant power

FFAFree fatty acid

GCGas chromatography

GC-FIDGas chromatography-flame ionization detector

GC–MSGas chromatography–mass spectrometry

GluCerGlucosylceramide

GLUTGlucose transporter

HDLHigh-density lipoprotein

HPHTHigh-pressure, high-temperature

HPLCHigh-performance liquid chromatography

HPLC-DADHigh performance liquid chromatography–diode array detection

HPLC–DAD/MSHigh-pressure liquid chromatography–diode array detector/mass spectrometry

HPLC–ESI-TOF/MSLiquid chromatography diode array detector–electrospray time-of-flight mass spectrometry

HR-MASHigh-resolution magic angle spinning

HRMSHigh resolution mass spectroscopy

HTHydroxytyrosol

ICAM-1Intercellular adhesion molecule 1

INEPTInsensitive Nuclei Enhanced by Polarization Transfer

IOCInternational Olive Council

IRInfrared

IRHIschemic reactive hyperemia

LABLactic acid

LacCerLactosylceramide

LCLiquid chromatography

LDALinear discriminant analysis

LDLLow-density lipoprotein

LLELiquid–liquid extraction

LPALysophosphatidic acid

LPCLysophosphatidylcholine

LPELysophosphatidylethanolamine

LPILysophosphatidylinositol

LPSLipopolysaccharide

LRP1Receptor-related protein

MAEMicrowave-assisted extraction

MAGMonoacyglycerol

MALDIMatrix-assisted laser desorption ionization

MARMacroporous resin

MCP1Monocyte chemoattractant protein

MdMedian defect

MFMicrofiltration

MFGMMilk fat globule membrane

MFGMilk fat globule

MFLAMonoaldehydic form of ligstroside aglycon

MFOAMonoaldehydic form of oleuropein aglycon

MIPMolecularly imprinted polymer

MSMass spectrometry

MUFAMonounsaturated fatty acid

MWCOMolecular weight cut-off

NACENonaqueous capillary electrophoresis

NFNanofiltration

NMRNuclear magnetic resonance

NOENuclear overhauser enhancement

OBPOlive biophenols

OHTyrHydroxytyrosol

OLOleuropein

OMWWOlive oil mill waste water

ORACOxygen radical absorbance capacity

OTPOlive tree pruning

PAPhosphatidic acid

PALL-phenylalanine ammonia-lyase

PCPhosphatidylcholine

PDOProtected designation of origin

PEPhosphatidylethanolamine

PESPolyethersulphone

PFGPulse field gradients

PGDProtected geographic denomination

p-HPEATyrosol

PIPhosphatidylinositol; Pigmentation index

PLEPressurized liquid extraction

PLPhospholipid; Polar lipid

PMLG5Phase-modulated Lee-Goldburg 5-step

PODPeroxidase

PPOPolyphenoloxidase

PSPhosphatidylserine

PVDFPolyvinylidene difluoride

RFRadiofrequency

RIRipeness index

ROReverse osmosis

RPLCReverse phase liquid chromatography

RSMResponse surface methodology

SFASaturated fatty acid

SFESupercritical fluid extraction

SMSphingomyelin

SPESolid-phase extraction

SWESuperheated water extraction

TAGTriacylglycerol

TEACTrolox equivalent antioxidant capacity

TGTriglycerides

TOPWTable olive processing wastewater

TQTriple quadrupoles

TRLsTriglyceride-rich lipoproteins

UFUltrafiltrate; Ultrafiltration

UNLPUniversidad Nacional de la Plata

URPEUltrasound-assisted boiling extraction at reduced pressures

VCAM-1Vascular cell adhesion molecule 1

VLDLVery-low-density lipoproteins

VOOVirgin olive oil

VSMCVascular Smooth Muscle Cell

w-PMLG5Windowed Phase-Modulated Lee-Goldburg 5-step

1

Olive Fruit, Table Olives, and Olive Oil Bioactive Constituents

Dimitrios Boskou,     Aristotle University, Department of Chemistry, Thessaloniki, Hellas

Introduction

Phenolic compounds, widely distributed in the plant kingdom and abundant in our diet, are today among the most talked about classes of phytochemicals. In the last two decades, much work has been presented by the scientific community; this work focuses on:

 Oxidation mechanisms and the real contribution of natural antioxidants in preventing free radical damage and oxidative stress

 The levels and chemical structure of antioxidant phenols in different plant foods, aromatic plants, and various plant materials

 The probable role of plant phenols in the prevention of various diseases associated with oxidative stress

 The dietary intake of phenolic compounds and its effect on lipoprotein metabolism, oxidative damage, inflammation, endothelial dysfunction, and blood pressure

 The potential of polyphenols in oncology based on the study of chemopreventive and cell-specific cytotoxic and apoptotic effects

 The clarification of molecular mechanisms accounting for the antioxidant, anti-inflammatory, and anticancer properties through gene transcription profiling

 The assessment and dissemination of strategies for the extraction of phenols and other bioactive compounds from fruits processing residues

Olive oil contains some minor constituents, including phenolic compounds, which contribute to the stability of the oil, antioxidant capacity, bitterness, and pungency, and have important biological properties. Published research work and ongoing studies are extended and their outcome may ultimately be used to make more specific dietary recommendations. These may regard the efficacy of olive oil phenolic fraction to counteract the burden of cardiovascular disease. One of the main factors that has delayed research on olive and olive oil phenols is the considerable diversity and complexity of their chemical structures. Therefore, new approaches should aim at integrating the results of past and future experiments in various disciplines, including food chemistry, biochemistry, pharmacology, and other biological sciences, and at validating most of the major individual phenols and other bioactives as new agents against various chronic and other human diseases.

Olive and Olive Oil Bioactive Ingredients

The Terms Polyphenols and Unsaponifiables

Olive and olive oil bioactives can be divided into two categories: (1) the nonpolar bioactives that are found in the unsaponifiable fraction of the oil, such as squalene, tocopherols, sterols, and triterpenic compounds, and (2) polar phenolic compounds usually characterized as olive oil polyphenols, which are contained in the fraction obtained by liquid–liquid extraction or solid phase extraction. The inaccurate term polyphenols is rather confusing because very few of the polar fraction compounds are polyphenols. Polyphenols contain two benzene rings joined by a linear three carbon chain. According to this definition, only flavonoids such as apigenin and luteolin are polyphenols. These compounds, however, are present only in trace amounts in olive oil. The rest of the olive oil polar phenols—hydroxytyrosol, tyrosol, dialdeydic forms of decarboxylmethyl elenolic acid linked to hydroxytyrosol and tyrosol, glycosides and aglycones, lignans, and phenolic acids—are not polyphenols. It is better to characterize them as olive oil bioactive phenols or as olive oil polar phenols to emphasize the difference from another class of phenols, tocopherols, which are the nonpolar phenols.

Unsaponifiables

In the literature, very often phenols such as hydroxytyrosol are categorized as unsaponifiables. This categorization is not correct because this term, according to IUPAC, is used for lipids. Unsaponifiables are components of an oily (oil, fat, wax) mixture that fail to form soaps when blended with sodium hydroxide. Squalene, sterols, tocopherols, linear alcohols, and triterpenic compounds are unsaponifiables. Polar phenols are not, because they are not lipids.

Phenolic Compounds Identified in Olives and Olive Oil

The composition of the polar phenolic fraction in olive fruit and virgin olive oil (VOO) is very complex, and there are minor compounds that have not yet been identified, despite a large number of studies aiming at elucidating their structure. The phenolic composition is not the same in every VOO because there is a variation due to agronomic, genetic, and environmental factors such as the variety of the olive fruit, the region and climatic conditions, the agricultural practices applied in the cultivation of the tree, the stage of maturity and harvest period, the mode of extraction, and storage and packaging (see Chapters 4, 5, 6, 7, 10).

Values reported in the literature vary significantly for another reason, too. Different methods are used (most of them not validated), peaks in the HPLC chromatograms are conventionally identified, some constituents have isomers, all the necessary standards needed for identification and quantitation are not available, and some of the constituents are present at very low levels. In the last two decades, remarkable progress has been observed in the preparation of the sample (solid-phase extraction techniques, semipreperative high-pressure chromatography, ultrasound-assisted emulsification–microextraction) and the identification and quantification of phenolic molecules. The latter are based on advanced techniques such as gas chromatography–mass spectrometry (GC–MS), high-pressure liquid chromatography–diode array detector/mass spectrometry (HPLC–DAD/MS), liquid chromatography diode array detector–electrospray time-of-flight mass spectrometry (HPLC–ESI-TOF/MS), and other hyphenated techniques and high-resolution mass spectrometry (HPLC-DAD-SPE-NMR/MS, ORBITRAP platform analyzers), as well as nuclear magnetic resonance techniques (see Chapters 6, 9, 10). So far, however, an official method for total phenolic content and individual phenols, which would be practical and would guarantee the comparison of results, does not exist. Therefore, some tables provided in the literature with sub-ppm quantities (micrograms per kg of oil), based mainly on conventional liquid–liquid extraction (for the preparation of sample) and high-performance liquid chromatography with UV detection (for identification and quantification of individual phenols), should be scrutinized for the parameters of analytical procedures (accuracy, repeatability, limit of detection) (Romero and Brenes, 2012). These remarks are important for oleuropein, which is encountered in sub-ppm quantities in olive oil and poses recovery problems; its quantification demands specific techniques such as isotope dilution method and specific mass spectroscopy providing the needed sensitivity and specificity (Sindona, 2010.)

Such analytical requirements should also be considered from the point of view of nutritional evaluation, taking into account the fact that since 2011, a European Food Safety Authority (EFSA) opinion exists that permits a health claim for olive oil phenols (hydroxytyrosol and its derivatives, e.g., oleuropein complex and tyrosol) and protection of blood lipids from oxidative stress (EFSA, 2011). Improvement of the terminology (polyphenols) used in this claim, based on the new chemical knowledge for the nature of the phenolic fraction and modified analytical approaches (Mastralexi et al., 2014; Purcaro et al., 2014; Romero and Brenes, 2012), seems to be necessary to avoid any misunderstanding concerning the actual compounds involved in the protection of low-density lipoprotein (LDL) cholesterol particles from oxidative damage and other possible unfavorable health effects.

The major phenolic compounds present in olive fruits are anthocyanins (cyanidin glucosides); flavonols (mainly quercetin-3-rutinoside); flavones (luteolin and apigenin glucosides); phenolic acids (hydroxybenzoic, hydroxycinnamic, others); phenolic alcohols (tyrosol, hydroxytyrosol, 3,4-dihydroxyphenylglycol); secoiridoids (oleuropein, demethyloleuropein, ligstroside, nuzhenide); verbascoside, a hydroxycinnamic acid derivative; and lignans and Oleoside-11-methylester (Alagna et al., 2012; Boskou, 2009; Franco et al., 2014a; Kanakis et al., 2013; Lama-Muñoz et al., 2013; Silva et al., 2006; Vinha et al., 2005).

In olive oil, the phenolic composition is different. Synthesis of phenolic compounds occurs when enzymes and substrates meet as olive fruit is crushed during the industrial process to obtain the oil. Lists of phenols and other molecules that often appear in the literature for the composition of VOO contain mainly phenolic acids (hydroxybenzoic, hydroxyphenylacetic, hydroxycinnamic); simple phenols (tyrosol, hydroxytyrosol); derivatives of phenolic alcohols (tyrosol acetate, hydroxytyrosol acetate); glycosides (oleuropein, ligstroside); aglyconic derivatives of glycosides (ligstroside aglycon, oleuropein aglycon); dialdehydic and monoaldehydic forms of decarboxymethyl elenolic acid linked to hydroxytyrosol and tyrosol; lignans [(+)-acetoxypinoresinol, (+)-pinoresinol, (+)-1-hydroxypinoresinol, syringaresinol]; flavonoids (apigenin, luteolin); hydroxy-isochromans [1-phenyl-6,7-dihydroxy-isochroman, 1-(3′-methoxy-4′hydroxy)phenyl-6,7-dihydroxy-isochroman]; other phenols (vanillin, 4-hydroxy-3-methoxybenzaldeyde, 4-ethylphenol (found in oils of second centrifugation, intended for refining); and nonphenolic compounds (cinnamic acid, elenolic acid, elenolic acid glycoside) (Bendini et al., 2007; Boskou, 2009; Christophoridou et al., 2005; Franco et al., 2014; Kanakis et al., 2013; Pérez et al., 2014; Saitta et al., 2010; Segura-Carretero et al., 2010). Bianco and his co-workers (2006) isolated from olive fruits the methyl acetal of the aglycone of ligstroside and the β-hydroxytyrosyl ester of methyl malate (Figure 1.1). They suggested that these microcomponents may be responsible for hedonistic-sensorial characteristics of olive products.

Figure 1.1 Methyl acetal of the aglycone of ligstroside (A) and hydroxytyrosol ester of methyl malate (B).

Levels of individual phenolic compounds differ from olive oil to olive oil, but generally the dialdehydic forms of elenolic acid linked to tyrosol (p-HPEA-EDA) and hydroxytyrosol (3,4-DHPEA-EDA), oleuropein and ligstroside aglycons, are the main phenols, followed by lignans, tyrosol, and hydroxytyrosol, whereas the concentrations of phenolic acids are low. The concentrations of tyrosol and hydroxytyrosol are usually low in fresh oils but increase during oil storage due to the hydrolysis of secoiridoids, which contain these phenols in their molecular structure.

Basic structural characteristics of major phenols from each class of phenolic compounds reported to be present in olive fruits, olive oil, and table olives are shown in Figures 1.2 through 1.8. More structural formulae are provided in other parts of the book. For minor constituents detected and diastereomers quantified in the phenolic fraction of olives and olive oil and other nonphenolic bioactives, see Chapter 10 and the following references: Kanakis et al. (2013), Mousouri et al. (2014), Pérez-Trujillo et al. (2010), Saitta et al. (2010), and Sivakumar and Ucella (2005, 2010).

Figure 1.2 Phenolic acids and derivatives.

Figure 1.3 Phenyl alcohols and derivatives.

Figure 1.4 Flavonoids.

Figure 1.5 Lignans.

Figure 1.6 Hydroxy isochromans.

Figure 1.7 Other phenols.

Figure 1.8 Nonphenolic compounds.

Other Bioactive Constituents and Nutritionally Important Compounds

Tocopherols

From the eight known E-vitamers, the alpha-homologue comprises 90% of the total tocopherol content. Low amounts (∼10–20 mg/kg) are reported for homologues β-tocopherol (∼10 mg/kg), δ-tocopherol, and γ-tocopherol (Ben-Hassine et al., 2013; Boskou et al., 2006; Cunha et al., 2006; Kalogeropoulos and Tsimidou, 2014).

The levels reported for α-tocopherol content indicate a wide range that depends on the cultivar potential and technological factors. Greek oils studied by Psomiadou et al. (2000) had very high levels of α-tocopherol, ranging from 98 to 370 mg/kg. Values ranging from 93 to 260 mg/kg have been reported for the Portugeese olive oil samples (Cunha et al., 2006). Unusually high levels of tocopherols have been reported for varieties Coratina, Arbequina, and Koroneiki cultivated in Egypt (above 600 mg/kg) (Benincasa et al., 2011). Variability of vitamin E in VOO by agronomical and genetic factors has been studied by Beltrán et al. (2010).

Olive oil can be a good source of vitamin E. The 23 g of the oil suggested per day for good health, with a mean value of 200 mg α-tocopherol/kg, provide 4.6 mg α-tocopherol, which is approximately 25% of the recommended dietary allowance. To retain a good level of α-tocopherol, olive oil should be stored carefully (Fregapane et al., 2013; Tsimidou, 2006). The contribution of α-tocopherol to the stability of VOO and combined autoxidation of α-tocopherol and phenols have been discussed by Baldioli et al. (1996), Bendini et al. (2006), Blekas et al. (1995), Franco et al. (2014b), Mancebo-Campos et al. (2014), Mateos et al. (2003), and Tsimidou (2010).

Nonphenolic Bioactive Compounds

Hydroxyterpenic Acids

Oleanolic (3β-hydroxyolean-12-en-28-oic acid) and maslinic acid (2α,3β)-dihy-droxyolean-12-en-28-oic acid) are the main triterpene acids present in olive oil (see Figure 1.9). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) and betulinic acid (3β-hydroxy- lup-20-[29]-en-28-oic acid) have also been identified. The levels of triterpenic acids in olive oil range between 40 and 185 mg/kg (Boskou et al., 2006). Much higher levels are found in olives and olive pomace oil.

Figure 1.9 Hydroxyterpenic acids and triterpene dialcohols.

Hydroxyterpenic acids and the triterpene dialcohols erythrodiol and uvaol (Figure 1.9) are bioactive compounds. Studies for their pharmacological potential focus on inflammation, cancer, cardiovascular pathology, and vasorelaxation (Herrera et al., 2006; Rodriguez-Rodriguez and Ruiz-Gutierrez, 2010; Valero-Muñoz et al., 2014).

Squalene

Squalene (2,6,10,15,19,23-hexamehyl-2,6,10,14,18,22-tetracosanehexaene) is an un- saturated terpene widely distributed in nature. Chemically, it is an all-trans isoprenoid containing six isoprene units. It occurs in high concentrations in the liver oil of certain sharks and in smaller amounts in olive oil. It is the major constituent of olive oil unsaponifiables. Its level in olive oil may range from 200 to 7500 mg/kg, although much higher levels (up to 12,000 mg/kg) have been reported.

Squalene may have a chemopreventive effect in some types of cancer, and it is beneficial for patients with heart disease and diabetes. In a 2013 report, Alvaro L. Ronco and Eduardo de Stéfani stressed that it would be desirable to have higher squalene concentrations in vegetable sources such as olive oil, taking into account the ecological impact of capturing marine species. Squalene can be recovered from olive oil deodorization distillates.

Phytosterols

Phytosterols are functional ingredients because they reduce the absorption of cholesterol in mammals. However, the concentration in olive oil is too low for such an effect. It is claimed that consumption of 1.5–2.0 g/day of phytosterols is needed for a hypocholesterolemic effect (commercial spreads have a high level of sterols or stanols [8%], in the form of esters with fatty acids). However, the possible small contribution to the effect from a natural source should not be overlooked. According to Cardeno et al. (2014), two constituents of the unsaponifiables of olive oil, β-sitosterol, and α-tocopherol have been shown to influence the reduction of reactive species as well as COX-2 activity.

Four classes of sterols occur in olive oil: common sterols (4α-desmethylsterols), 4α-methylsterols, triterpene alcohols (4,4-dimethylsterols), and triterpene dialcohols (for review, see Boskou et al., 2006). Sterol composition and total sterol content are affected by cultivar, crop year, degree of fruit ripeness, storage time of fruits prior to oil extraction, and geographic factors. Olive harvesting practices and processing also affect individual sterols’ composition. Total sterol content and the composition of the sterolic fraction are important indices for checking olive oil authenticity.

Carotenoids

Lutein, zeaxanthin, and beta-carotene are plentiful in olives. Methods of extraction, temperatures used during extraction, and sequence of extraction can all cause significant differences in the final carotenoid content of the oil (Sanz et al., 2007). The main carotenoids present in olive oil are beta-carotene and lutein. Xanthophylls such as violaxanthin, neoxanthin, and others have also been reported to occur in very small quantities. Total carotenoids in olive oil may range between 1 and 20 mg/kg, but usually values do not exceed 10 mg/kg.

Carotenoids are singlet oxygen quenchers and protect the oil from photo-oxidation. There is a relation between carotenoids and the mode of action of polar phenols and α-tocopherol. In addition to providing its own carotenoids, olive oil may be able to help humans absorb carotenoids from other foods.

Phenolic Compounds and Olive Oil Quality and Origin

In addition to its health properties, phenolic compounds are important because they provide the oil with a remarkable stability to oxidation and they are related to valuable organoleptic properties. Therefore, the total phenol content and antioxidant activity are routinely measured and intercorrelated in the plants in which there is an interest in building an excellence of quality or in defining protected designation of origin (PDO) typicality. It is important for the producers and the consumers to understand the role of minor components in commercial olive oil categories during their shelf life. This information can indicate the individual quality of a specific oil product beyond the conventional commercial category standards. Novel analytical techniques have been developed to monitor quality and check authenticity, varietal differences, geographical origin, and denomination of protected origin. Near infrared spectroscopy, gas chromatography, nuclear magnetic resonance spectroscopy, high-performance liquid chromatography and hyphenated methods, and DNA-based methods have been proposed, and the literature is extended. Even phenols occurring in sub-ppm quantities in olive oil can be used as markers of quality and identity. According to Sindona (2010), oleuropein can be monitored to differentiate oil from destoned drupes or the effect of filtering at the end of the manufacturing process. Fraudulent addition of leaves during olive milling, which raises the content of oleuropein, can be also verified by determining oleuropein-related molecules identified only in leaf extracts.

Bitterness and Pungency

Organoleptic properties of VOO are very important for the evaluation of quality. To qualify as extra virgin, the oil must pass a sensory test by a trained tasting panel in addition to chemical examination with the official methods. The panel, recognized by the International Olive Council, should indicate that the oil is free from defects and exhibits some fruitiness.

The standard method, also known as a panel test, involves a group suitably trained to identify and evaluate the intensity of positive and negative attributes. Tasters work under fixed facilities with controlled temperature and moisture in the tasting room and rank the intensity of the sensations they experience on a scale from 0 to 10. Data provided is then subjected to statistical processing.

Definitions Established by Regulating Bodies

Median defect (Md) demanded for extra virgin olive oil is 0, and fruity median (Mf) is greater than 0. For virgin olive oils, the limits are Md ≤ 3.5 and Mf > 0.

 Fruity: Having pleasant spicy fruit flavors characteristic of fresh ripe or green olives. Ripe fruit yields oils that are milder, aromatic, buttery, and floral. Green fruit yields oils that are grassy, herbaceous, bitter, and pungent. Fruitiness also varies by the variety of olive.

 Bitter: Creating a mostly pleasant acrid flavor sensation on the tongue.

 Pungent: Creating a peppery sensation in the mouth and throat.

Specific Sensations Caused by Various Olive Oil Phenols

Each single component can contribute to different sensory perceptions. It is well-established that specific phenolic compounds are responsible for bitterness and pungency. Andrewes et al. (2003) isolated phenol fractions from olive oil and measured sensation intensities. Almost all fractions were described as bitter and astringent. However, the fraction containing deacetoxy-ligstroside aglycon (p-HPEA-EDA) produced a strong burning pungent sensation at the back of the throat, whereas the fraction containing the analogous deacetoxy-oleuropein aglycon (3,4-DHPEA-EDA) produced only a slight burning/numbing sensation perceived more on the tongue. Beauchamp et al. (2005) measured the pungent intensity of p-HPEA-EDA isolated from different VOOs, confirming that this molecule is the principal compound responsible for throat irritation. These researchers also tested the throat-irritant properties of its synthetic form (named oleocanthal). Sensory properties of VOO have been extensively discussed by Bendini et al. (2011) and Favati et al. (2013).

The intensity of bitterness of olive oils can be high or low. Bitterness largely defines the olive oil’s style, and therefore how it is used. VOOs are classified as being mild, medium, or robust. Robust olive oils tend to have a total phenol level above 300 mg/k, conventionally expressed as caffeic acid equivalents, whereas oils perceived as mild have levels below 180 mg/kg.

Desirable levels of bitterness in extra virgin olive oils (EVOOs) may be correlated to factors such as conditions of cultivation, harvesting time, and extraction system. Due to the correlation of bitter compounds to the oil oxidative stability and the bitterness–human health link, there is an increasing consumer interest in oils with specific bitterness intensity characteristics. The gap between the quality perceived by consumers and that established by expert tasters for VOOs having high concentration of phenol compounds is getting smaller and smaller. As a result, bitterness evaluation is becoming a very important area in olive oil research.

Esti et al. (2009) measured relationships between bitter or pungent sensations and phenolic compounds. Oleuropein and ligstroside derivatives were found to be good predictors of taste, bitterness, and pungency. Dierkes et al. (2012) correlated areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method with the bitterness and pungency. Six compounds—oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester—showed high correlations to bitterness and pungency.

A rapid evaluation of bitterness intensity is based on the spectrophotometric absorbance K225 values of oil samples (Favati et al., 2013; Inarejos-Garcia et al., 2009). This method, however, has the drawback of interferences from elenolic acid, which is not a phenol. Favati et al. (2013) attempted to build and validate a bitterness predictive model by using K225 values. The results indicated that the proposed predictive model could be a useful complementary tool in the characterization of EVOO samples in the routine analysis on the basis of perceived bitterness intensity.

Oxidative Stability and Phenolic Compounds as Markers of Freshness

There are many studies of the stability of olive oil and the role of minor compounds, especially the combined oxidation of phenols and α-tocopherol in autoxidation (for review, see Frankel, 2010).

Significant decreases observed in secoiridoid derivatives or other phenols and α-tocopherol may be used as markers of freshness of VOO (Fregapane et al., 2013; Morello et al., 2004). Secoiridoid