Crystallization of lipids fundamentals and applications in food, cosmetics and pharmaceuticals 9781118593899, 1118593898, 9781118593905, 1118593901, 9781118593912, 111859391X, 9781118593929

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Crystallization of Lipids

Crystallization of Lipids Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals

Edited by Kiyotaka Sato

Hiroshima University, Higashi‐Hiroshima, Japan

This edition first published 2018 © 2018 John Wiley & Sons Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Kiyotaka Sato to be identified as the author of the editorial material in this work has been asserted in accordance with law. Registered Office(s) John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print‐on‐demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging‐in‐Publication Data Names: Sato, Kiyotaka, 1946– editor. Title: Crystallization of lipids / edited by professor Kiyotaka Sato, Hiroshima University,  Higashi-Hiroshima. Description: First edition. | Hoboken, NJ, USA : Wiley, [2018] | Includes bibliographical   references and index. | Identifiers: LCCN 2017044562 (print) | LCCN 2017046371 (ebook) | ISBN 9781118593912 (pdf ) |   ISBN 9781118593899 (epub) | ISBN 9781118593929 (cloth : alk. paper) Subjects: LCSH: Lipids. | Crystal growth. Classification: LCC QP751 (ebook) | LCC QP751 .C78 2018 (print) | DDC 612/.01577–dc23 LC record available at https://lccn.loc.gov/2017044562 Cover Design: Wiley Cover Images: Courtesy of Laura Bayés-García, Barcelona, Spain Set in 10/12pt Warnock by SPi Global, Pondicherry, India 10 9 8 7 6 5 4 3 2 1

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Contents Preface  xiii List of Contributors  xv 1

Introduction: Relationships of Structures, Properties, and Functionality  1 Kiyotaka Sato

1.1 ­Introduction  1 1.2 ­Lipid Species  1 1.2.1 Hydrocarbons 1 1.2.2 Fatty Acids  2 1.2.3 Alcohols and Waxes  4 1.2.4 Acylglycerols 4 1.3 ­Physical States and the Functionality of Lipid Products  5 1.4 ­Formation Processes of Lipid Crystals  7 1.5 ­Polymorphism  9 1.6 ­Aging and Deterioration  11 1.7 ­Trans‐Fat Alternative and Saturated‐Fat Reduction Technology  13 ­References  15 2

Polymorphism of Lipid Crystals  17 Kiyotaka Sato

2.1 ­Introduction  17 2.2 ­Thermal Behavior of Polymorphic Transformations  17 2.3 ­Molecular Properties  20 2.3.1 Subcell and Chain‐Length Structures  20 2.3.2 Conformation of Hydrocarbon Chains  24 2.3.3 Glycerol Conformations  25 2.3.4 Polytypism 26 2.4 ­Fatty Acids  27 2.4.1 Saturated Fatty Acids  27 2.4.2 Unsaturated Fatty Acids  32 2.5 ­Monoacylglycerols and Diacylglycerols  37 2.5.1 Crystal/Molecular Structures  37 2.5.2 Polymorphic Behavior  39 2.6 ­Triacylglycerols (TAGs)  41 2.6.1 Crystal/Molecular Structures  42

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2.6.2 Polymorphic Behavior  46 2.7 ­Conclusions  54 ­ References  54 3

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals  61 Eckhard Floeter, Michaela Haeupler, and Kiyotaka Sato

3.1 ­Introduction  61 3.2 ­Thermodynamic Considerations  63 3.2.1 Framework for Engineering Calculations  63 3.2.2 Phase Behavior of Co‐Crystallizing Components  66 3.2.3 Governing Principles for Phase Boundaries  70 3.3 ­Effects of Molecular Structures on the Phase Behavior  70 3.3.1 Aliphatic Chain‐Chain Interactions: n‐Alkanes  71 3.3.2 Mixtures of Fatty Acids  72 3.3.3 Mixtures of Partial Glyceride Fatty‐Acid Esters  81 3.3.4 Mixtures of TAGs  82 3.4 ­Mixing Behavior of TAGs in Natural and Interesterified Fats  92 3.4.1 Cocoa Butter  93 3.4.2 Palm Oil  94 3.4.3 Coconut Oil  95 3.4.4 Milk Fat  95 3.4.5 Interesterified Fats  96 3.5 ­Crystallization Properties  97 3.6 ­Conclusions  98 ­ References  100 4

Fundamental Aspects of Crystallization of Lipids  105 Hironori Hondoh, Satoru Ueno, and Kiyotaka Sato

4.1 ­Introduction  105 4.2 ­Physical and Structural Properties of Lipid Liquids  105 4.2.1 Preheating Effects  106 4.2.2 Liquid Phases of Triacylglycerols  109 4.3 ­Driving Forces for Crystallization  112 4.4 ­Nucleation  114 4.4.1 Homogeneous versus Heterogeneous  114 4.4.2 Polymorph‐Dependent Nucleation Kinetics  118 4.4.3 Secondary Nucleation  121 4.4.4 Crystal Seeding  122 4.5 ­Kinetics of Crystal Growth  125 4.5.1 Mechanism of Crystal Growth  125 4.5.2 Crystal Growth Rate  127 4.5.3 Polymorph‐Dependent Growth Rate  129 4.5.4 Spherulite 130 4.5.5 Epitaxial Growth  132 4.5.6 Morphology of Crystals  133 4.6 ­Conclusions  135 Acknowledgment  136 ­ References  136

Contents

5

Supramolecular Assembly of Fat Crystal Networks from the Nanoscale to the Mesoscale  143 Fernanda Peyronel, Nuria C. Acevedo, David A. Pink, and Alejandro G. Marangoni

5.1 ­Introduction  143 5.2 ­Cryo‐TEM  144 5.2.1 Challenges Associated with the Microscopic Observation of Fat Microstructure  144 5.2.2 Sample Preparation for Cryo‐TEM  145 5.2.3 Nanoscale Structure Characterization  146 5.2.4 Effects of External Fields on Fat Nanostructure  148 5.3 ­Physical Interactions, Models, and Mathematical Methods  154 5.3.1 Models in General  155 5.3.2 Coarse‐Grained Interactions: Nano‐ to Mesoscale  156 5.3.3 Models Using Spheres  157 5.3.4 Introduction to Modeling the Statics and Dynamics of Aggregates  157 5.3.5 Static Structure Functions  158 5.3.6 Application 1: CNP Aggregation. Tristearin Solids in Triolein Oil  158 5.3.7 Application 2: Complex Oils. Tristearin Solids in Complex Oils  161 5.3.8 Application 3: Nanoscale Phase Separation in Edible Oils  162 5.4 ­Ultra Small Angle X‐Ray Scattering (USAXS)  164 5.4.1 Principles of X‐Ray Scattering  164 5.4.2 USAXS Instrumentation at the APS  167 5.4.3 Sample Preparation  168 5.4.4 Unified Fit and Guinier‐Porod Models  168 5.4.5 Experimental Results  170 5.5 ­Concluding Remarks  174 Acknowledgments  175 ­ References  175 6

Effects of Dynamic Temperature Variations on Microstructure and Polymorphic Behavior of Lipid Systems  183 Laura Bayés‐García, Teresa Calvet, and Miquel À. Cuevas‐Diarte

6.1 ­Introduction  183 6.2 ­Influence on the Polymorphic Behavior in Bulk State  183 6.2.1 Single TAG Components  183 6.2.2 Binary Mixtures of TAGs  189 6.3 ­Colloidal Dispersion States  193 6.3.1 Emulsions 193 6.3.2 Organogels 196 6.4 ­Role of Thermal Treatments on End Food Products Properties  198 6.4.1 Milk Fats  198 6.4.2 Other Dairy Products  199 6.4.3 Cocoa Butter  200 6.4.4 Vegetable Fats  204 6.5 ­Conclusions  206 ­ References  207

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7

Lipid Crystal Networks Structured under Shear Flow  211 Farnaz Maleky and Gianfranco Mazzanti

7.1 ­Introduction  211 7.2 ­Overview of the Formation of Fat Crystals  212 7.3 ­Temperature Gradients and Optimal Supercooling  213 7.4 ­Basic Concepts on Shear Flow  214 7.5 ­Fat Crystallization under Shear  216 7.5.1 Shear Affects Polymorphic Transformations  216 7.5.2 Crystalline Orientation Induced by Shear Flow  219 7.5.3 Shear Affects Fat Structural Properties at the Micro‐ and Nano‐Length Scales  224 7.5.4 Physicochemical Properties of Sheared Fat Matrices  227 7.5.5 Effects of Shear Flow on Mass Transfer Dynamics of Crystallizing and Crystallized Materials  231 7.6 ­Concluding Remarks  233 ­ References  234 8

Tailoring Lipid Crystal Networks with High‐Intensity Ultrasound  241 Yubin Ye, Peter R. Birkin, and Silvana Martini

8.1 ­Introduction  241 8.2 ­Fundamentals of Sonication  242 8.2.1 Acoustic Driving Force  242 8.2.2 Acoustic Cell Characteristics  243 8.2.3 Cavitation 244 8.2.4 Experimental Conditions  245 8.3 ­Tailoring Lipid Crystal Networks  246 8.3.1 Crystallization Kinetics  246 8.3.2 Inferential Mechanism  249 8.3.3 Postsonication Changes  250 8.4 ­Practical Considerations  255 8.4.1 Oxidation 255 8.4.2 Scale Up  257 8.4.3 Combination with Other Processing Methods  258 8.5 ­Conclusions and Future Research  258 ­ References  259 9

Effects of Foreign and Indigenous Minor Components  263 Kevin W. Smith and Kiyotaka Sato

9.1 ­Introduction  263 9.2 ­Basic Understanding  264 9.3 ­Effects of Foreign Components  265 9.3.1 Emulsifiers 265 9.3.2 Indigenous Minor Components  276 9.4 ­Other Additives  276 9.5 ­Conclusions  278 ­ References  279

Contents

10

Crystallization Properties of Milk Fats  283 Christelle Lopez

10.1 ­Introduction  283 10.2 ­Milk Fat: A Wide Diversity of Fatty Acids and Triacylglycerols (TAGs)  284 10.3 ­Crystallization Properties of Bovine Anhydrous Milk Fat (AMF)  285 10.3.1 Thermal Properties  285 10.3.2 Effect of Cooling Rate on AMF Crystals  286 10.3.3 Effect of Shear on AMF Crystals  295 10.3.4 Effect of Minor Lipid Compounds  295 10.4 ­Crystallization of TAGs in Bovine Milk Fat Globules and Emulsion Droplets  296 10.4.1 Effect of Cooling Rate and Tempering  298 10.4.2 Effect of the Size of Milk Fat Globules and Lipid Droplets  304 10.5 ­Crystallization Properties of Milk Fat in Dairy Products  306 10.6 ­TAG Compositions Affecting Crystallization Properties of Milk Fat  308 10.6.1 Technological Process: Dry Fractionation  308 10.6.2 Dietary Manipulations  312 10.6.3 Milk Fat from Various Mammal Species  315 10.7 ­Liquid TAG Phase  316 10.8 ­Conclusions  317 ­ References  318 11

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications  323 Maria L. Herrera and Silvana Martini

11.1 ­Introduction  323 11.2 ­High Stearic High Oleic Sunflower Oil  324 11.2.1 Fractionation of HSHO‐SFO  324 11.2.2 Crystallization Behavior  326 11.2.3 Polymorphic Behavior  329 11.3 ­Blends of Sunflower Oil and Milk Fat  337 11.3.1 Chemical Composition  340 11.3.2 Physical Properties  340 11.3.3 Addition of Palmitic Sucrose Ester  344 11.4 ­HSHO‐Based CBE  347 11.5 ­Conclusions  348 ­ References  348 12

Physical Properties of Organogels Developed with Selected Low‐Molecular‐Weight Gelators  353 Jorge F. Toro‐Vazquez, Flor Alvarez‐Mitre, and Miriam Charó‐Alonso

12.1 ­Introduction  353 12.2 ­Basic Aspects of LMOGs: From Molecular Architecture to Functional Assemblies  355

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Contents

12.3 ­Why Developing Organogels with Vegetable Oils?  356 12.3.1 Vegetable Oils as Solvent in the Development of Organogels with LMOGs  357 12.3.2 Relationship between Molecular Structure of LMOGs and Physical Properties of Organogels  367 12.4 ­Organogels of Candelilla Wax  373 12.4.1 Rheological Properties of Candelilla Wax Organogels Developed Applying Shear Rate  373 12.4.2 Applications of Candelilla Wax Organogels  377 12.5 ­Conclusions  377 ­ References  379 13

Formation and Properties of Biopolymer‐Based Oleogels  385 Ashok R. Patel

13.1 ­Introduction  385 13.2 ­Formation of Polymer‐Based Oleogels  386 13.2.1 Polymer Oleogelation through Direct Methods  387 13.2.2 Polymer Oleogelation through Indirect Methods  389 13.3 ­Properties of Polymer‐Based Oleogels  393 13.3.1 Mechanical Properties  393 13.3.2 Temperature Sensitivity  394 13.3.3 Stability in Presence of Water  397 13.4 ­Potential Applications of Polymer‐Based Oleogels  397 13.4.1 Replacement of Beef Fat in Frankfurters  397 13.4.2 Heat‐Resistant Chocolates  397 13.4.3 Polymer Oleogels as Alternative to Full‐Fat Shortenings  397 13.4.4 Bakery Applications of Ethyl Cellulose Oleogels  398 13.5 ­Conclusions: Opportunities and Challenges  398 Acknowledgments  401 ­ References  402 14

Lipid Crystallization in Water‐in‐Oil Emulsions  405 Nicole L. Green and Dérick Rousseau

14.1 ­Introduction  405 14.2 ­Basics of Emulsion Properties  406 14.3 ­Emulsifier Effects on W/O Emulsions  408 14.3.1 Mono‐ and Diacylglycerols (E471)  409 14.3.2 Sucrose Fatty‐Acid Esters (E473)  411 14.3.3 Lecithins (E322)  412 14.3.4 Sorbitan Esters and Polyesters (E491‐E496)  413 14.3.5 Polyglycerol Esters (E475 – E476)  415 14.4 Stabilization Modes of W/O Emulsions  415 14.4.1 Pickering Stabilization  416 14.4.2 Network Stabilization  420 14.4.3 Combined Pickering and Network Stabilization  421 14.5 ­Conclusions  423 ­ References  424

Contents

15

Crystallization of Lipids in Oil‐in‐Water Emulsion States  431 John N. Coupland

15.1 ­The Basic Concepts  431 15.2 ­Surface Nucleation  432 15.3 ­Polymorphic Transitions in Droplets  436 15.4 ­Morphology of Crystalline Droplets  437 15.5 ­Colloidal Stability of Crystalline Droplets  439 15.6 ­Conclusions  442 ­ References  443 16

Lipid Crystals and Microstructures in Animal Meat Tissues  447 Michiyo Motoyama, Genya Watanabe, and Keisuke Sasaki

16.1 ­Introduction  447 16.2 ­Depot Fat and Crystalline State  448 16.2.1 Adipose Tissue  448 16.2.2 Triacylglycerol (TAG) Compositions of Animal Fats  449 16.3 ­Fat Crystals and Quality of Porcine Adipose Tissue  450 16.3.1 Polymorphism of Extracted Porcine Fat Crystals  450 16.3.2 Fat Crystals and Macroscopic Meat Quality  454 16.3.3 Application to Actual Meat and Meat Products  455 16.4 ­Crystal Microstructures in Adipose Tissues  460 16.5 ­Concluding Remarks  462 Acknowledgments  462 ­ References  462 17

Conventional and New Techniques to Monitor Lipid Crystallization  465 Annelien Rigolle, Koen Van Den Abeele, and Imogen Foubert

17.1 ­Introduction: What Would Be a Perfect Technique?  465 17.2 ­Conventional Techniques (and Advances Made)  466 17.2.1 Pulsed Nuclear Magnetic Resonance  466 17.2.2 Differential Scanning Calorimetry  469 17.2.3 X‐Ray Diffraction  472 17.2.4 Rheology 474 17.2.5 Microscopy 476 17.3 ­“New” Techniques with Potential for Online Monitoring  478 17.3.1 Ultrasonic Techniques  478 17.3.2 Laser Backscattering  484 17.3.3 Near‐Infrared and Raman Spectroscopy  485 17.4 ­Conclusions  485 Acknowledgments  486 ­ References  487 Index  493

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xiii

Preface This text presents new and emerging knowledge, techniques, and applications of lipid crystals, which are major hydrophobic ingredients of semi‐solid soft matters—along with liquid oils, water, emulsifiers, and other minor components—used in food, cosmetic, and pharmaceutical industries. In various semi‐solid lipids, the lipid crystals exhibit invaluable physical and chemical properties: solubilization and controlled release of oil‐soluble nutrients, drugs and flavoring ingredients, hardness, consistency, melting behavior, spreadability, structuring of liquid oil, water and air cells. This text covers recent advances in the research of polymorphic structures, molecular interactions, nucleation and crystal growth, and crystal network formation of lipid crystals in bulk and emulsion states. Specific efforts have been made to relate two problems of trans‐fat alternative and saturated‐fat reduction technology to lipid crystallization. Although the latter is still debated in academia and industry, the two problems are the most significant in the edible application of lipids, and one of the key solutions must be present in ideas to improve the crystallization processes of various lipid materials. In my mind, this text is an evolution of previous publications, Crystallization and Polymorphism of Fats and Fatty Acids and Crystallization Processes of Fats and Lipid Systems, which were published in 1988 and 2001, respectively. During the last decades, three driving forces may have prompted me to publish this text. First, global trends in many lipid‐related industries have rapidly changed and developed, and they have pushed us to elucidate knowledge and technology of lipid crystallization. Some of these trends are consumers’ preferences for more natural and healthy lipid materials, ethics and sustainability of raw lipid materials production, and innovation and hybridization of functional end products. Second, driven by these trends, the quantity and quality of the research and technology of lipid crystallization has advanced quickly. Now is the time for a text that can comprehensively review the current studies and foresee the future developments of our research fields. Third, the scientific methods used to analyze the crystallization processes and structures of lipids have also developed (e.g., synchrotron radiation X‐ray diffraction and scattering, ultrasonic, laser scattering, near infrared and Raman spectroscopic techniques) and deserve to be reviewed. The 17 chapters in this text may be categorized in four groups. The first of these begins with Chapter 1, which introduces the readers to the world of lipids, and continues with Chapters 2, 3, and 4 in which the fundamental aspects of lipid crystals and crystallization are further described. In the second group, Chapters 5 through 9, the formation of lipid crystal networks under various external influences of thermal

xiv

Preface

fluctuation, ultrasound irradiation, shear, and additives is discussed. In the third group, Chapters 10, 11, and 16 review the crystallization properties of lipids in milk fats, sunflower oils, and animal meat tissues, and in Chapters 12 to 15, the lipid crystals in oleogel and emulsion states are discussed. In the final group and chapter, traditional and cutting‐edge research tools to analyze lipid crystallization are highlighted. I hope that this text can be a valuable resource for novel and creative knowledge for R&D technologists in food, cosmetic, and pharmaceutical industries; for professors and graduate students in departments of food science, bioengineering, and life materials science; and all of those who are working with the lipid crystals. Kiyotaka Sato

xv

List of Contributors Nuria C. Acevedo

John N. Coupland

Department of Food Science and Human Nutrition Iowa State University Ames, Iowa, United States

Department of Food Science Pennsylvania State University University Park, Pennsylvania, United States

Flor Alvarez‐Mitre

Miquel À. Cuevas‐Diarte

Facultad de Ciencias Químicas‐CIEP Universidad Autónoma de San Luis Potosí San Luis Potosí, México Laura Bayés‐García

Facultat de Ciències de la Terra Universitat de Barcelona Barcelona, Spain Eckhard Floeter

Facultat de Ciències de la Terra Universitat de Barcelona Barcelona, Spain

Department of Food Technology and Food Chemistry Technical University of Berlin Berlin, Germany

Peter R. Birkin

Imogen Foubert

Department of Chemistry University of Southampton Highfield, Southampton United Kingdom Teresa Calvet

Facultat de Ciències de la Terra Universitat de Barcelona Barcelona, Spain Miriam Charó‐Alonso

Facultad de Ciencias Químicas‐CIEP Universidad Autónoma de San Luis Potosí San Luis Potosí, México

Food & Lipids Katholieke Universiteit Leuven Kulak Kortrijk, Belgium Nicole L. Green

Department of Chemistry and Biology Ryerson University Toronto, Ontario, Canada Michaela Haeupler

Department of Food Technology and Food Chemistry Technical University of Berlin Berlin, Germany

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List of Contributors

Maria L. Herrera

Ashok R. Patel

Institute of Polymer Technology and Nanotechnology (ITPN) National Research Council of Argentina (CONICET) University of Buenos Aires Buenos Aires, Argentina

International Iberian Nanotechnology Laboratory Braga, Portugal

Hironori Hondoh

Graduate School of Biosphere Science Hiroshima University Higashi‐Hiroshima, Japan Christelle Lopez

INRA, Science et Technologie du Lait et de l’OEuf Rennes, France Farnaz Maleky

College of Food, Agricultural and Environmental Sciences Department of Food Science and Technology Ohio State University Columbus, Ohio, United States

Fernanda Peyronel

Department of Food Science University of Guelph Ontario, Canada David A. Pink

Department of Physics St. Francis Xavier University Antigonish, Nova Scotia, Canada Annelien Rigolle

Group Long Term Research and Services Lab (GRS) Puratos, Groot‐Bijgaarden I, Belgium Dérick Rousseau

Department of Chemistry and Biology Ryerson University Toronto, Ontario, Canada

Alejandro G. Marangoni

Department of Food Science University of Guelph Ontario, Canada Silvana Martini

Department of Nutrition Dietetics and Food Sciences Utah State University Logan, Utah, United States Gianfranco Mazzanti

Process Engineering and Applied Science Dalhousie University Halifax, Nova Scotia, Canada

Keisuke Sasaki

Institute of Livestock and Grassland Science National Agriculture and Food Research Organization (NARO) Tsukuba, Ibaraki, Japan Kiyotaka Sato

Hiroshima University Higashi‐Hiroshima, Japan Kevin W. Smith

Fat Science Consulting Limited Bedford, United Kingdom

Michiyo Motoyama

Institute of Livestock and Grassland Science National Agriculture and Food Research Organization (NARO) Tsukuba, Ibaraki, Japan

Jorge F. Toro‐Vazquez

Facultad de Ciencias Químicas‐CIEP Universidad Autónoma de San Luis Potosí San Luis Potosí, México

List of Contributors

Satoru Ueno

Genya Watanabe

Graduate School of Biosphere Science Hiroshima University Higashi‐Hiroshima, Japan

Institute of Livestock and Grassland Science National Agriculture and Food Research Organization (NARO) Tsukuba, Ibaraki, Japan

Koen Van Den Abeele

Wave Propagation and Signal Processing Department of Physics Katholieke Universiteit Leuven Kulak Kortrijk, Belgium

Yubin Ye

Nestlé Development Center Marysville, Ohio, United States

xvii

1

1 Introduction: Relationships of Structures, Properties, and Functionality Kiyotaka Sato

1.1 ­Introduction This chapter presents a comprehensive sketch of the lipid species and functionality of lipid crystals present in various end products by outlining different stages of crystallization. In doing so, topics will be highlighted that will be elaborated further in chapters of this book. At the end of this chapter, a particular effort is made to relate trans‐fat alternative and saturated‐fat reduction technology to lipid crystallization because these two issues are the most significant problems in the edible‐application technology of lipids and one of the key solutions is lipid crystallization.

1.2 ­Lipid Species Lipids are a class of compounds that contain long‐chain aliphatic hydrocarbons and their derivatives (O’Keefe 2008). There is a wide variety of lipid materials such as hydrocarbons, fatty acids, acylglycerols, sterols and sterol esters, waxes, phospholipids, plasmalogens, sphingolipids, and so on. Typical lipids whose crystallization properties have critical implications in food and other industries include hydrocarbons, fatty acids, alcohols, waxes, and acylglycerols. Because the lipid species of natural lipids of vegetable or animal resources vary from one to another, the understanding of the crystallization, melting, and physical properties must be based on the effects of major and minor lipid components included in every lipid material. In this section, we take a brief look at the chemical structures of these typical lipid molecules. 1.2.1 Hydrocarbons Hydrocarbons comprise a group of the simplest lipid molecules and are composed of hydrogen and carbon atoms. A typical molecular shape of hydrocarbons containing all saturated carbon–carbon bonds is expressed as CH3‐(CH2)n‐2‐CH3, in which n is the Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, First Edition. Edited by Kiyotaka Sato. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

2

Crystallization of Lipids

number of carbon atoms. Hereafter, we use nc as the number of carbon atoms in the all‐hydrocarbon chains. In nature, even‐numbered and odd‐numbered hydrocarbons occur, depending on whether nc is even or odd. Molecular interactions operating among the hydrocarbon molecules are van der Waals forces, and these comprise the major molecular interactions among lipid molecules when they contain hydrocarbon chains as hydrophobic moieties. When the number of carbon atoms exceeds four, structural isomers occur (e.g., straight chains or branched chains). The straight‐chain hydrocarbons are called n‐alkanes as illustrated for n‐octadecane with nc=18 (Fig. 1.1a). 1.2.2  Fatty Acids

–

Fatty acids are formed by replacing one end of –CH3 in n‐hydrocarbons with a carboxyl group (–COOH). In contrast, dicarboxilic acids are formed when both end groups of –CH3 in n‐hydrocarbons are replaced with –COOH. There are saturated and unsaturated fatty acids, depending on whether double bonds are included and stereoisomers of cis or trans unsaturated fatty acids occur. In nature, a wide variety of fatty acids is present, differing in nc, the number of double bonds having cis or trans conformations or the positions of the double bonds at the hydrocarbon chains. Similarly to hydrocarbons, even‐ and odd‐numbered fatty acids occur. The principal fatty acids abundantly occurring in nature are summarized in Table 1.1. Although standard (IUPAC) systematic names are given to fatty acids, the common names and abbreviations presented in the table will be used throughout this book. (a) As typical fatty acids having nc=18, stearic acid is a saturated fatty acid, oleic acid is a mono‐unsaturated fatty acid having a cis (b) ­double bond at the 9–10 carbon atoms, and COOH elaidic acid is a mono‐unsaturated fatty acid (c) having a trans double bond at the 9–10 carbon atoms, as seen in Fig. 1.1(b, c, and d). The melting temperatures (Tm) of the three fatty COOH acids in their most stable polymorphic forms (d) are 69° C (stearic acid), 44° C (elaidic acid), COOH and 16.1° C (oleic acid). This typically repre(e) R sents the relationships between Tm and the O molecular shapes of the fatty acids in the following aspects. CH2–CH–CH2 –

O

●●

–

– R

–

– O

R

Fig. 1.1  Typical lipid molecules. (a) n‐Octadecane, (b) stearic acid, (c) oleic acid, (d) elaidic acid, and (e) triacylglycerol. In (a)–(d), carbon atoms are shown except for COOH groups for fatty acids. In (e), R is fatty acid moiety.

●●

 t a fixed number of nc, Tm decreases with A increasing numbers of double bonds, and the conformation of the double bonds changes from trans to cis. As for saturated fatty acids, Tm increases with increasing nc, although the values of Tm for fatty acids with an odd‐numbered nc is a bit lower than those with an

Introduction: Relationships of Structures, Properties, and Functionality

Table 1.1  Systematic, common, and shorthand names of principal fatty acids. Systematic

Common

Shorthand

Abbreviation

Octanoic

Caprylic

8:0

Ca

Decanoic

Capric

10:0

C

Dodecanoic

Lauric

12:0

L

Tetradecanoic

Myristic

14:0

M

Hexadecanoic

Palmitic

16:0

P or PA

Heptadecanoic

Margaric

17:0

Ma

Octadecanoic

Stearic

18:0

S or SA

Nonadecanoic

Nonadecanoic

19:00

No

Eicosanoic

Arachidic

20:0

A

Docosanoic

Behenic

22:0

B

c‐9‐Hexadecenoic

Palmitoleic

16:1, Δ9‐ω7

POA

c‐9‐Octadecenoic

Oleic

18:1, Δ9‐ω9

O or OA

c‐12‐Octadecenoic

Petroselinic

18:1, Δ6‐ω12

PSA

t‐9‐Octadecenoic

Elaidic

18:1, Δ9‐ω9

E

c‐11‐Octadecenoic

Asclepic

18:1, Δ11‐ω7

APA

12‐hydroxy, c‐9‐Cctadecenoic

Ricinoleic

18:1, Δ9‐ω9

R

t‐11‐Octadecenoic

Vaccenic

18:1, Δ11‐ω7

V

c‐9, c‐12‐Octadecadienoic

Linoleic

18:2‐ω6, 9

Li

c‐9, c‐12‐ c‐15‐Octadecatrienoic

α‐Linolenic

18:3‐ω3, 6, 9

ALA

c‐6, c‐9‐ c‐12‐Octadecatrienoic

γ‐Linolenic

18:3‐ω6, 9, 12

GLA

c‐11‐Eicosanoic

Gondoic

20:1, Δ11‐ω9

GOA

c‐5, c‐8, c‐11, c‐14, c‐17‐Eicosapentanoic

Eicosapentanoic

20:5, ω3, 6, 9, 12, 15

EPA

c‐13‐Docosenoic

Erucic

22:1, Δ13‐ω9

Er

c‐4, c‐7, c‐10, c‐13, c‐16, c‐19‐Docosahexanoic

DHA

22:6, ω3, 6, 9, 12, 15, 18

DHA

Saturated

Unsaturated

even‐numbered nc–1. For example, Tm of margaric acid nc=17 (palmitic acid, nc=16) is 61° C (63° C). This is ascribed to the instability of molecular packing at the lamellar interfaces, where CH3‐CH3 end groups are stacked against each other, of odd‐numbered fatty acids compared to that of even‐numbered fatty acids. These relationships apply to other lipids containing fatty acid chains as their hydrophobic moieties. The –COOH group is hydrophilic (water soluble), and the hydrocarbon chains are hydrophobic (oil soluble). Therefore, the hydrophobicity or hydrophilicity of a fatty acid molecule as a whole depends on nc. Fatty acids with nc ≤6 become water soluble, whereas

3

4

Crystallization of Lipids

they are sparingly water soluble when nc exceeds 6. Molecules having a hydrophobic moiety in one part and a hydrophilic moiety in another part are called amphiphilic, as revealed in other lipids: alcohols, mono‐ and di‐acylglycerols, phospholipids, emulsifiers, and so on. 1.2.3  Alcohols and Waxes Alcohols are formed by replacing one –CH3 end of n‐hydrocarbons with –OH. Similarly to fatty acids, the alcohols become liphophilic as nc increases above 6, and even‐numbered and odd‐numbered alcohols occur. There are narrow and broad categories of “waxes.” The former refers to the esters of long‐chain fatty acids and alcohols. The latter represents “waxy matter” abundantly occurring in nature as epidemic lipids, which include hydrocarbons, ketones, and aldehydes. Here we limit the waxes to the esters of long‐chain fatty acids and alcohols. The nc for constructing naturally occurring waxes vary widely from one wax to another. For example, candellila wax is made of fatty acids with nc = 16–34 and alcohols with nc = 22–34, whereas rice bran wax is made of fatty acids with nc = 16–32 and alcohols with nc = 24–38. 1.2.4 Acylglycerols Acylglycerols are formed by esterification of the hydroxyls in glycerol molecules (CH2OH‐CHOH‐CH2OH) with fatty acids. Monoacylglycerols (MAGs), diacylglycerols (DAGs), and triacylglycerols (TAGs) are formed when one hydroxyl, two hydroxyls, or three hydroxyls, respectively, are esterified, as summarized in Fig. 1.2. TAGs (Fig. 1.1 e) are the principal lipids that construct animal adipose tissues, vegetable and edibles fats, and oils. The term used, fat or oil, depends solely on whether the (a) HO

CH2OH C

H sn-2

CH2OH

(b) HO

CH2OR C*

H

CH2OH

(c) RO

CH2OH C

H

CH2OH

(d) HO

CH2OH C*

sn-1

H

CH2OR

(e) R2O

sn-3

CH2OR1 C*

H

CH2OH

(f)

CH2OR1 HO

C*

H

CH2OR2

(g) R1O

(h) R2O

CH2OH C*

H

CH2OR2 CH2OR1 C*

H

CH2OR3

Fig. 1.2  Structure models of acylglycerols. (a) Stereospecific numbering of glycerol, (b) 1‐monoacyl‐sn‐glycerol, (c) 2‐monoacyl‐ sn‐glycerol, (d) 3‐monoacyl‐sn‐glycerol, (e) 1,2‐diacyl‐sn‐glycerol, (f ) 1, 3‐diacyl‐ sn‐glycerol, (g) 2, 3‐diacyl‐sn‐glycerol, and (h) triacylglycerol. C*: chiral carbon; R, a fatty acid moiety; sn: stereospecific number.

Introduction: Relationships of Structures, Properties, and Functionality

TAG melts at room temperature (~25° C); at that temperature, fat is in a crystalline state and oil is in a liquid state. MAGs are intermediate products formed during enzymatic decomposition of TAGs during digestion. In addition, MAGs are industrially synthesized and used as emulsifiers because of their strong amphiphilic properties. DAGs are present as relatively minor components in natural oils and fats and are also industrially produced and used as edible fats and oils. There is no chiral center in a glycerol molecule as seen in Fig.  1.2(a). However, it becomes chiral when for MAGs, a fatty acid is esterified either at the sn‐1 or at the sn‐3 positions (Fig. 1.2b and d), for DAGs, two fatty acids are esterified at the sn‐1 (or sn‐3) and sn‐2 positions (Fig. 1.2e and g) or different fatty acid moieties are esterified at the sn‐1 and sn‐3 positions (Fig. 1.2f ), and for TAGs, the three fatty‐acid moieties are all different or different fatty acid moieties are esterified at the sn‐1 and sn‐3 positions (Fig.  1.2g). Instead of a numbering method using the sn‐positions, an alternative description using Greek letters has been employed, as in α‐monoacyl‐sn‐glyverol (1‐ monoacyl‐sn‐glycerol), β‐monoacyl‐sn‐glycerol (2‐monoacyl‐sn‐glycerol), α, β‐diacyl‐ sn‐glyverol (1, 2‐diacyl‐sn‐glycerol), α, α’‐diacyl‐sn‐glycerol (1, 3‐diacyl‐sn‐glycerol), etc. Optical isomers can occur for chiral acylglycerols, and the mixing‐phase behavior of the chiral molecules affects the structural and physical properties in natural lipids when racemic mixtures are present. TAGs can be simply described by using the abbreviated names of the fatty acids listed in Table 1.1. For example, we have tristearoylglycerol (SSS), 1,3‐dipalmitoyl‐2‐stearoyl‐ sn‐glycerol (PSP), and 1,3‐distearoyl‐2‐oleoyl‐ sn‐glycerol (SOS). Chiral TAGs can also be described by using the abbreviated names of the fatty‐acid moieties. For example sn‐POS is 1‐palmitoyl‐2‐oleoyl‐3‐stearoyl‐sn‐glycerol. An equal mixture of both stereoisomers of the chiral TAGs can be described as rac (e.g., rac‐POS), which means that there are equal amounts of sn‐POS and sn‐SOP. Lipid species can be precisely described by highlighting the atomic‐level crystal structures in Chapter 2.

1.3 ­Physical States and the Functionality of Lipid Products The crystallization and functionality of crystallized lipids are complicatedly influenced by the physical states where the lipids are crystallized, as seen in Fig. 1.3. Before going into the details of the crystallization in various physical states, which will be presented in forthcoming chapters, let us briefly view the relationship between the functionality of lipid products and the physical states presented in the figure. The liquid state simply refers to an oil phase, as represented by frying oil and biofuel, whose functionality is in heat transfer, viscosity, oxidation stability, and so on. The crystallization process in liquid‐state materials may occur as a deterioration of the end product (e.g., the clouding of cooking oils during storage in a refrigerator or precipitation causing an increase in the pouring point for biofuels at chilled temperatures). Therefore, retardation or prohibition of the crystallization of minor‐component lipids becomes critical in these products. Lubricants made of vegetable oils also require similar physical properties for optimum functionality. The crystalline state in a bulk sample signifies that the major portion of the material is composed of lipid crystals, as typically represented in confectionery fat (chocolate).

5

6

Crystallization of Lipids Physical States

Typical Products

Typical Functionality

Frying oil Biofuel Lubricant

Heat transfer Viscosity Oxidation stability

Chocolate Hard lipstick

Crispy touch Hardness Sharp melting

Gel

Water barrier film Soft lipstick

Hardness/Softness Spreadability Oil-binding property

Emulsion

Cream Mascara Drug carrier

Spreadability Texture Stability

Foam

Whipped cream Ice cream

Dispersibility (air) Stabillity Thermal conductivity

Liquid

Bulk

Fig. 1.3  Relationships between physical states and functionality of lipid products.

Fine particles of sugar, cocoa mass, and milk powder are suspended in the continuous phase of cocoa butter crystals, which comprise about 30 wt.% of the total mass of chocolate. Crispy touch, hardness, and sharp melting are typical functionalities of chocolate, which are mostly brought about by the lipid crystals comprising the major matrices of the products. Lipid crystal–based hard lipsticks require the functionalities of hardness, spreadability, gloss, anti‐sweating, and anti‐blooming of the products. Such properties are also determined by the network of lipid crystals, in which pigments, fragrance materials, and biologically active substances (vitamins, hormones, amino acids, etc.) are dispersed. The gel state is defined as a two‐phase colloidal system consisting of solid components along with water (hydrogels) or oil (oleogels or organogels), in which the solid behavior prevails over the sol state. Oleogels may be defined as lipophilic liquids and solid mixtures in which solid lipid materials (gelators) with lower concentrations can entrap bulk liquid oil by forming a network of gelators in the bulk oil. The gelators can be grouped into two categories: self‐assembly systems and crystal‐particle systems. Water‐barrier films and soft lipsticks are typical products made of oleogels. The morphology, size, density, and crystal networks of lipid crystals are the dominant factors that influence the physical functionalities of the gel state, such as hardness/softness and spreadability. An emulsion is defined as a two‐phase colloidal system consisting of water and oil along with emulsifiers that reduce the water–oil interfacial energy. There are two types of emulsions, water‐in‐oil (W/O) and oil‐in‐water (O/W). Butter, margarine, and

Introduction: Relationships of Structures, Properties, and Functionality

spread (W/O) and whipped (O/W) systems are typical emulsion systems consisting of lipid crystals, in which the physical properties of the emulsion, such as the spreadability, texture, and stability, are influenced by the lipid crystals present in the continuous phase of the W/O emulsion or in the dispersed phase of the O/W emulsion. Both the W/O and O/W emulsions are widely employed in the food, cosmetics, and pharmaceutical industries. In particular, nanometer‐sized lipid droplets are employed as carrier systems for poorly water‐soluble drugs. Aerated colloidal systems, also known as foams, are widely applied in the cosmetics, food, and porous material production industries. Foams have the significant advantages of shape retention, soft texture, the ability to act as a thermal barrier, and low calorie content. Aqueous foams contain air bubbles in a continuous aqueous phase, like whipped cream and ice cream. Nonaqueous foams are formed by dispersing air bubbles in oil phases and are important for foamed plastics, whipped butter, and confections. In both cases, the dispersibility and stability of air bubbles are major functionalities that are partly governed by the lipid crystals surrounding the air bubbles together with other ingredients such as proteins and starches. In the lipid crystal–based products displayed in Fig. 1.3, the lipid crystals play critically important roles in revealing the firmness, gloss, melting/crystallization, texture, rheology, and stabilization of water droplets (W/O emulsion) and air cells (foams) by themselves alone or together with emulsifiers, proteins, starch, and so on.

1.4 ­Formation Processes of Lipid Crystals The basic principles underlying the formation processes of lipid structures are common to the physical states displayed in Fig. 1.3, including the microscopic and macroscopic features in Fig. 1.4. Polymorphic structures and primary particles of lipid crystals comprise the microscopic features, whereas the formation of flocs and networks of lipid crystals determines the macroscopic features. The molecular structures of lipids are revealed in polymorphism and primary‐particle formation. Polymorphism remarkably influences the macroscopic properties of fat products. For example, there are three polymorphic forms in TAG crystals, α, β’, and β. In margarines and fat spreads, lipids are first crystallized in the least stable form (α) by rapid cooling of the molten materials. However, the α crystals are very short‐lived and do not exist in the finished products, in which metastable β’ crystals are formed as the most desired polymorphic form. This is because β’ crystals are relatively small and can incorporate a large amount of semi‐solid oil phases and

External factors

Molecules (polymorph)

Primary particles

Flocs

Crystal network

Fig. 1.4  External factors affecting formation processes of lipid crystals.

Macroscopic structures

7

8

Crystallization of Lipids

water droplets within the crystal network. Thermodynamic stabilization, however, causes the transformation from the metastable β’ form to the most stable β form during storage or other shelf‐life conditions. The β crystals tend to grow into large needle‐like agglomerates, which results in a sensation of sandiness in the mouth. In contrast, cocoa butter in chocolate should be crystallized in a β polymorph (more correctly, Form V of a β‐type polymorph, see Chapter 3) because of its high density and optimal melting point, resulting in the desired sharp melting of chocolate. As β crystals crystallize too slowly compared with the α and β’ forms, the use of a special processing of crystallization called tempering is necessary for producing cocoa‐butter‐ based chocolate. External factors can produce many of the desired microscopic features of lipid crystals, and knowledge of the relationship between their molecular structures, their particle formation along different dimensions, and their spatial networks under internal and external factors gives us optimal ways of designing materials with the desired functionality. Typical factors that have already been applied, or have high potential to be applied, to the actual industrial processing include the following. a) Internal factors ●● Interesterification (chemical, enzymatic) ●● Fractionation (dry, solvent, detergent) ●● Blending b) External factors ●● Intentionally varying the temperature ●● Applying shear ●● Applying hydrostatic pressure ●● Adding foreign materials (additives) ●● Applying ultrasound waves ●● Encapsulating of lipids into small droplets (O/W emulsion) These external factors are thoroughly discussed in this book. The details of the formation of lipid crystal networks vary from one physical state to another. For example, crystallization in a bulk sample proceeds without the effects of oil–water interfaces, whereas interfacial crystallization in the O/W and W/O emulsion states plays a critical role in creating the lipid crystal network (see Fig. 1.3). The basic streams, however, of the formation of a lipid crystal network can be drawn as in Fig. 1.5, which includes the formation of crystal nuclei (nucleation), the subsequent growth of crystal nuclei (crystal growth), the aggregation of crystal particles, and the formation of a crystal network (network formation). All of these processes should be enabled only when a given set of external conditions (e.g., temperature, pressure, and concentration) provides the driving forces for crystallization as expressed by supercooling or supersaturation. Supercooling (ΔT) is defined as the difference in temperature between the melting point (Tm) and the crystallization temperature (Tc), that is, ΔT = Tm – Tc. Supersaturation (S) is defined as the ratio of the actual solute concentration X in solution to the solubility (Xs) at T = Tc, that is, S = X/Xs. The former refers to crystallization from neat liquid (melt), and the latter to crystallization from solution.

Introduction: Relationships of Structures, Properties, and Functionality

(a)

(b)

(c)

Fig. 1.5  A model of formation processes of lipid crystal network. (a) Nucleation, (b) crystal growth, and (c) network formation.

1.5 ­Polymorphism Almost all lipids possess two or more different crystal structures under a given set of thermodynamic conditions. This multiplicity of crystalline structures of the same substance is called polymorphism. The polymorphic behavior of lipid crystals is basically determined by their molecular structure, thermodynamic stability, and phase transformations. The thermodynamic stability of polymorphic forms is illustrated by the relationship of their Gibbs energy values, G = H  –  TS, where H, S, and T are the enthalpy, entropy, and temperature. Polymorphic forms with greater G values are less stable than those with lower ones, which have higher solubility and lower melting points. Polymorphic transformations occurring during and after crystallization are also quite important. Two types of transformations can occur from less stable forms to more stable polymorphic forms (e.g., from α or β’ forms to β’ or β forms for TAGs). Solid‐state transformation occurs when the metastable form is stored below its melting temperature in the crystalline state. Another type of polymorphic transformation is melt‐ mediated transformation, which occurs as the temperature rises just above the melting

9

Crystallization of Lipids Driving force Nucleation Cluster

Monomer

Crystallization

Nucleus

Step Crystal growth

Kink

Crystal

Solid state Polymorphic transformation

Melt mediation

Gibbs Energy

10

α

Liquid

βʹ β

T

Fig. 1.6  Elementary processes of crystallization of lipids.

point of a metastable form, where melting of the metastable form and successive crystallization of more stable forms occur. Figure. 1.6 summarizes the elementary processes of the polymorphic crystallization of lipids. We may consider that the nucleation and crystal growth are relatively straightforward in accordance with the theory of nucleation and crystal growth. Complicated events, however, must occur during the formation of lipid crystal networks in the actual production stages of the lipid products because the methods of distribution and aggregation of the crystal particles differ greatly from those occurring in the initial stages of nucleation and crystal growth. Network formation may be affected by the following processes. ●●

●●

●●

Nucleation and crystal growth to form primary particles, in which tiny crystals having different sizes and polymorphic forms are present. In addition, the multiple lipid components comprising the lipid products are mixed either in miscible or immiscible phases, depending on the molecular shapes of the lipid components and crystallization. Recrystallization of primary crystal particles through Ostwald ripening, polymorphic crystallization, and transformation, as well as variations in the mixing behavior and successive crystallization of different lipid materials. Particle–particle interactions including sintering (Fig. 1.5c) may lead to the formation of crystal networks.

One must recall that lipid materials are produced in factory‐scale machines under external factors, which particularly affect the nucleation and crystal growth. Recrystallization proceeds during the aging period between factory‐scale production and storage in warehouses.

Introduction: Relationships of Structures, Properties, and Functionality

1.6 ­Aging and Deterioration The principal flows in the production of lipid materials may be simply drawn as in Fig. 1.7. Raw materials of lipids and water, salts, sugar, protein, starch, and emulsifiers are mixed and then melted or dissolved at elevated temperatures. They are then cooled or evaporated to cause lipid crystallization, which usually is conducted under stirring or shearing conditions for efficiency of heat exchange, emulsification, and aeration. After the dynamic production process has ceased, the lipid materials are kept in storage at optimal temperature ranges over certain periods (days to months) before releasing them into the markets. Throughout the processes shown in Fig. 1.7, including the commodity circulation of final products at the consumer end, the roles of physical and chemical control may be summarized as follows: sustaining the high value of raw materials, stabilizing the final products, and revealing the functionality of the products. From the viewpoint of stabilization, it is worthy to note that almost all lipid products are actually in thermodynamically metastable conditions when they retain highly functional properties. In contrast, conversions into thermodynamically stable conditions lead to degradation of the functionality, so this stabilization must be prohibited. The conversion proceeds in accordance with thermodynamic laws, so it is impossible to ultimately shut it off; but practical technology is used to retard the conversion as much as possible by proper methods. For example, chocolate is in a thermodynamically metastable condition because fine particles of ingredients (e.g., cacao mass, sugar, and others) are dispersed in the crystal networks of cocoa butter and other specialty fats (i.e., suspension). The metastability of chocolate is revealed in many aspects. Specifically, the fine particles of ingredients and fat crystals have sizes ranging from submicrons to several tens of microns, and the fats are not simple components but rather are mixtures of different fat components that differ in melting temperatures. Furthermore, the fat crystals in chocolate are in metastable polymorphic forms (e.g., Form V of cocoa butter and β’ of cocoa butter substitute [CBS]). Stabilization may lead to Ostwald ripening, which causes the growth of large particles at the expense of small particles (coarsening) during long‐period storage. Different fat fractions can separate when the mixtures of component fats are eutectic. Furthermore, polymorphic transformations from Form V to Form VI of cocoa butter or β’ to β of CBS may cause fat blooming. These degradations cause inferior mouth feel, loss of gloss, and so on. Another example is W/O emulsions like margarine and fat spread, which are in metastable states, as water droplets are forced to disperse in the semi‐solid fat phase with the aid of emulsifying reagents by applying a high shear force (emulsification). Also, the fats have multiple components with crystals in metastable β’ polymorph form. When the Processing Raw materials

Mix Melt Dissolve

Cool Stir Shear

Fig. 1.7  Flows of production of lipid materials.

Storage

Products

11

Crystallization of Lipids

Fig. 1.8  Aging and deterioration of lipid products. A and B are metastable states, C is the most stable state.

A Free Energy

12

Aging

B

Deterioration

C

Time

measures to sustain such metastable condition are not used, various degradations occur  (e.g., coalescence of water droplets, separation of oil and water phase in an extreme case, or granulation of fat crystals, which causes a loss of gloss, decrease in spreadability, etc.). In any case, metastability and stability during production, commodity circulation, and consumption stages vary from one product to another. However, one may consider the stabilization of lipid products in terms of the diagram of free energy and time given in Fig. 1.8. There are two stages of a metastable state, A and B. A is the stage between factory production and storage, and B is the stage between storage and circulation in the commodity. Both are metastable, but A is less stable than B. Optimal functionality of the products must be revealed in stage B. However, the most stable state is C, where degradation begins to occur because of stabilization. We may think that aging is the process of transforming from A to B, and that deterioration corresponds to the transformation from B to C. Technological innovation focuses on how to promote the aging and retard the deterioration. Therefore, the physical properties of lipid crystals affecting the microscopic and macroscopic mechanisms of aging and deterioration in lipid products should be clarified. In this respect, our particular concerns are focused on the kinetic processes involved in forming lipid crystal networks as revealed in the changes from A to B, which follow nucleation and crystal growth under various external factors noted in Fig. 1.5 (a and b). To summarize, the crystallization of lipids that occurs in the various production stages is quite dynamic (e.g., the time‐size scales differ greatly from one process to another, as shown in Table 1.2). For example, achieving the driving forces for crystallization (e.g. supercooling) may require seconds to minutes, depending on the size of the production system and the rate of cooling. Nucleation begins soon after the driving force is achieved, and the sizes of the crystal nuclei may be on the order of several to several tens of nanometers. Crystals may then grow in several to several tens of minutes. The formation of lipid crystal networks may take longer during aging, typically

Introduction: Relationships of Structures, Properties, and Functionality

Table 1.2  Time‐size scales of crystallization processes. Phenomena

Time

Size

Supercooling

Seconds–minutes

—

Nucleation

Minutes

~50 nm

Crystal growth

Minutes–hours

200 nm–1 µm

Aging

Hours–days

1 µm–10 µm

Deterioration

Weeks–months

20 µm–100 µm

hours to days, and deterioration such as fat bloom formation in chocolate occurs in weeks to months, depending on the temperature and its fluctuations during the distribution and consumption stages.

1.7 ­ Trans‐Fat Alternative and Saturated‐Fat Reduction Technology Trans‐fats can be defined as fats containing trans‐unsaturated fatty acids, in which the local conformation of hydrocarbon chains at the double bonds is trans‐type, as illustrated for elaidic acid in Fig. 1.1(d). Trans‐fats are rather uncommon in nature except for meat and dairy products from ruminants, but they are produced industrially as partially hydrogenated oils (PHOs) from liquid oils containing cis‐unsaturated fatty‐acid moieties. Trans‐fats have been used in margarine, snack foods, and confections since the 1950s. However, recent nutritional studies have claimed that, depending on the method and quantity of its intake, trans‐fat can be associated with increased risk of coronary heart disease. Therefore, reduction of trans‐fat has been a major concern of national and local governments, consumer organizations, and private enterprises (Kodali 2014; Wang et al. 2016). In addition, the intake of saturated fatty acids (SAFAs) and the reduction of SAFAs have also been a critical nutritional concern, although debates about SAFA‐related issues are still continuing. We do not intend to discuss the nutritional issues surrounding trans‐fat and SAFAs in this chapter, but we do take a closer look into the necessity to cope with the issues around trans‐fat and SAFAs from the viewpoint of lipid crystallization technology. There are rational reasons for the long‐time use of trans‐fat: it exhibits such high functionality when employed for edible applications, it is low cost, easily produced, β’‐ tending and β’‐stable crystals, fine crystal network formation, high rates of crystallization, sharp melting behavior, and high oxidation stability. Therefore, so‐called “trans‐fat alternative” technology must satisfy the following requirements. a) Functionality ●● Maintaining flakiness, firmness, crispiness, melting, and appearance ●● Stabilization of end products (e.g., anti‐oxidation) b) Availability ●● Easily available ●● Smooth processing

13

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Crystallization of Lipids

c) Economics ●● Not highly expensive Along these lines, typical examples of trans‐fat alternative technology are listed. a) Zero‐trans‐fat resources ●● High‐oleic sunflower, high‐oleic canola, high‐oleic soybean ●● Semi‐solid fats (palm, palm kernel, coconuts, fully‐hydrogenated fats) ●● Organogels (low‐molecular/macromolecular organogelators) b) Zero‐trans fat resources + processing ●● Molecular design (esterification) c) Zero‐trans fats + processing+ reduced‐SAFA ●● Blending of high‐oleic soybean/palm/fully‐hydrogenated fats ●● Algal oil (high oleic >90%) ●● Fractionation of palm oil, palm kernel oil, coconut oil, high‐oleic high‐stearic sunflower oil, etc. ●● Esterification (high‐oleic soybean + palm fraction, etc.) d) Efficient uses of additives ●● Emulsifiers Similarly to trans‐fat alternative technology, SAFA‐reduction technology will have to satisfy the following requirements. a) Maintaining functionality with reduced SAFA ●● Firmness and gloss ●● Melting and crystallization ●● Texture, rheology, and spreadability ●● Stabilization (water droplets, air cells) b) Production conditions ●● Availability ●● Economics ●● Minimum changes in processing c) Limitations ●● Hardness of chocolate ●● Softness of cookies Along these lines, typical examples of SAFA‐reduction technology are as follows. a) Increasing oleic‐acid moiety ●● High‐oleic sunflower, canola, and soybean ●● Oleic‐rich TAGs (POO, SOO, OPO, OSO, etc.) b) SAFA‐alternative materials ●● Organogels (see above) ●● Fat replacers ●● Increasing starch c) Modifying crystallization conditions ●● (See above.)

Introduction: Relationships of Structures, Properties, and Functionality

To summarize, we are confident that research on lipid crystallization will play a critically significant role in trans‐fat alternative and SAFA‐reduction technologies. Many chapters of this book share the same aims and desires.

References Kodali, D. R. (ed.) (2014) Trans Fats Replacement Solutions. Urbana, IL, AOCS Press. O’Keefe, S. F. (2008) Nomenclature and classification of lipids. In: Akoh, C. C., Min, B. D. (eds.) Food lipids: Chemistry, nutrition and biotechnology, 3rd ed. Boca Raton, FL, CRC Press, pp. 3–37 Wang, F. C., Gravelle, A. J., Blake, A. I., & Marangoni, A. G. (2016) Novel trans fat replacement strategies. Current Opinion in Food Science. 7, 27–34.

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2 Polymorphism of Lipid Crystals Kiyotaka Sato

2.1 ­Introduction Many lipid crystals possess two or more different structures, categorized as polymorphism and polytypism, under a given set of thermodynamic conditions. ­ The  polymorphic behavior of lipid crystals is basically determined by the molecular structure, thermodynamic stability, and phase transformation. This chapter begins with a basic discussion of the thermodynamic stability of polymorphic forms and their transformation behavior. The polymorphic structures and transformation of fatty acids and acylglycerols will then be discussed.

2.2 ­Thermal Behavior of Polymorphic Transformations The thermodynamic stability of polymorphic forms is illustrated by the relationship of their Gibbs energy values, G = H – TS, where H, S, and T are enthalpy, entropy, and temperature, respectively. One can usually determine the G‐T relationship by measuring the temperatures and enthalpy of the polymorphic transformation and the temperature variation of the solubility of the polymorphic forms. Polymorphic forms with greater G values are less stable than those with lower values, which have higher solubility values and lower melting points. The general behavior of polymorphic‐phase transformations of lipids can be discussed for two polymorphs, A and B, whose G‐T relationships are depicted in Fig. 2.1. Based on Fig.  2.1, possible pathways of crystallization and subsequent transformations during cooling and heating of the melt phase are revealed by the differential scanning calorimetry (DSC) cooling and heating patterns illustrated in Fig. 2.2. For simplicity, crystallization and transformation in the solution phase are not fully discussed except for special cases. a) Enantiotropic Polymorphism: Form A (B) is a high‐melting (low‐melting) polymorphs because the G value of A is smaller than that of B around the melting Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, First Edition. Edited by Kiyotaka Sato. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

Crystallization of Lipids

(a)

(b) A

A

Gibbs Energy

Gibbs Energy

L B

TA–B

L

B

Tm (A)

Tm (B) Tm (A)

Temperature

Tm (B)

Temperature

Fig. 2.1  Thermodynamic stability of two polymorphic forms of A and B, showing (a) enantiotropic and (b) monotropic behavior.

Cooling

Heating 1–1–1

Exothermic

18

1–1–2

1–1

1–1–3

1–2

1–2–1

TA–B Tm (B) Tm (A) Temperature

TA–B Tm (B) Tm (A) Temperature

Fig. 2.2  Hypothetical DSC cooling and heating patterns of two polymorphs of A and B grown from melt phase, revealing enantiotropic polymophism.

temperatures. The G values, however, are reversed in lower‐temperature ranges, making a cross point at TA‐B. 1) Case 1: No Solid‐State Transformation between A and B. Form A crystallizes by cooling below Tm(A) and remains kinetically stable in low‐temperature ranges where B is thermodynamically stable, then A melts at Tm(A), which is pattern 1‐1‐1. This apparently means “no polymorphism” because of steric hindrance prohibiting solid‐state transformation between A and B. However, B can occur in the solution phase because of solution‐mediated transformation.

Polymorphism of Lipid Crystals

2) Case 2 No Solid‐State Transformation from A to B below TA‐B, with Solid‐State Transformation from B to A below TA‐B. Two possibilities arise. First, A crystallizes by cooling below Tm(A) and transforms to B below TA‐B during exothermic heating, B then transforms to A at TA‐B with endothermic heating, and finally A melts at Tm (A), which is pattern 1‐1‐2. Second, A crystallizes by cooling below Tm (A) and transforms to B below TA‐B during exothermic heating. B then does not transform to A at TA‐B but melts at Tm (B), and A crystallizes soon after the melting of B (melt‐mediated transformation). Finally, A melts at Tm (A), which is pattern 1‐1‐3. 3) Case 3 Solid‐State Transformation between A and B at All Temperature Ranges. Two exothermic peaks (crystallization of A and transformation from A to B) are formed during cooling, solid‐state transformations between B and A occur around TA‐B, and A melts at Tm (A), which are patterns 1‐2 and 1‐2‐1. In this case, hysteresis phenomena may appear as “supercooling” and “superheating” for the solid‐state transformations from A to B below and from B to A above TA‐B, respectively, when serious steric hindrance retards the transformation. In this case, the dynamic molecular rearrangement of lipid molecules must be accompanied by the transformations. b) Monotropic Polymorphism: Form A (B) is a low‐melting (high‐melting) polymorph because the G value of A is always larger than that of B at all temperature ranges. In this type of polymorphism, the crystallization and transformation behavior largely depend on how crystallization occurs during the cooling process. As a general tendency, the metastable form A crystallizes at a very high rate of cooling, whereas the most stable form B crystallizes at a very low rate of cooling. Cooling at moderate rates causes concurrent crystallization of A and B. Fig. 2.3 illustrates various DSC

Exothermic

Cooling

Heating 2–1–1

2–1

2–1–2

2–2–1 2–2 2–2–2

2–3–1 2–3 Tm (A) Temperature

Tm (B)

Tm (A)

Tm (B)

Temperature

Fig. 2.3  Hypothetical DSC cooling and heating patterns of two polymorphs of A and B grown from melt phase, revealing monotropic polymophism.

19

20

Crystallization of Lipids

patterns of this type of polymorphism. Many acylglycerol crystals exhibit monotropic polymorphism, and their crystallization and transformation pathways can be recognized in the patterns in Fig. 2.3. 1) Case 1: Crystallization of Low‐Melting Form A. The DSC cooling pattern of the crystallization of form A is shown in pattern 2‐1. Two types of transformation may occur with heating, depending on whether or not the solid‐state transformation from A to B occurs. If there is no solid‐state transformation from A to B, A melts at Tm (A) soon after B crystallizes (melt‐mediated transformation) and B melts at Tm (B), which is pattern 2‐1‐1. By contrast, if A transforms to B above Tm (A) with exothermic heating, B melts at Tm (B), which is pattern 2‐1‐2. 2) Case 2: Concurrent Crystallization of A and B. Two exothermic DSC peaks appear, which is pattern 2‐2, and two types of transformation may occur on heating. A melt‐ mediated A–B transformation occurs at Tm (A) and B melts at Tm (B), when there is no solid‐state transformation from A to B, which is pattern 2‐2‐1. If A transforms to B above Tm (A) with exothermic heating, B melts at Tm (B), which is pattern 2‐2‐2. 3) Case 3: Crystallization of Low‐Melting Form A. A single exothermic peak and a single endothermic peak of crystallization and melting of A appear as seen in patterns 2‐3 and 2‐3‐1. It should be noted here that a single melting peak of B appears when the first‐crystallized A transforms to B by isothermal incubation below Tm (A) without explicit exothermic DSC peaks. In this case, cooling DSC patterns 2‐1 and 2‐2 may result in the heating DSC pattern of 2‐3‐1 when the isothermal stabilization is completed. The melt‐mediated transformation explained here has technological significance because the tempering process performed during fat crystallization in food engineering corresponds to a melt‐mediated transformation. Furthermore, a solution‐mediated transformation also occurs from A to B below Tm(A)—see Fig. 2.1(b)—even when steric hindrance prohibits a solid‐state transformation and the two forms are present in nearly saturated solution. This transformation has practical significance in the formation of granular crystals in fat crystal networks when minor low‐melting ingredients are melted at elevated temperatures and form an oil matrix in which fat crystals are embedded. Crystallization of a high‐melting (low‐solubility) polymorph can increase at the expense of low‐melting (high‐solubility) polymorph in the oil matrix. The driving forces for this type of solution‐mediated transformation are the differences in the solubilities of the two polymorphs. The solution (oil)‐mediated transformation may act as a partial role of fat‐bloom formation in cocoa butter–based chocolate (Sato & Koyano 2001).

2.3 ­Molecular Properties 2.3.1  Subcell and Chain‐Length Structures It is necessary for us to define the technical terms used to characterize the crystal ­structures that are applied to most lipid crystals. A unit cell is the smallest group of atoms or molecules comprising a crystal. Repetition of unit cells at regular intervals in three dimensions can construct the crystal lattices

Polymorphism of Lipid Crystals

of a material. The number of molecules included in a unit cell and the dimension of the unit cell are expressed by Z and lattice parameters (a, b, c, α, β and γ). An asymmetric unit is a part of a unit cell. We can construct a complete unit cell with an asymmetric unit using the symmetry of the space group. A crystal system refers to a class of space group that is divided into seven groups: triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal, and cubic. Actually, triclinic, monoclinic, orthorhombic, and hexagonal crystal systems often occur in lipid crystals. Having clarified the crystallographic definitions, let us define fundamental concepts about the molecular properties of lipid crystals by taking as an example the TAG (1, 3‐ dipalmitoyl‐2‐oleoyl‐glycerol [POP]) presented in Fig. 2.4. POP is one of the most complicated lipid substances because it is a saturated–unsaturated mixed‐acid TAG. Therefore, using POP as an example of lipids, we may discuss most of the important concepts about the molecular properties of lipid crystals. TAG molecules and other lipid crystals are often indicated by bars as symbols representing long‐chain molecules (Fig. 2.4a). Because of the strong interactions of the molecules along lateral directions compared with those along longitudinal directions, TAGs and other lipid crystals construct layered structures. This principle is applied to POP. The term lamella refers to laterally packed POP molecules. We can construct the entire structure of a POP crystal by stacking unit lamellae as depicted in Fig. 2.4(a). The thickness of the lamellae is revealed in X‐ray diffraction (XRD) long‐spacing patterns, which appear in small‐angle diffraction patterns using powder crystal samples. In the most stable polymorphic form of POP, a lamella is composed of POP molecules whose palmitic acid chains and oleic acid chains are placed in different lamellar planes (c)

Subcell

Leaflet

Palmitic

Oleic

Glycerol group

Palmitic

Double bond

Oxygen atom Carbon atom Hydrogen atom

Fig. 2.4  Definition of molecular structures of a triacylglycerol: POP.

Δ chain

lamella

(b)

Leaflet ω chain

(a)

21

22

Crystallization of Lipids

(Fig.  2.4b). This is because steric hindrance among straight palmitic acid chains and bent oleic acid chains does not allow them to lie within the same lamellar plane. To differentiate each fatty‐acid chain in the lamella, the term leaflet represents a sublayer composed of single fatty‐acid chains (Fig. 2.4c). The lamellar structure of POP depicted in Fig. 2.4(c) is composed of three leaflets. Glycerol groups are placed at the interior parts of the lamellae, and the interfaces among different lamellae are made of methyl end groups. Polar groups are placed in the interior part. In lipid crystals containing unsaturated fatty‐acid chains, the number, position, and conformation (cis or trans) of the double bonds are the primary factors that influence their physical and chemical properties. In an oleic acid leaflet of POP, a double bond having cis conformation is at the central part of the leaflet. The hydrocarbon chains of the oleic acid leaflet are divided into two segments by introducing the double bond. In the case of a mono‐unsaturated fatty acid like oleic acid, the chain segment between the CH3 group and the double bond is called the ω‐chain, and the other side between the double bond and polar groups (hydroxyl, carboxyl, and glycerol groups) is called the Δ‐chain, as noted in Fig. 2.4(c). Subcell structures are defined as the cross‐sectional packing mode of the zigzag hydrocarbon chains used to characterize the molecular packing, as illustrated in Fig.  2.4(c) and Fig.  2.5(a). More than nine types of subcell structures have been (a)

H

O′⊥

O⊥ bs

bs

as

as M//

T// bs

O// bs

bs as

as

(b)

Single

Inter digitated

(c)

Double

O′//

bs as

as

Triple

Quatro

Hexa

Lamellar interface

Fig. 2.5  (a) Subcell structures, (b) chain‐length structures, and (c) chain inclination of lipid crystals.

Polymorphism of Lipid Crystals

identified in lipids (Pascher et al. 1992; Dorset 2005), and the seven subcells depicted in Fig. 2.5(a) are often found in lipid crystals. Because the subcell structures refer to local arrangements of hydrocarbon chains, the symmetry of the subcell and the unit cell can be different. For example, an orthorhombic perpendicular subcell (O┴) and a triclinic parallel subcell (T//) can be formed in a crystal having a monoclinic crystal system. All of the hydrocarbon zigzag planes are parallel in T// and are thought to be the densest packing of aliphatic chains. The O┴ subcell consists of zigzag planes that are perpendicular to the planes of its neighbors. Two subcells, O’// and M//, contain parallel‐arranged hydrocarbon zigzag chains. There is also a hexagonal (H) subcell in which the hydrocarbon chains do not assume a specific orientation. Instead, they undergo torsional motion with an aliphatic gauche conformation, making the H subcell less stable than the others. There is diversified polymorphism in acyglycerol crystals, whose independent polymorphic forms are called α, β’, and β, based on the subcell structures (e.g., α, β’, and β possess H, O┴, and T//, respectively). The subcell structures are revealed in XRD short‐ spacing patterns, as indicated for triacylglycerol crystals in Fig. 2.6. In addition to α, β’, and β, sub‐α appeared in acyglycerols when α was cooled, having XRD short‐spacing peaks of 0.37–0.38 nm and 0.41–0.42 nm, as reported for tristearoy‐glycerol (SSS; Akita et  al. 2006), and 1, 3‐distearoyl‐2‐oleoyl‐glycerol (SOS) and 1, 3‐dioleoyl‐2‐stearoyl‐ glycerol (OSO; Yano et  al., 1999). A chain‐length structure comprises a repetitive sequence of the leaflets involved in unit lamella along the long‐chain axis, as seen in Fig. 2.5(b). This property is critically important for TAG crystals because a TAG may contain fatty‐acid moieties whose chemical structures largely differ from each other (e.g., long‐chain and short‐chain or saturated‐chain and unsaturated‐chain). A single chain‐length structure is a lamella composed of one leaflet. A double chain‐ length structure is formed when the chemical natures of the acyl chains are the same or (a)

α

0.415

β′

0.413

β

0.459

0.380

0.386

0.368

(b) 0.415

0.459 0.413 0.380

0.386

0.368

Fig. 2.6  (a) X‐ray diffraction short spacing patterns and (b) subcell structures of α, β’, and β poylmorphs of triacylglycerol crystals. Unit, nm.

23

24

Crystallization of Lipids

very similar. “Double” means that two leaflets are present within a unit lamella together with glycerol groups. A triple‐chain‐length structure having three leaflets is formed when the chemical nature of one or two of the three hydrocarbon chains largely differ from the others. The POP in Fig. 2.4(c) is a triple chain‐length structure. Higher‐ordered chain‐length structures of quarto‐chain‐length and hexa‐chain‐length can occur. One unique chain‐length structure is called interdigitated, in which methyl end groups and polar groups are alternatively placed in the same lamellar plane side by side. The interdigitated structure is found in the crystals of cis‐unsaturated fatty acid (oleic acid β form) and mixed‐acid TAGs (see below). The chain inclination is defined as the angle between the long‐chain axis and the lamellar interface (angle of gradient; see Fig. 2.5c). The chain inclination is often different among the polymorphic forms of the lipid as a result of the combined effects of the subcell structures and the methyl end stacking. When single‐crystal structure analysis cannot be done, the degree of the chain inclination can be assessed by dividing the XRD long‐spacing value by the total chain length. In TAG crystals, for example, the TAG molecules are arranged normal to the lamellar interface in α, whereas the angle of gradient becomes small in β’ and β. 2.3.2  Conformation of Hydrocarbon Chains The molecular packing of the lipids in crystals largely influences their physical properties (e.g., the density of crystals, melting point, and enthalpy and entropy of melting and crystallization, and thereby the rate of nucleation, etc.). The conformation of hydrocarbon chains is defined by a rotation angle (τ) as illustrated in Fig. 2.7. The rotation around the C‐C axis of CH2(1) and CH2(2) noted by a dotted arrow in Fig. 2.7(a) is defined by the angle of rotation (τ) of the C‐C bond between CH2(2) and CH2(B) with respect to the C‐C bond between CH2(A) and CH2(1). The conformations are defined as follows: cis (C) for τ = 0°, ±gauche (G and G’) for = ±60°, ±skew (S and S’) for = ±120° and trans (T) for =180°. The values of τ observed in real lipid crystals differ slightly from the standard values shown because of the bond rotation energy, which varies from one structure to another. In saturated fatty‐acid chains, the conformations of cis and ± skew are too unstable to occur under usual conditions. The conformation of ± gauche (trans) is metastable (stable). In the all trans conformations, the zigzag chain of carbon atoms makes a planar structure. The increase in gauche conformation, however, makes the hydrocarbon chains more flexible and disordered, causing the hydrocarbon chains to be nonplanar structures. Deviations from the planar trans conformation are indicated by defects appearing in the molecular chains. Fig. 2.8 illustrates how hydrocarbon chains are converted from planar to nonplanar conformations by (a) (b) A introducing gauche conformations. A (2) In cis‐unsaturated fatty‐acid chains, there are CH2 CH2 τ B different conformations of hydrocarbon chains CH2 CH2 adjacent to the double bond, expressed as  skew‐ (1) B 1, 2 cis‐skew’ (S‐C‐S’), skew‐cis‐skew (S‐C‐S), and trans‐cis‐trans (T‐C‐T; Fig.  2.9). The conformaFig. 2.7  (a) Hydrocarbon chains and tions of S‐C‐S’ and S‐C‐S are considered to be sta(b) rotation angle (τ) around C‐C bond ble. Although these two conformations cause the between CH2(1) and CH2(2)

Polymorphism of Lipid Crystals G

G G′ G′

G

T

(a)

(b)

(c)

(d)

Fig. 2.8  Molecular conformation defects in hydrocarbon chains. (a) All trans, (b) end gauche, (c) kink, and (d) double‐end gauche. Closed circles, carbon atoms.

skew-cis-skew′ (S-C-S′)

skew-cis-skew (S-C-S)

trans-cis-trans (T-C-T)

Fig. 2.9  Conformations of hydrocarbon chains adjacent to a cis double bond, viewing normal direction of hydrocarbon chains.

bent geometry of the hydrocarbon chain, there are clearly different forms in which the ω chain and Δ chains are placed in the same plane in the S‐C‐S’ conformation and normal to each other in the S‐C‐S conformation. The two conformations were widely observed in oleic acid, erucic acid, and other mono‐unsaturated fatty acids, whereas the T‐C‐T conformation is unique and was observed in the most stable polymorph of oleic acid (see below). 2.3.3  Glycerol Conformations The conformations of the glycerol groups in the crystals of acyglycerols are important in understanding the molecular arrangements, chain‐packing modes, and lateral interactions of lipids in biomembranes (Pascher 1996). In addition, one may assume that they are strongly related to the occurrence of different subcell structures in the acylglycerol crystals. To date, two types of glycerol conformations have been observed in TAG crystals: the “tuning fork” and the “chair” (Fig. 2.10). In the tuning fork conformation, hydrocarbon chains at the sn‐1 and sn‐3 positions connected to a glycerol group are packed in the same direction with respect to the glycerol group, and the hydrocarbon chain at the sn‐2 position is arranged in the opposite direction. The term tuning fork was coined by Jensen and Mabis (1963) when they

25

26

Crystallization of Lipids sn-3

sn-1

c3

c1

sn-3

c3

c2

c1

sn-2

sn-1 Tuning fork

c2

sn-2 Chair

Fig. 2.10  Conformations of glycerol groups (●, carbon atom; ○, oxygen atom; zigzag line, fatty‐acid moieties).

reported the crystal structure of TAG (the β form of tricapriroyl‐glycerol) for the first time. In the chair conformation, the hydrocarbon chains at the sn‐1 and sn‐2 positions are packed in the same leaflet, and the hydrocarbon chain at the sn‐3 position is arranged in the opposite direction. The direction of the C1‐C2‐C3 bond of a glycerol group is parallel to the lamellar interface in the tuning fork conformation, whereas it makes a right angle in the chair conformation. This indicates that there is no general rule about how two types of glycerol conformation will occur, though previous studies do indicate that mono‐acid TAGs and symmetric mixed‐acid TAGs exhibit the tuning fork conformation and that asymmetric mixed‐acid TAGs exhibit the chair conformation (see below).

2.3.4 Polytypism In addition to polymorphism, there is another higher‐order structural variation called polytypism, which is caused by a different stacking sequence of lipid layers. Fig. 2.11 illustrates two polytypes observed in fatty‐acid crystals having the polymorphic structure of a monoclinic system. In Mon, the layers having a monoclinic crystal system are simply stacked in such a way that the chain axes of the fatty‐acid molecules in the adjacent layers are arranged in the same direction. By contrast, the chain axes of the fatty‐acid molecules in the adjacent layers are rotated by 180 degrees along the normal to the lamellar interface in Orth II. The name of Orth II was given to differentiate it from the usually employed orthorhombic crystal system. Thus, the nomenclature of Orth II was given to the polytypic structures to distinguish them from polymorphic orthorhombic structures. Polytypic structures have been widely observed in the crystals of n‐alkanes and saturated and unsaturated fatty acids. Interestingly, Orth II can be more thermodynamically stable than Mon because of the stabilization of the lamellar interface. This property will be elaborated on in the next section. Fig. 2.11  Structure models of polytypism. An arrow means a unit repeating lamellae.

Mon

Orth II

Polymorphism of Lipid Crystals

2.4 ­Fatty Acids Here we discuss the polymorphic structures of the principal saturated and unsaturated fatty acids. Fatty acids are the main hydrophobic moieties of lipids present in biotissues and are also employed for multiple purposes in the food, pharmaceutical, and polymer industries (Chow 1992). Saturated fatty acids exhibit a straight chain configuration, allowing for melting points higher than those of unsaturated fatty acids with the same number of carbon atoms. The inclusion of a double bond reduces the melting point in an unsaturated fatty acid, depending on its conformation (cis or trans) and its position in the carbon chains. It also introduces multiplicity in molecular conformations of chain segments separated by a double bond, which results in the complex polymorphic structures of unsaturated fatty acids. The polymorphic properties of saturated and unsaturated fatty acids must affect the physical properties of lipid materials composed of fatty acids and their esters, but they also affect the physical properties of acylglycerols containing fatty acids as their hydrophobic moieties. 2.4.1  Saturated Fatty Acids Table 2.1 summarizes the polymorphic forms of saturated fatty acids with nc values of 12 through 18 (Kaneko 2001; Moreno et al. 2007). Triclinic (monoclinic) forms commonly occur in even‐(odd)‐numbered saturated fatty acids. In addition to polymorphism, polytypism was found for the monoclinic B and E forms of stearic acid and arachidic acid (C20; Kaneko 2001; Moreno et al. 2007). In this chapter, we refer to the polytypic structures as B (Mon), B (Orth II), E (Mon), and E (Orth II). 2.4.1.1  Crystal/Molecular Structures

Table  2.2 summarizes the lattice parameters of polymorphic and polytypic forms of selected saturated fatty acids. Fig. 2.12 portrays three different types of triclinic crystals of lauric acid and myristic acid. The peculiar properties of the three triclinic structures are revealed in their lamellar interfaces, called segregated and nonsegregated layer structures (Kaneko 2001). In the segregated layer, the (COOH)2 terminals and the CH3 terminals are separated from each other, whereas the (COOH)2 terminals and the CH3 terminals are placed on the same lamellar interface, neighboring each other. The A1 and A‐super forms of lauric Table 2.1  Polymorphism in major saturated fatty acids. nc

Even‐Numbered

nc

Odd‐Numbered

12

A1(t), A‐super(t), C(m)

13

A’(t), C’(t)

14

A2(t), B(m), C(m)

15

A’(t), B’(t), C’(m)

16

A2 (t), B(m), C(m), E(m)

17

A’(t), B’(t), C’(m)

18

A(t), B(m), C(m), E(m)

m, monoclinic; nc, number of hydrocarbon atoms; t, triclinic.

27

28

Crystallization of Lipids

Table 2.2  Lattice parameters of polymorphic and polytypic forms of selected saturated and unsaturated fatty acids. Fatty Acid: Form

Space Group Z a (nm)

b (nm)

c (nm) α (deg.) β (deg.) γ (deg.)

Lauric acid: A1

p1

2 0.745

0.540

1.747

96.53

113.8

81.7

Lauric acid: A‐super

A1

6 0.5415 2.596

3.518

69.82

113.14

121.15

Lauric acid: C

P21/a

4 0.9524 0.4966 3.539

90

129.21

Myristic acid: A2

P1

2 0.7377 0.5457 3.794

Stearic acid: B(Mon)

P21/a

4 0.5587 0.7386 4.933

90

Stearic acid: B(Orth II)

Pbca

8 0.7404 0.5591 8.766

90

Stearic acid: E(Mon)

P21/a

4 0.5603 0.7360 5.0789

90

105.60

93.49 117.24 90 119.40

83.96 90 90 90

Stearic acid: E(Orth II)

Pbca

8 0.7359 0.5609 8.841

90

Stearic acid: C

P21/a

4 0.936

90

128.24

90

Erucic acid: γ

P21/a

4 0.9564 0.4683 4.949

90

93.65

90

Oleic acid: β1

P1

4 0.9317 0.5543 3.5284

87.90

82.82

86.18

0.495

5.07

90

90

90

Petroselinic acid: LM(Orth II) Pbca

8 0.7311 0.5565 8.801

90

90

90

Elaidic acid

8 9.848

90

92.57

90

C2/c

(a)

0.4938 0.7182

(b)

(c) a

c

c/2

c

a a

Fig. 2.12  Crystal structures of (a) A1 form of lauric acid, (b) A‐super form of lauric acid, and (c) A2 form of myristic acid. Closed circle, carbon atom; hydrogen atom is not shown; open circle, oxygen atom.

acid, both of which have an interdigitated structure, form the nonsegregated layer structures. The subcell structure of the A1 form of lauric acid is T// and the conformation of the hydrocarbon chains are all trans (Fig. 2.12a). It is worth noting that the c‐axis of a unit cell corresponds to a one‐molecule length. The A‐super form of lauric acid also has a T// subcell and an all‐trans conformation (Fig. 2.12b), but it is a modified structure of A1 in which three lauric acid molecules (indicated by arrows in Fig. 2.12b) comprise the a‐axis, as a superlattice. A segregated‐layer triclinic structure is formed in the A2 form of myristic acid (Fig. 2.12c), in which the length of the c‐axis equals that of the dimer of myristic acid. In this case, the chain‐length structure is double. The occurrence of A2 in palmitic and stearic acid and of A‐super in myristic and palmitic acid was confirmed (Moreno et al. 2007).

Polymorphism of Lipid Crystals

(c)

(b)

(a)

c

c

a

c

a

a

Fig. 2.13  Crystal structures of (a) C form, (b) B form, and (c) E form of stearic acid. Closed circle, carbon atom; hydrogen atom is not shown; open circle, oxygen atom.

Crystal structural analyses of stearic acid polymorphs using single crystals have been performed for C (Malta et al. 1971), B (Mon; Goto & Asada 1978), and E (Mon; Kaneko et al. 1990; Fig. 2.13). Forms C and E contain straight hydrocarbon chains, yet the gauche conformation exists near the COOH groups in B, indicated by an arrow in Fig. 2.12(b). The three forms have an O┴ subcell, and the molecular volume in the crystal is greatest for C, smallest for B, and between those values for E. The crystal structures of B (Orth II; Kaneko et al. 1994a) and E (Orth II) (Kaneko et al. 1994b) were determined to have the higher‐order structures illustrated in (a) Fig. 2.11. O2 C12 The odd‐numbered saturated fatty acids have three forms (A’, B’, and C’). The subO1 cell structures are T// in A’, whereas O┴ O3 occurs in B’ and C’. 12‐hydroxystearic acid (12‐HSA, (b) Fig. 2.14a) has been used as an organogel‐ O3 forming compound (see Chapter  12). As O3 12‐HSA is optically active, chiral 12‐HSA and racemic 12‐HSA are formed. It has been known that solution‐grown chiral 12‐HSA crystals and racemic DL‐12‐HSA crystals exhibit a twisted fiber and plate‐like shape, respectively (Tachibana & Kambara a 1969; Uzu & Sugiura 1975). A recent study showed that chiral 12‐HSA crystals formed a gel, whereas DL‐12‐HSA crystals did not b form any gels in organic solution (Sakurai et al. 2010). Fig. 2.14  (a) Molecular shape of 12‐ Solution‐grown racemic DL‐12‐HSA hydroxystearic acid (12‐HSA) and (b) a crystals were subjected to single‐crystal schematic model of the c‐plane projection of X‐ray structure analysis (Kuwahara et  al. c‐c plane and hydrogen bonding (dotted lines).

29

30

Crystallization of Lipids

1996). The structure data for the monoclinic space group of P21/a of racemic DL‐12‐ HSA crystal are a = 0.5488 nm, b = 0.7831 nm, c = 4,424 nm, β = 93.64°, Z = 4. Fig. 2.14(b) illustrates the packing of DL‐12‐HSA without H atoms viewed nearly along the acyl chain, which has an approximately all‐trans conformation with a subcell structure. The molecular structure of DL‐12‐HSA is similar to that of the E form of stearic acid. However, the torsion angles between carbon atoms in the aliphatic chains of stearic acid E are all 180 degrees, resulting in a straight chain, whereas the torsion angle between C10‐C11‐C12‐C13 of DL‐12‐HAS is 173 degrees. This is because such bending in 12‐HSA is needed to fill out the space expanded by the 12‐hydroxyl group. The hydrogen bonding sequences formed by the 12‐hydroxy groups extend zigzag along the a‐axis as illustrated in Fig. 2.14(b). 2.4.1.2  Polymorphic Behavior

Only the C form crystallized from the melt phase at ambient cooling rates for the all‐ even‐numbered saturated fatty acids. However, crystallization of the A, B, C, and E forms of stearic acid from organic solution phases was observed (Sato & Boistelle 1984; Kaneko et al. 1992). Their crystallization behavior largely depends on the crystallization temperature (Tc) and the cooling rate. In general, the C form occurred at high Tc and high cooling rates, whereas the A, B, and E forms crystallized at low Tc and low cooling rates. On heating, all forms exhibited a solid‐state transformation to C (Moreno et al. 2007). Recently, high supersaturation favored the crystallization of E over C when the crystallization of stearic acid was examined by depressurization of an expanded liquid organic solution (DELOS) and gas antisolvent (GAS) crystallization techniques, both based on CO2‐expanded solvents as solvent media (Sala et al. 2010). The relationships involved in the thermodynamic stability of C, E(Mon), E(Orth II), B(Mon), and B(Orth II) of stearic acid were experimentally determined. Fig. 2.15 schematically illustrates the Gibbs energy (G) of the five forms as a function of temperature. The relative stability of the triclinic A form was not determined precisely, although it frequently crystallized from the solution phase. The crystallization and transformation behavior of these forms are summarized in the following.

C E (Mon)

Liquid

E (Orth II) B (Mon) G

B (Orth II)

23 32

69

Temperature (° C)

Fig. 2.15  Thermodynamic stability relationships of polymorphic/polytypic structures of stearic acid.

Polymorphism of Lipid Crystals

No solid‐state transformation occurred from C to B or E on cooling at any temperature range. However, solid‐state transformations from B (two polytypes) to C, from A to C, and from E (two polytypes) to C occur upon heating and have been studied on a molecular level. B(Orth II) is thermodynamically more stable than B(Mon), as determined by careful solubility measurements of B(Orth II) and B(Mon; Sato et al. 1988). The solubilities of B(Orth II) and C cross at 32 ° C, whereas those of B(Mon) and C cross at 23 ° C. This result is explained by the contribution of the free energy of the lattice vibration longitudinal to the lamella interface, which lowers the total Gibbs energy of B(Orth II) compared to B(Mon; Kobayashi et  al. 1984). Quite similar results were observed for two polytypes of monoclinic‐polymorph of n‐hexatriacontane (n‐C36H74). Orth II‐polytype is more stable than Mon‐polytype, as confirmed by solubility and inelastic neutron‐ scattering measurements (Kubota et al. 2005). It is interesting to note that the solid‐state transformations from B to C occurred above the crossing points of the G values (superheating). For example, B(Mon) and B(Orth II) transformed to C in the solid state at 40–50 ° C, higher than the crossing points of the G values (23 ° C and 32 ° C). This “overheating” is caused by steric hindrance against the dynamic molecular rearrangements involved in B‐C transformations, in which polytypic‐polymorphic transformations of B(Orth II)–C and B(Mon)–C of stearic acid occur at the same time. Two types of molecular rearrangements occur: conformational changes from gauche (B form) to trans (C form) near the COOH group, and chain inclination because C is more tilted than B with respect to the lamellar plane (see Table 2.2). Fig. 2.16(a) depicts an alternative rotation of stearic acid molecules that occurred in adjacent lamellae around the c‐axis of B(Orth II) during the B(Orth II)–C transformation, keeping the subcell arrangements unchanged. In contrast, displacements during the B(Mon)–C transformation are caused by a collective inclination of the molecules within the lamellar plane followed by deformation of the subcell of the aliphatic chain, keeping the symmetry axis (c‐axis) unchanged (Larsson et  al. 2006). (a)

(b)

as cs

bs

as cs

bs

cs bs

c

c

bs

as

cs

c

a

b

B(Orth II)

C

c

b

b B(Mon)

as

C

Fig. 2.16  Molecular rearrangements in solid‐state transformations of (a) B(Orth II) → C and (b) B(Mon) → C.

31

32

Crystallization of Lipids

Polymorphic/polytypic forms were also observed in n‐alkane crystals, and precise studies were performed by vibrational spectroscopy with a focus on their formation conditions, thermodynamic stability, and molecular motion (Kaneko & Kubota 2011). The solid‐state transformation from E to B revealed unique behavior, as examined with infrared (IR) oblique transmission (Kaneko et al. 1992). The crystal structures of the two polymorphs are quite similar except for local hydrocarbon conformations close to COOH (see Table 2.2 and Fig. 2.12). Therefore, the crystal habit and transparency of single crystals and the polarization in the infrared bands because of the CH2 rocking and scissoring modes did not change during the transition, indicating that the E‐B transformation is achieved through small structural changes occurring in localized parts of the crystals of E. It was clarified that the E‐B transformation occurred through heterogeneous nucleation of B on the (001) faces of single crystals of E at the first stage, and subsequent solid‐state transformation including reorientation of dimerized carboxyl groups accompanied by conformational change from trans to gauche at the C2‐C3 bond, with most of acyl chain maintaining its original orientation. Such a transformation including nucleation‐growth processes can be called topotaxy, which is widely observed in organic, inorganic, and mineral crystals (Figlarz et al. 1990; Bowes et al. 1996). The E‐B transformation was the first case in lipid crystals. An important question about the polymorph/polytype structures of stearic acid remains unanswered: why E and B exhibit two polytypes, but C does not, despite the fact that B(Mon) and E(Mon) and C are monoclinic with the same space group of P21/a and the same O┴ subcell. The answer may lie in the interactions between the chain tilting and methyl end stacking structure, and this problem may deserve to be explored in the future. 2.4.2  Unsaturated Fatty Acids The polymorphism of unsaturated fatty acids differs significantly from that of saturated fatty acids in terms of their crystal/molecular structures and transformation behavior (Kaneko et  al. 1998). Tables  2.3 and 2.4 summarize the structures and polymorphic transformations of selected unsaturated fatty acids in which the crystal structures were determined by single‐crystal XRD analyses (Kaneko 2001). We discuss here the overall characteristics of the polymorphic behavior of the principal unsaturated fatty acids. 2.4.2.1  Crystal/Molecular Structures

Table 2.2 lists the lattice parameters of the polymorphic forms of selected unsaturated fatty acids. The major properties of the crystal structures of monounsaturated fatty acids can be summarized as follows. a) The COOH groups and the double bonds of neighboring molecules are located in the same plane normal to the chain axes, as indicated in the γ form of erucic acid (Fig. 2.17a). The intermolecular polar and π–π interactions may be stabilized by this arrangement. Basically, the same structures were observed in the α and γ forms of oleic acid, the LM form of petroselinic acid, the LM form of elaidic acid, and the α, α1, and γ1 forms of erucic acid. One exception was the β1 form of oleic acid, which has an interdigitated chain‐length structure made of two independent molecules in

Polymorphism of Lipid Crystals

Table 2.3  Molecular properties of polymorphism in unsaturated fatty acids. Subcell Fatty acid

Form

Oleic acid (18:1, Δ9‐ω9)

γ α β2 β1

Petroselinic aid (18:1, Δ6‐ω12)

LM

Elaidic acid (18:1, Δ9‐ω9)

LM

Erucic acid (22:1, 13‐ω9)

γ α α1 γ1

Local Conformation Near Double Bond*,†

Δ chain

ω chain

S‐C‐S’ S‐C‐T Unclear A:(174°,cis,173°) (T‐C‐T) B:(175°,cis,175°) (T‐C‐T)

O’// O’// //‐type T//

O’// O⊥‐like //‐type T//

157°, C, –160°

O⊥

O⊥

S‐T‐S’

O⊥

O⊥

S‐C‐S’ S‐C‐T S‐C‐S’ S‐C‐S

O’// O’// T// T//

O’// O⊥‐like T// T//

* S‐C‐S, skew‐cis‐skew; S‐C‐S’, skew‐cis‐skew’; S‐C‐T, skew‐cis‐trans ; S‐T‐S’, skew‐trans‐skew’; T‐C‐T, tans‐cis‐trans. †  Rotation angles are noted

Table 2.4  Subcell parameters of oleic acid β1 form (unit, nm) Subcell Parameter

Subcell 1

Subcell 2

as

4.09

4.18

bs

5.36

5.54

cs

2.54

2.54

α (deg.)

81.4

72.2

β (deg.)

106.5

109.7

γ (deg.)

120.8

123.5

an asymmetric unit (Fig. 2.17b; Kaneko et al., 1997b). The interdigitated structure is formed in such a way that the methyl group of molecule A and the carboxyl group of molecule B (or vice versa) are located in the same plane. This stacking mode is unusual because fatty acids usually form a separated lamellar structure in which the methyl terminals are segregated from the carboxyl groups, except for the triclinic A1 and A‐super forms (Fig. 2.12a,b). b) Parallel‐type subcell structures are found in the γ and β1 forms of oleic acid and the γ, α1, and γ1 forms of erucic acid. However, O┴ was found in the LM forms of petroselinic acid and elaidic acid (trans‐unsaturated acid). In the α forms of oleic acid and erucic acid, the Δ chain is O’//, but the ω chain is O┴‐like. The β1 form of oleic acid has two subcells of T//, indicated as 1 and 2 in Fig. 2.17(b). Subcell 1 is formed of the ω chain of molecule A and the Δ chain of molecule B, and subcell 2 is formed

33

34

Crystallization of Lipids

(a)

(b) A

B

Subcell 1 c/2 Subcell 2

b

a

c

a

Fig. 2.17  Crystal structures of (a) erucic acid g form and (b) oleic acid b1 form. Closed circle, carbon atom; hydrogen atom is not shown; open circle, oxygen atom.

of the ω chain of molecule B and the Δ chain of molecule A. The lattice parameters of two subcells in oleic acid β1 are listed in Table 2.4. c) The olefinic conformations of S‐C‐S, S‐C‐S’ and S‐C‐T were observed in many cis‐ monounsaturated fatty acids, except for T‐C‐T in the β1 form of oleic acid. d) The formation of an interdigitated lamellar structure of oleic acid β1 can be explained as follows. The number of carbon atoms of the ω and Δ chains of oleic acid is same. If the lengths of the ω and Δ chains differ, as in erucic acid and petroselinic acid, the interdigitated structure should form a void at either methyl end. A void formed at the carboxyl terminals may cause excess energy for the formation of hydrogen bonding, and this structure should not form, except for oleic acid. However, the mechanisms involving the unique molecular structures of oleic acid β1 such as the interdigitated chain length, the T‐C‐T olefinic conformation, and the two subcells have not been fully identified. e) Although petroselinic and oleic acids are structural isomers of C18‐cis‐monounsaturated fatty acid, the polymorphic structures of petroselinic acid are different from those of oleic acid, but quite similar to stearic acid in terms of subcell structure (O┴) and the occurrence of Orh II and Mon polytypes in the LM polymorph (Kaneko et al. 1997a). This difference has not been clearly explained, but one possibility may be the effect of methyl end stacking caused by even‐numbered ω chains of petroselinic acid, which may resemble those of stearic acid. f ) The linear chain structure of the trans‐double bond of elaidic acid is quite similar to that of the stearic acid E form (Low et al. 2005). There have been no explanations as to why and how cis‐unsaturated fatty acids exhibit the extensive diversity in molecular structures shown here. One factor must be the introduction of a cis‐double bond, whose position at the aliphatic chain may modify three major stabilization factors of the crystal structures of fatty acids: aliphatic chain‐chain interaction, the methyl end‐stacking mode, and polar interactions at COOH groups.

Polymorphism of Lipid Crystals

Table 2.5  Thermodynamic and structural properties in polymorphic transformations of unsaturated fatty acids. ΔHtr

Fatty Acids

Form

Transformation

Oleic acid

γ

→α

–2.2

8.8

α

→liquid

13.3

39.6

β2

→liquid

16.0

48.9

β1

→liquid

Asclepic acid (18:1, Δ11‐ω7)

γ

→α

α

→liquid

Petroselinic acid

LM

→HM

Palmitoleic acid (16:1, Δ9‐ω7) Erucic acid

Linoleic acid

α‐linolenic acid

Ttr

16.3

57.9

–15.4

7.8

13.8

39.8

—

—

→liquid

28.5

—

HM

→liquid

30.5

47.5

γ

→α

α

→liquid

γ

→α

–1.0

8.8

α

→α1

31.3

5.4

γ1

→α1

α1

→liquid

LT MT

–18.4

7.5

2.0

32.1

9.0

8.9

34.0

54.0

→MT

–51.3

2.6

→HT

–35.4

0.27

HT

→liquid

LT

→HT

–60.2

HT

–7.2

33.6 0.11

→liquid

–13.0

27.8

Conjugated linoleic acid (9‐cis, 11‐trans)

→liquid

14.9

38.7

Conjugated linoleic acid (10‐trans, 12‐cis)

→liquid

19.8

35.6

Ttr, transformation temperature (°C); ΔHtr, enthalpy of transformation (kJ/mol).

2.4.2.2  Polymorphic Behavior

As indicated in Table  2.5, the polymorphic transformation pathway varies from one acid to another in a quite diversified manner. Even so, some common features appear among cis‐monounsaturated fatty acids. To discuss this, we choose oleic acid and petroselinic acid as representatives of cis‐mono‐unsaturated fatty acids. Compared to transformations occurring during the heating process, the crystallization of the cis‐ mono‐unsaturated fatty acids during the melt and solution phases was tremendously diversified because of kinetic effects on the occurrence of multiple polymorphic forms, and therefore, we do not discuss it here. Fig. 2.18(a) illustrates the Gibbs energy relationships of four polymorphic forms of oleic acid. The α and γ forms exhibit a reversible transformation of an enantiotropic nature (Suzuki et al. 1985). A study using vibrational spectroscopic and thermal techniques showed that the ω chain exhibits conformational transformation from all‐trans ordered (γ) to gauche‐rich disordered (α) upon heating, like “partial melting,” whereas

35

36

Crystallization of Lipids

(a)

(b) γ β2

G

Liquid

α

Liquid

β1

–10

G

0 Temperature (° C)

10

20

LM HM

0

10

20

30

Temperature (° C)

Fig. 2.18  Thermodynamic stability relationships of polymorphic forms of (a) oleic acid and (b) petroselinic acid.

the ordered conformation of the Δ chain did not change (Kobayashi et al. 1986). This transformation changed the subcell structure of the ω chain from O’// to O┴‐like and the local conformation near the double bond from S‐C‐S’ to S‐C‐T, while the subcell structure of O’// of the Δ chain is unchanged (Table  2.3). The same transformation was observed in erucic acid (Kaneko et al. 1996), asclepic acid (Yoshimoto et al. 1991), palmitoleic acid (Hiramatsu et  al. 1990), and gondoic acid (Sato et  al. 1997). One may conclude that partial melting of the ω chain is a typical structural characteristic in cis‐ mono‐unsaturated fatty acids caused by the introduction of the cis‐double bond. In Fig. 2.18(a), two β forms of oleic acid had a monotropic nature because no solid‐ state transformation occurred among α, β2, and β1, and the three forms melted independently. Fig. 2.18(b) depicts the thermodynamic stability relation of two polymorphs of petroselinic acid (Sato et al. 1990). The LM and HM forms crystallized at the same time when cooled from the melt phase, and the ratio of the occurrence of the two forms depended on the cooling rate. No transformation from HM to LM occurred below 18.7 °  C (supercooling) and the transformation from LM to HM was kinetically hindered (superheating). Therefore, the crossing point of the G values of the two forms, 18.7 ° C, was determined by measuring the solubility values of the two forms at different temperatures (Sato et al. 1990). Above 18.7 ° C, two unique transformations occurred from LM to HM. The first one is a melt‐mediated transformation from LM to HM at the melting temperature of LM, 28.5 ° C. The other one is a martensitic transformation from LM(Mon) to HM, in which a molecular jump and cooperative displacement of petroselinic acid occurred when mechanical stress was applied to the LM crystal when it was rapidly heated to above 28.5 ° C. This transformation did not occur from LM(Orth II) to HM because the molecular motions from the double‐layered polytype of LM to HM are too large to cause collective displacements for the martensitic transformation (Kaneko et al. 1997a). The polymorphic behavior of linoleic acid and α‐linolenic acid is summarized in Table 2.5. The transformation temperatures and enthalpy values of the transformation of LT(→MT) → HT, and HT → liquid decreased with an increase in the number of cis‐ double bonds. An XRD study indicated that the HT and LT forms of the two acids exhibited the same diffraction patterns as those of the γ and α forms of oleic acid.

Polymorphism of Lipid Crystals

Therefore, it can be concluded that the order‐disorder transformation observed in many cis‐mono‐unsaturated fatty acids also occurs in polyunsaturated fatty acids (Ueno et al. 2000). A temperature‐dependent Fourier transform‐infrared (FT‐IR) study showed that structure changes in the LT → MT transition of linoleic acid were rather localized around the cis‐diene group, while they occurred in the larger portion between the cis‐ olefin group and the methyl end in the γ‐α transformation of oleic acid (Pi et al. 2011). Conjugated linoleic acid (CLA) occurs naturally and can be industrially manufactured by alkali‐induced conjugation of linoleic acid‐rich oils in the presence of propylene glycol. After conjugation, one can obtain a mixture consisting of almost equivalent amounts of the isomers of 9‐cis,11‐trans‐CLA (c9t11), and 10‐trans, 12‐cis‐CLA (t10c12), which have two double bonds (cis and trans) at different positions on the aliphatic chains. A study of the crystallization of two CLA isomers from melt and solvent phases showed that there is no polymorphism in either acid, and that the 9c11t isomer has a higher melting temperature than the 10t12c isomer (Table 2.5; Uehara et al. 2008). XRD and FT‐IR measurements indicated O┴ subcell packing in the crystals of c9t11 and t10c12, and long‐spacing values of 4.22 nm for c9t11 and 3.88 nm for t10c12.

2.5 ­Monoacylglycerols and Diacylglycerols Monoacylglycerols (MAGs) and diacylglycerols (DAGs) are polar lipids that form crystals in lamellar structures in which glycerol groups connected side‐by‐side by hydrogen bonding are placed at the inner positions. All MAGs are amphiphilic and easily hydrated in the presence of water, exhibiting quite diversified lyotropic liquid crystalline phases with different amounts of excess water at different temperatures (Kulkarni 2012). This section, however, does not discuss lyotropic liquid crystals; instead. the polymorphic behavior of distilled MAGs will be discussed. The precise molecular structures and transformation behavior of MAGs and DAGs depend on the fatty‐acid moieties and the sn‐positions of the glycerol carbon atoms at which the fatty acids are esterified. 2.5.1  Crystal/Molecular Structures The crystal structures of selected MAGs and DAGs are summarized in Table 2.6. The precise crystal structure of the orthorhombic β1’ form of 1‐monostearoyl‐sn‐glycerol is presented in Fig. 2.19(a) (Goto et al., 1988). Two independent molecules, A and B, are present in an asymmetric unit, with different glycerol conformations for A and B. Although the unit cell is orthorhombic, the stearoyl chains are inclined with respect to Table 2.6  Lattice parameters of monoacylglycerol and diacylglycerol crystals. Forms

Space Group Z a (nm) b (nm) c (nm) α (deg.) β (deg.) γ (deg.)

1‐ monostearoyl‐sn‐glycerol β1’ P212121

8 0.4943 0.8585 10.105 90

90

90

1‐ monolauroyl‐rac‐glycerol β1 P21

8 0.9247 7.431

0.4952 90

97.51

90

1, 2‐dilauroyl‐sn‐glycerol

2 0.546

3.42

90

93.1

90

90

91.46

90

P21

1‐stearoyl‐3‐oleoyl‐sn‐glycerol Cc

0.759

4 0.9362 0.5495 7.792

37

38

Crystallization of Lipids

the lamellar interface. The intermolecular hydrogen bonding is formed in a zigA A1 B1 zag manner connecting the two glycerol groups, parallel to the lamellar plane. The subcell structure is O┴. Fig. 2.19(b) illustrates the crystal strucB ture of the β1 form of 1‐monolauroyl‐ rac‐glycerol (Goto & Takiguchi 1985). b/2 Because of the formation of racemic compound crystals, an asymmetric unit c/2 contains four independent molecules A1, A2, B1, and B2, in which A1/A2 and B1/B2 are mirror‐image isomers. Hydrogen bonding is formed among the glycerol groups, making “bridges” between –OH A2 B2 A groups connecting ‐A2‐B1‐A1‐B2‐. The a size of the polar group is larger than that of enantiomer crystals because of racemic compound formation as seen in the B lattice parameters (Table 2.6). The subb cell structure is M//. Fig.  2.20(a) illustrates the crystal Fig. 2.19  Crystal structures of s ­ tructure of 1, 2‐dilauroyl‐sn‐glycerol (a) 1‐monostearoyl‐sn‐glycerol β1’ form and (b) 1‐monolauroyl‐rac‐glycerol β1. Closed circle, (sn‐1,2‐LL‐DAG) of double‐chain‐length carbon atom; hydrogen atom is not shown; structure (Pascher & Sundell 1981). Two open circle, oxygen atom. lauric acid chains are packed in the same leaflet of the double‐chain‐length structure, resulting in a chain inclination with respect to the lamellar interface of 53.5 degrees. The subcell structure is O┴ and the crystal system is monoclinic (Table 2.6). (a)

(b)

(a)

(b)

2

c

1 3

c

3 2

b

1

b

Fig. 2.20  Crystal structures of (a) 1, 2‐dilauroyl‐sn‐glycerol and (b) 1‐stearoyl‐3‐oleoyl‐sn‐glycerol (sn‐1, 3‐SODG). Closed circle, carbon atom; dotted lines, hydrogen bonds; hydrogen atom is not shown; open circle, oxygen atom.

Polymorphism of Lipid Crystals

The important property is that the lauric acid chain at the sn‐2 position is directed normal to the lamellar plane, parallel to the lauric acid chain at the sn‐1 position. This arrangement is enabled by forming the trans conformation of C1‐C2‐C3‐O in the glycerol group. Hydrogen bonding occurs among neighboring DAGs between C3‐O of a glycerol and C = O of the sn‐1 chain, parallel to the lamellar plane. No hydrogen bonding is formed among the DAGs packed in different leaflets along the direction normal to  the lamellar plane. The crystal structure of 1, 2‐dipalmitoyl‐sn‐glycerol ((sn‐1,2‐ PP‐DAG) is essentially identical to that of 1, 2‐dilauroyl‐sn‐glycerol (Dorset & Pangborn 1988). In contrast, the stearoyl and oleoyl chains were packed in separate leaflets of the ­double‐chain‐length structure in the stable form of 1‐stearoyl‐3‐oleoyl‐sn‐glycerol (sn‐1,3‐SO‐DAG) as seen in Fig. 2.20(b) (Goto et al. 1995). The molecules formed an extended V‐shaped conformation with the oleoyl and stearoyl chains coming off the two ends of the glycerol group with an angle of 94 degrees between their planes. The stearoyl chain is roughly straight and packed in the T// subcell. The ω chain and Δ chain of the oleoyl leaflet are also packed in the T// subcell. The combination of the T// subcell and the trans‐skew‐cis‐skew‐skew‐trans (T‐S‐C‐S‐S‐T) conformation at the oleoyl carbons of C7 to C13 in the oleic acid leaflet is of particular interest. Hydrogen bonding is formed between the oxygen atom of the glycerol sn‐2 carbon and the ‐C=O group of the sn‐3 oleoyl chain. This hydrogen bonding stabilizes the glycerol layers. It should be noted that the overall molecular conformations of sn‐1,2‐LL‐DAG and sn‐1,3‐SO‐DAG have significantly different arrangements of the two fatty‐acid chains and glycerol groups, the direction of hydrogen bonding, the subcell structure, and the chain inclination. A problem arises: what are the main approaches to distinguishing the different arrangements of the two fatty‐acid chains segregated in different leaflets or packed side‐by‐side in the same leaflet? Whether the two chains are saturated acids or a mixture of saturated and unsaturated acids may not be the key. This is because 1, 2‐ stearoyl‐oleoyl‐sn‐glycerol (1, 2‐sn‐SO‐DAG; Di & Small 1993) and 1, 3‐sn‐DAG containing thiadecanoic acid (Larsson 1963) exhibit the same structures as those of 1, 2‐sn‐LL‐DAG and 1, 3‐sn‐SO‐DAG, respectively. It can be assumed that the glycerol conformation driven by hydrogen bond formation may determine whether the two fatty‐ acid chains are packed together in the same leaflet or segregated in different leaflets. Interesting properties were observed in the crystal structures of three types of saturated‐unsaturated mixed‐acid DAGs: sn‐1,2‐SO‐DAG (Di & Small 1993), sn‐1, 3‐SO‐ DAG (Goto et al. 1995), and 1‐stearoyl‐2‐ linoleoyl‐ sn‐glycerols (sn‐1, 2‐SLi‐DAG; Di & Small 1995). Molecular structures of DAGs composed of one saturated and one unsaturated chain make up the hydrophobic core of many biological membranes. Therefore, the interactions of the acyl chains in the membrane bilayer are of great interest because saturated and unsaturated chains have marked difficulty in packing together in the crystalline state (Small 1984). 2.5.2  Polymorphic Behavior The Tm values of sub‐α, α, β of MAGs having different saturated fatty‐acid moieties were determined (Table 2.7), and Tm for the β form of monooleoyl‐glycerol was 37 °C (Vereecken et al. 2009). A few reports have discussed the long‐spacing (LS) value of the most stable β form of the MAGs having saturated fatty‐acid moieties with different nc

39

Crystallization of Lipids

Table 2.7  Melting temperature (° C) of different polymorphs of monoacylglycerols with saturated fatty acid moieties. Fatty‐Acid Moiety

Sub‐α

α

β

Lauric

28.99

45.90

63.60

Myristic

37.13

58.65

72.44

Palmitic

42.47

67.12

77.99

Stearic

49.40

73.64

82.65

Arachidic

58.27

82.12

86.12

Behenic

65.22

86.56

87.32

values (Kodali et al. 1985; Krog 2001; Vereecken et al. 2009). It has been indicated that LS (nm) is expressed as 0.208nc + 1.253 and that the chain inclination angle can be determined to be 54.97 degrees by dividing the slope of the line by 0.254, which is the increase in the LS values per CH2 unit having the all‐trans conformation of the double‐ chain‐length structure (Vereecken et al. 2009). Different opinions about the occurrence and nomenclature of the independent polymorphic forms of DAGs have been reported in the literature, but the Tm values of the α, β’, and β forms of 1, 3‐DAGs and the α and β’ forms of 1, 2‐sn‐DAGs with different even‐numbered saturated fatty‐acid moieties are given in Fig.  2.21(a) by Hagemann (1988) and Fig. 2.21(b) by Kodali et al. (1990a), respectively. As to 1,2‐DAGs, the Tm values of β’ of chiral DAGs are higher than those of racemic DAGs. An interesting point is that the Tm values of the most stable forms of MAG, DAGs, and TAG with stearic acid moiety are 82.65 ° C (β of MAG), 78.0 ° C (β of 1, 3‐DAG), 77.2 ° C (β’ of 1, 2‐sn‐DAG), and 73.5 ° C (β of TAG). This means that the contribution of hydrogen bonding at the polar group is to increase the Tm values. Interesting features were observed in the polymorphism of mixed‐acid 1, 2‐sn‐diacylglycerols (1, 2‐sn‐DAGs), in which stearic acid is placed at the sn‐1 position and different (a)

(b)

1, 3-DAGs 80

1, 2-DAGs

80 Tm(° C)

Tm(° C)

40

60 β β′ α

40 10

12

14 Cn

16

18

60 40

β′ (chiral) β′ (racemic) α

20 12

20

16 Cn

Fig. 2.21  Melting temperatures (Tm) of polymorphic forms of saturated diacylglycerols (DAGs) with different number of carbon atoms (Cn) of fatty‐acid chains. R, chiral (R); RS, racemic.

24

Polymorphism of Lipid Crystals

Table 2.8  Polymorphism of mixed‐acid 1, 2‐sn‐diacylglycerols with stearic acid (S) at the sn‐1 position. Fatty‐Acid Moieties at sn‐2 Position Polymorph

Linoleic Acid (SLi‐DAG)

Oleic Acid (SO‐DAG)

Stearic Acid (SS‐DAG)

α  Tm

11.6

16.4

61.6

  ΔHm

31.4

28.4

70.2

  ΔSm

109.9

98.2

209.8

16.1

25.7

77.2

β’  Tm   ΔHm

64.4

49.7

127.9

  ΔSm

223.2

166.4

364.9

ΔHm, enthalpy of melting (kJ/mol); ΔSm, entropy of melting (kJ/mol/K); Tm, melting temperature (° C).

fatty acids are placed at the sn‐2 positions (Table 2.8). Quite reasonably, the Tm values of α and β’ decreased in the order of SS‐DAG, SO‐DAG, and SLi‐DAG in accordance with the decreasing Tm values of stearic, oleic, and linoleic acid moieties. However, the values of ΔHm and ΔSm of SLi‐DAG are larger than those of SO‐DAG, meaning that the chain packing of SLi‐DAG is tighter than that of SO‐DAG. This result is discussed in the following. Sn‐1,2‐SO‐DAG exhibits eight polymorphic forms in a dry state, γ2, γ1, α, β4, β3, β2, β1, and β’ (Di & Small 1993). Interestingly, the most stable form of sn‐1,2‐SO‐DAG is β’ with Tm = 25.7 ° C, and the second most stable form is β1 with Tm = 23.1 ° C. A structure model of β’ of sn‐1,2‐SO‐DAG was constructed based on the depiction in Fig. 2.20, in which there is certain steric hindrance between the stearoyl and oleoyl chains packed in the same leaflet. Sn‐1,2‐SLi‐DAG possesses four polymorphs in the dry state, α, sub‐α1, sub‐α2 and β’ (Di & Small 1995). Two sub‐α forms are metastable low‐temperature polymorphs, and the melting points of α and β’ are 11.6 ° C and 16.1 ° C. Hydrated sn‐1,2‐ SLi‐DAG possesses three forms, αw, sub‐αw1, and sub‐ωw2. The subcell packing is hexagonal for α and O┴ for β. It appears that stearic and linoleic chains of sn‐1,2‐SLi‐ DAG are more efficiently packed than stearic and oleic chains of sn‐1,2‐SODG because of the presence of two cis‐double bonds in sn‐1,2‐SLi‐DAG. The polymorphism of MAGs and DAGs is monotropic, and therefore every polymorphic form has its own melting temperature. Melt‐mediated transformations from meta‐ stable to more stable forms occurred on rapid heating, whereas isothermal incubation or slow heating caused a transformation to more stable forms in the solid state. These complicated phenomena were observed in MAGs (Vereecken et al. 2009) and DAGs (Kodali et al. 1985).

2.6 ­Triacylglycerols (TAGs) TAGs can be divided into three classes with respect to their fatty‐acid compositions: monoacid TAGs containing only one type of fatty acid and diacid or triacid TAGs containing two or three types of fatty acids, both of which are referred to as mixed‐acid

41

42

Crystallization of Lipids

TAGs. Mixed‐acid TAGs are further divided into two types, symmetric and asymmetric. Chiral properties are seen in the asymmetric mixed‐acid TAGs. The physical properties of TAGs are determined by the types of their fatty‐acid moieties (e.g., saturated and unsaturated chains, cis‐ and trans‐double bonds, short and long chains, even and odd numbers of carbons in the hydrophobic chains, and the esterified positions of fatty acids with glycerol carbon atoms). In principle, we can modify the molecular shapes of fats through hydrogenation, interesterification, fractionation, or genetic engineering to obtain desirable physical properties for fat‐based products by knowing the compositions of fatty acids and TAGs and their polymorphic behavior. In fact, however, polymorphic structures and their transformation pathways are quite complicated because of the diversity in chemical structures of the TAGs as discussed previously. This section highlights crystal and molecular structures and polymorphic behavior by selecting TAGs representing saturated mono‐acid and mixed‐acid TAGs and saturated‐unsaturated mixed‐acid, with specific attention to the influence of fatty‐acid moieties esterified at different glycerol carbons. 2.6.1  Crystal/Molecular Structures Very few studies on atomic‐level structure analyses with XRD using single crystals have been successful. This is simply because growing single crystals of TAGs from solution phase is quite difficult because the growth rate is very low. In addition, different types of stacking defects such as twins are present, even when one can obtain crystals whose sizes are suitable for XRD structure analysis. In particular, complicated twinning makes the atomic‐level structure analysis quite difficult and uncertain. An alternative method is to use polycrystals, which are subjected to powder XRD experiments to get limited numbers of unique X‐ray diffraction data and to draw the structure models with the help of modeling such as Rietveld refinement methods (Young 1993; McCusker et al. 1999; van de Streek & Neumann 2014). Such approaches are quite significant in understanding the structures of the metastable polymorphic forms of TAGs because growing single crystals of metastable polymorphic forms is extremely difficult. A group at the University of Amsterdam conducted systematic powder XRD experiments to determine the crystal structures of saturated TAGs (van Langevelde et al. 1999a, 1999b, 2000, 2001a, 2001b; Helmholdt et  al. 2002) and saturated‐unsaturated mixed‐acid TAGs (Peschar et al. 2004; van Mechelen et al. 2006a, 2006b, 2007, 2008a, 2008b, 2008c, 2009). We here focus primarily on single‐crystal‐based crystal structures of TAGs, briefly referring to the results of the powder XRD analysis. The first single‐crystal‐based structure determination was performed for the β form of tricaproyl‐glycerol (CCC; Jensen & Mabis 1963, 1966). Its crystal system is triclinic and the chain‐length structure is double, having a T// subcell structure as seen in Fig. 2.22(a) and Table 2.9. This glycerol conformation is called the tuning fork (Fig. 2.23), which was described in a historic paper as follows (Jensen & Mabis 1963: p. 682). Two hydrocarbon chains with two ester oxygen atoms and two of the glycerol carbon atoms forming one long, nearly straight chain. The third hydrocarbon chain branches off from the remaining glycerol carbon atom, by way of the ester oxygen atom, and bends and packs in parallel to and between the other two chains of adjacent molecules.

(a)

(b)

c

c

1 2

3

3

2

1

θ b θ b

Fig. 2.22  Crystal structures of (a) β form of tricaproyl‐glycerol (CCC) and (b) β form of 1, 2‐ dipalmitoyl‐3‐acetoyl‐sn‐glycerol (PP2). Table 2.9  Lattice parameters and density (ρ) of triacylglycerol (TAG) crystals. Space Group Z ρ (g/cc) a (nm) b (nm) c (nm)

Forms

α (deg.) β (deg.) γ (deg.)

tricaproyl‐glycerol (CCC): β P1

2 1.047

0.5488 1.2176 2.693

85.35

87.27

79.28

1, 2‐dipalmitoyl‐3‐acetoyl ‐sn‐glycerol (PP2): β

P21

2 1.065

0.5375 0.8286 4.296

90

93.3

90

1‐2‐dipalmitoyl‐3‐myristoyl C2 ‐sn‐glycerol (PPM): β2’

8 1.018

1.6543 0.7537 8.1626

90

90.28

90

1, 3‐caproyl‐2‐lauroyl‐sn ‐glycerol (CLC): β’

8 1.04

5.7368 2.2783 0.56945 90

90

90

2 0.994

1.2092 4.5705 5.446

Iba2

trielaidoyl‐glycerol (EEE): β P1 CCC: β

O3 O1 C1

PPM: β2′

PP2: β

C3 C2 O2

Tuning fork

O1

O2

C1 O3

C2 C3

Chair

O2 C2 C1

O3 C3

89.79

100.47 102.09 CLC: β′

O1 C1 C3

C2 O2

O1

Chair

O3

Chair

Fig. 2.23  Arrangements of glycerol groups with respect to the lamellar plane (dotted line).

Crystallization of Lipids 6 : Odd-numbered Even-numbered Long Chain Axis (nm)

44

5

4

3 10

12

14

16

18

cn

Fig. 2.24  Variation of length of longest axis of saturated monoacid TAGs with cn.

The same structure was observed in β of trilauroyl‐glycerol (LLL; Larsson 1964) and in even‐ and odd‐numbered saturated mono‐acid TAGs examined with powder XRD methods (van Langevelde et al. 1999a, 2001a, 2001b; Helmholdt et al. 2002). The length of the long‐chain axis of β of the odd‐numbered and even‐numbered TAGs increased linearly with increasing cn with an increment of 0.51 nm for Δnc = 2 (Fig.  2.24; Helmholdt et al. 2002). Goto et al. (1992) determined the crystal structure of the β form of diacid TAG, 1, 2‐dipalimitoy, 3‐acetyl‐sn‐glycerol (PP2) as seen in Fig. 2.22(b). Two molecules are ­present in a monoclinic unit cell whose lattice parameters are given in Table 2.9. The chain‐length structure is triple with an interdigitated nature because short sn‐3 acetoyl leaflets are sandwiched between two leaflets of the sn‐1 and sn‐2 palmitoyl chains. The subcell structure is T// for both the palmitoyl and acetoyl leaflets. The sn‐2 palmitoyl chain bends around the glycerol C2–C3 bond, so that the sn‐1 and sn‐2 palmitoyl chains are packed in the same leaflet. Therefore, the glycerol conformation is chair (Fig. 2.23). The crystal structure of the β form of trans‐unsaturated monoacid TAG, trielaidoyl‐ glycerol (EEE) was determined by Culot et al. (2000). The lattice parameters of EEE are summarized in Table  2.9. The hydrocarbon chains are in the all‐trans conformation except near the trans double bond, where skew‐trans‐skew’ with torsional angles of (120°, 180°, and –120°) is formed. The subcell structure is T//, and the glycerol conformation is tuning fork. Therefore, the crystal structure of EEE is similar to that of the saturated mono‐acid TAGs. Many researchers have worked on the β’ structure (Hernqvist & Larsson 1982; Hernqvist 1988; Birker et  al. 1991; Birker & Blonk 1993; van Langevelde et  al. 2000; van  de Streek et  al. 1999). Structure analyses of β’ crystal using single crystals were ­conducted on symmetric diacid TAG, 1, 3‐caproyl‐2‐lauroyl‐sn‐glycerol (CLC; van Langevelde et al. 2000) and asymmetric diacid TAG, 1,2‐palmitoyl‐3‐ myristol‐sn‐gycerol (PPM; Sato et al. 2001) as seen in Fig. 2.25(a and b, respectively). The subcell structures of the two β’ forms are of the O? type, and the glycerol conformation was chair (Fig. 2.23). The lattice parameters are given in Table 2.9.

Polymorphism of Lipid Crystals

(a)

(b)

-PPP-

c θ1

c

c Leaflet II

-PMMa

c

θ2

a

Leaflet I

-PMM-

-PPP-

c

b

a

b

Fig. 2.25  Crystal structures of (a) β’ form of 1, 3‐caproyl‐2‐lauroyl‐sn‐glycerol (CLC) and (b) β’2 form of 1, 2‐dipalmitoyl‐3‐myristoyl‐sn‐glycerol (PPM).

The differences in the crystal structures between β in saturated mono‐acid TAGs and the β’ forms of CLC and PPM are summarized in the following. a) The subcell structures are T// in β and O┴ in β’. b) The glycerol conformations are tuning‐fork in β and chair in β’. c) The chain‐length structures are double in β whereas they are quarto in two β’ forms. In β’, two double‐layer leaflets were combined end‐to‐end in the unit lamellae, and the chain axes were alternately inclined against the lamellar interface. The fact that the β’ form is stabilized when a TAG contains different fatty‐acid moieties (diacid TAGs) such as CLC and PPM may indicate a general tendency. Milk fat is a natural fat that is fairly stable in the β’ form; its stability may be attributed to the presence of a high concentration of mixed‐acid TAGs (see Chapter 10). de Man (1999) indicated that the following factors are prerequisites for stabilization of the β’ form: (1) Fatty‐acid chain‐length diversity, (2) TAG carbon‐number and diversity, (3) TAG structure, (4) concentration of liquid oil, and (5) temperature fluctuation. The first three factors are of a molecular nature, which may be partially explained by stabilization due to methyl‐end stacking (as revealed in PPM β’2) and/or bending at the glycerol group (as revealed in CLC β’). Therefore, the following mechanisms may explain the solid‐state β’‐β transformation, based on the crystal structures of β’ in Fig. 2.25 and of β in Fig. 2.22(a) for CCC. The transformation of CLC from β’ to β may require conformational changes in the glycerol groups combined with rotation of the half leaflets between the glycerol and methyl‐end groups around the lamella plane normal, as well as conversion of the subcell structure from O┴ to T//. The transformation of PPM from β’2 to β may require rotation of double‐layer leaflets I and II around the lamella plane normal and conversion of the subcell structure from hybrid orthorhombic to T//. Clarification of these processes, however, associated with the β’‐β transformation will require further basic research. Furthermore,

45

46

Crystallization of Lipids

diversity in the β’ structure should be clarified because there are several TAGs whose most stable polymorph is β’: β’1 of PPM whose vibrational spectroscopic data showed the presence of an O┴ subcell (Yano et al. 1997), asymmetric saturated‐unsaturated mixed‐ acid TAGs such as 1, 2‐dipalmitoyl‐3‐oleoyl‐rac‐glycerol (rac‐PPO; Minato et al. 1997), 1‐ steaoryl, 2,3‐dioleoyl‐sn‐ glycerol (sn‐SOO; Zhang et al. 2009), and so on (see below). 2.6.2  Polymorphic Behavior It is hardly possible to investigate the occurrence and transformation pathways of the diversified polymorphic forms of many TAGs in this section because of the limited space. Therefore, selected TAGs will be highlighted with an emphasis on the effects of fatty‐acid moieties and thermal treatments on the polymorphic behavior. 2.6.2.1  Thermal Behavior

The stable polymorphic forms of principal saturated and saturate‐unsaturated TAGs are summarized in Fig. 2.26 and Table 2.10, in which the following common properties can be noted. a) β‐2 in mono‐acid TAGs (SSS and OOO), symmetric saturated mixed‐acid TAGs (PSP and SPS), and symmetric saturated/trans‐unsaturated mixed‐acid TAGs (PEP). b) β‐3 in symmetric saturated/unsaturated mixed‐acid TAGs (SOS, OSO, and S‐ALA‐S) and asymmetric chiral saturated mixed‐acid TAGs (sn‐PP2, sn‐PP6, and sn‐PPC). c) β’‐4 in asymmetric saturated mixed‐acid TAGs (sn‐PPM) and β’‐3 in saturated/ unsaturated mixed‐acid TAGs (rac‐SSO and sn‐SOO) d) γ‐3 in symmetric saturated mixed‐acid TAG (CaSCa) and symmetric saturated/ unsaturated TAG (SLiS). SSS β-2

PEP β-2

PSP β-2

SOS β-3

SPS β-2

CaSCa γ-3

rac-SSO β′-3

sn-PP2 β-3

SLiS γ-3

sn-PP6 β-3

S-ALA-S β-3

OSO β-3

Fig. 2.26  Diversity in polymorphic structures of different TAGs.

sn-PPC β-3

sn-PPM β′-4

sn-SOO β′-3

OOO β-2

Polymorphism of Lipid Crystals

Table 2.10  Melting temperatures (Tm, ° C) of stable forms of selected triacylglycerols (TAGs). SSS*

PSP†

SPS*

CaSCa‡

sn‐PP2§

sn‐PP6§

sn‐PPCa‖

sn‐PPM§

Form

β‐2

β‐2

β‐2

γ‐3

β‐3

β‐3

β‐3

β2’‐4

Tm

73.5

66

68.0

33.5

51.0

43.0

47.0

58.8

PEP†

SOS¶

rac‐SSO**

SLiS††

S‐ALA‐S‡‡

OSO§§

sn‐SOO‖‖

OOO*

Form

β‐2

β1‐3

β’‐3

γ‐3

β‐3

β‐3

β1’‐3

β‐2

Tm

54

43.0

41.4

34.5

40.1

25

27

5.5

*

 Hagemann (1988),  van Mechelen et al. (2009), ‡  Sato (unpublished), §  Kodali et al. (1984), ‖  Kodali et al. (1989), ¶  Sato et al. (1989), **  Takeuchi et al. (2002), ††  Takeuchi et al. (2000), ‡‡  Sato et al. (2009), §§  Kodali et al. (1987), ‖‖  Zhang et al. (2009) Fatty‐acid moieties: 2, acetic; 6, hexanoic; ALA, α‐linolenic; Ca, capric; E, elaidic; Li, linoleic; M, myristic; O, oleic; P, palmitic; S, stearic. †

Methyl end stacking Aliphatic chain packing Glycerol conformation

Fig. 2.27  Major molecular interactions affecting the polymorphic structures of TAGs.

In addition, we may consider three major molecular interactions among TAGs as influential factors affecting the complicated polymorphic occurrences (Fig. 2.27). a) Aliphatic chain packing within the lamella/leaflets, which may affect the subcell structures and chain‐length structures. b) Glycerol conformation among the glycerol groups, which may affect the total ­configuration (straight or bent) of the TAG molecules. c) Methyl end‐stacking, whose influence may play an important role in organizing the chain‐length structures.

47

Crystallization of Lipids

(a)

(b) 70

Tm (° C)

80 Tm (° C)

48

60 40 20 10

12

14

16 cn

18

20

22

60 50 40

0

4

8 cn

12

16

Fig. 2.28  Melting temperature (Tm) of (a) saturated mono‐acid TAGs with even and odd carbon number (Cn) and (b) 1, 2‐dipalmitoyl‐3‐acyl‐sn‐glycerols with different even carbon number of the sn‐3 chains (Cn).

These three factors may interrelate in a complicated manner. For example, stabilization of the aliphatic chain packing with the T// subcell (β form) may lead to the separation of saturated‐unsaturated chains and saturated long/short chains to form a triple‐chain‐length structure, as revealed in SOS, OSO, S‐ALA‐S, sn‐PP2, and sn‐PP6. Stabilization of the glycerol conformation and aliphatic chains may stabilize the O┴ subcell (β’ form) of the triple‐chain‐length structure, as revealed in rac‐SSO and sn‐ SOO. In all cases, the total stabilization of the molecular packing must be reflected in the melting behavior. Fig.  2.28(a) depicts the variation in melting temperature (Tm) of even‐numbered mono‐acid TAGs (C2mC2mC2m) and odd‐numbered mono‐acid TAGs (C2m+1C2m+1C2m+1; Lutton & Fehl 1970). All of the saturated mono‐acid TAGs form a?‐2 structure, whereas the alternation of Tm is revealed in such a way that the value of Tm for C2m is higher than that for C2m+1. This is because the aliphatic chain packing in the odd‐ and even‐numbered mono‐acid TAGs are identical, but the methyl end‐stacking among adjacent TAG layers in the even‐numbered TAGs is more stabilized than in the odd‐numbered TAGs, as precisely studied on the methyl end‐stacking structures (van Langevelde et al. 2001b). Fig. 2.28(b) depicts the variation in Tm of mixed‐acid TAGs of PPn, which is a series of TAGs with palmitic acid at the sn‐1 and sn‐2 positions and even‐numbered (Cn: 0 to 16) saturated fatty acids at the sn‐3 position (Kodali et al. 1984, 1990b). As Cn increases from 0 to 16, Tm decreases from 60 ° C for PP0 (1, 2‐dipalmitoyl‐sn‐glycerol) to 43 ° C for PP4, does not vary from around 45–47 ° C for PP6 through PP10, and increases to 56 ° C (PP12) and 68 ° C (PPP). The decrease in Tm for PP2–PP10 compared with PP0, PP12, and PPP is caused by the unstable leaflet‐leaflet stacking of β‐3, in which the methyl end group of the sn‐3 chain makes direct contact with the glycerol group, as illustrated for sn‐PP2 and sn‐ PP6 in Fig.  2.26. This stacking mode must increase the excess lattice energy near the methyl end and glycerol groups and decrease Tm. Tm increases with increasing Cn from 12 to 16, as the chain‐length structure changes from triple to double, and thereby the instability in the methyl end‐stacking is diminished. Similar behavior of the variation in Tm was reported for a series of SnS TAGs containing sn‐1, 3‐acyl chains (stearic) and sn‐2‐chains with Cn of 18–2 (Lovegren & Gray 1978). The Tm values changed from 73 ° C for SSS to 53 ° C for S6S and 62.8 ° C for S2S. Although not examined, the chain‐length structures of SnS may change with changing Cn quite similarly to PPn.

Polymorphism of Lipid Crystals

2.6.2.2  Polymorphic Transformations

Table 2.11 summarizes the polymorphic occurrences, melting temperatures, and subcell structures of stearic‐acid containing TAGs, in which oleic, ricinoleic, and linoleic acids were placed at different carbon positions of the glycerol groups in SOS, SRS, and SLiS, respectively. The subcell structures of SOS were precisely analyzed using Table 2.11  Polymorphism of SSS, SOS, SSO, SRS, SLiS and OSO. Sub‐α forms are not shown. TAG

Form

Tm (° C)

Subcell structure

SSS* α–2

55.0

H

β’‐2

61.6

O┴

β–2

73.0

SOS†

T// Stearoyl leaflet

Oleoyl leaflet

α–2

23.5

H

H

γ–3

35.4

//‐type

H

β’‐3

36.5

O┴

H

β2‐3

41.0

T//

T// or O’ //

β1‐3

43.0

T//

T//

rac‐SSO‡ α–3

31.6

H

β’‐3

41.4

O┴

α–2

25.8

H

γ‐3

40.6

//‐type

β’2‐3

44.3

O┴

β’1‐3

48.0

O┴

SRS§

SLiS‖

α–2

20.8

H

γ‐3

34.5

//‐type

α–2

–6

H

β’‐2

—

O┴

β–2

25

T//

OSO¶

* Hagemann (1988), †  Sato et al. (1989), ‡  Takeuchi et al. (2002), §  Boubekri et al. (1999), ‖  Takeuchi et al. (2000), ¶  Kodali et al.(1987). Fatty‐acid moieties: Li, linoleic; O, oleic; R, ricinoleic; S; stearic.

49

Crystallization of Lipids

(a)

(b) α

Liquid

α

β′

Gibbs Energy

Gibbs Energy

50

β

50

60 70 Temperature (° C)

Liquid

γ β′ β2 β1

20

30 40 Temperature (° C)

Fig. 2.29  Gibbs energy compared with temperature diagrams of polymorphic forms of (a) SSS and (b) SOS.

polarized FT‐IR methods (Yano et al. 1993, 1999), and the main results were supported by a study using a DSC‐ Raman method (Sprunt et al. 2000). Three forms of α, β’, and β with a monotropic nature occurred in SSS, quite similarly to other mono‐acid TAGs including OOO, as seen in Fig.  2.29(a). Replacement of stearic acid at the sn‐2 position with oleic, ricinoleic, and linoleic acids caused drastic changes. Five forms occurred in SOS, whose thermodynamic stability of monotropic nature is revealed in Fig. 2.29(b). The chain‐length structure varied from double to triple in the other four forms, and the Tm values increased from α to other forms. The most stable form of SOS was β1, with T// subcell structures in both the stearoyl and oleoyl leaflets. The polymorphic behavior of asymmetric rac‐SSO, however, was different from that of SOS because α‐3 and β’‐3 occurred but β did not. The polymorphic behavior of symmetric SRS and SLiS was partly similar to SOS, but the most stable form of SRS was β2’‐3 and that of SLiS was γ‐3. Symmetric OSO revealed α, β’, and β forms. As a low‐temperature form, sub‐α also occurred in SOS and OSO (Yano et al. 1999), but it is disregarded in this discussion. Fig. 2.30(a) illustrates typical features of polymorphic transformation from α to γ, β’, β2, and β1 of SOS. The chain‐length structure converted from double (α) to triple (γ, β’, β2 and β1). The presence of two β forms of SOS was confirmed by powder XRD (van Mechelen et al. 2007). Five polymorphs of SOS also occurred in homologous symmetric mixed‐acid saturated‐oleic‐saturated TAGs, in which the fatty‐acid moieties at the sn‐1, 3 positions varied among palmitic, arachidic, and behenic acids (Wang et al. 1987; Arishima et al. 1991; Koyano et al. 1991; Arakawa et al. 1998; van Mechelen et al. 2007). The polymorphic transformation behavior in SOS can be summarized as follows. In α, the stearic and oleic chains are packed in the same leaflet. This can be enabled by reducing the steric hindrance between stearoyl and oleoyl moieties due to nonspecific chain packing of the subcell of H, where metastable disordered conformations are revealed in both stearoyl and oleoyl chains. Along with the structural stabilization, α‐2 transforms to γ‐3, in which the oleoyl and stearoyl leaflets are separated as a result of chain sorting. The stearoyl leaflet is assumed to be parallel packed, and the oleoyl leaflets retain the hexagonal subcell structure. The total SOS molecules in γ‐3 are arranged normal to the lamella

Polymorphism of Lipid Crystals

(a)

α-2

γ-3

β′-3

β2-3, β1-3

(b)

β2

β1

(c)

α-2

β′-2

β-3

Fig. 2.30  Structure models of polymorphic transformations in (a) SOS, (b) β2 and β1 of SOS, and (c) OSO.

interface. The triple chain‐length structure is maintained in β’, in which the stearoyl leaflet is packed according to the O┴ subcell, and the hexagonal subcell structure is retained in the oleoyl leaflet. The SOS chains are inclined with respect to the lamella plane. Finally, β’‐3 transforms to two β forms, which reveal an inclined chain arrangement against the lamellae interface. An analysis of the electron‐density profiles of the chain‐length structures of SOS polymorphs, using long‐spacing XRD patterns, supported the structure models depicted in Fig. 2.30(a) (Mykhaylyk & Hamley 2004; Mykhaylyk et al. 2004). A polarized FT‐IR technique was applied to observe the different conversions of the subcell structure between the stearoyl and oleoyl leaflets in SOS, using infrared CH2 scissoring and CH2 rocking regions as indicators of subcell packing (Yano et al. 1999). The results are summarized in Table  2.11. The bands of oleoyl leaflets overlapped those of stearoyl leaflets for the usual hydrogenated specimen. Therefore, partial deuteration in SOS was attempted so that the stearoyl chains were deuterated (D‐stearoyl) and the oleoyl

51

52

Crystallization of Lipids

chains were hydrogenated (H‐oleoyl). Thus, the FT‐IR scissoring and rocking spectra of the D‐stearoyl and H‐oleoyl leaflets could be separated. Based on the shapes of the absorption peaks, it was decided that both the D‐stearoyl and H‐oleoyl leaflets in α are packed in the H subcell. However, a sharp single band of the D‐stearoyl leaflet and a broad band of the H‐oleoyl leaflet were observed in γ, suggesting that the D‐stearoyl leaflet forms parallel packing and the H‐oleoyl leaflet packs in a hexagonal subcell. The scissoring and rocking spectra of the D‐stearoyl leaflet in β’ exhibited two components, indicating the O┴ subcell, whereas parallel packing was indicated in the H‐oleoyl leaflet in β’ because no splitting occurred. Both the D‐stearoyl and H‐oleoyl leaflets were packed in the T// subcell in β1. It is assumed that the subcell structure of the D‐stearoyl leaflet of β2 is similar to T//, but the molecular conformation of the H‐oleoyl leaflet may differ from that of β1. As to the differences in the crystal structures between β2 and β1 of saturated‐oleic‐ saturated TAGs, van Mechelen et al. (2006a, b) claimed to observe the occurrence of triple‐layer and hexa‐layer structures in β2 and β1, respectively, based on their powder XRD structure analyses as depicted in Fig. 2.30(b). Further research is needed to more precisely determine the two β structures of SOS, highlighting the chain‐length structures, conformation of glycerol groups, methyl end‐stacking structures, local structures around the cis‐double bonds of the oleoyl leaflet, and so on. These studies have a critical implication for cocoa butter polymorphism because the two forms are believed to be identical to Form V and Form VI of cocoa butter and the transformation from Form V to VI is the main cause of fat‐bloom formation. The polymorphic structures depicted in Fig. 2.30(a) for SOS are in part common to stearic/unsaturated diacid TAGs having ricinoleic acid (SRS; Boubekri et al. 1999) and linoleic acid (SLiS; Takeuchi et al. 2000) at the sn‐2 position instead of oleic acid. There is one remarkable difference between SOS, SRS, and SLiS: SRS has α, γ, and two β’ forms, and SLiS has α and γ forms. It is assumed that the hydrogen bonding in the ricinoleoyl chains in SRS was so tight that the O┴ subcell was stabilized through the carbonyl groups at the acyl chain, probably making β’ the most stable. Interactions among linoleoyl chains having two cis‐double bonds may stabilize the γ form in SLiS, prohibiting transformation into the more stable forms of β’ and β (see SLiS in Fig. 2.26). In this connection, interesting results were observed in the polymorphism of stearic‐polyunsaturated fatty‐acid (PUFA)‐stearic acid TAGS: 1,3‐stearoyl‐2‐α‐linoleoyl‐ glycerol (S‐ALA‐S), 1,3‐stearoyl‐2‐eicosapentaeneoyl‐glycerol (S‐EPA‐S), and 1,3‐stearoyl‐ 2‐docosahexaenoyl‐glycerol (S‐DHA‐S; Sato et al. 2009). Table 2.12 lists the values of Tm, long spacing, and the enthalpy of melting (ΔHm) of the γ and β forms of S‐ALA‐S and the β form of S‐EPA‐S and S‐DHA‐S. All of the crystals form a triple‐chain‐length structure in which the stearic‐acid moiety and PUFA‐moiety are stacked in different leaflets (see S‐ALA‐S in Fig. 2.26). The values of Tm for the three crystals are surprisingly higher than those of PUFAs in a free fatty‐acid state. In addition, the values of ΔHm of the three crystals are comparable with those of β1‐3 of SOS (151 kJ/mol) and γ‐3 of SLiS (137.2 kJ/ mol; Sato et al. 2009). We can assume that strong van der Waals interactions between the stearic acid layers of the triple‐chain‐length structure may force the ALA, EPA, and DHA leaflets to exhibit an extended chain conformation, producing long‐spacing values around 6 to 7 nm and high values of Tm and ΔHm. The polymorphic transformation pathways in OSO are similar to those in SOS, although the arrangements of the oleoyl and stearoyl leaflets with respect to the lamellar plane are opposite (Fig. 2.30b). Compared to SOS, few studies have been done on the

Polymorphism of Lipid Crystals

Table 2.12  Polymorphism of mixed‐acid triacylglycerols (TAGs) containing stearic and poly‐unsaturated fatty‐acid moieties. S‐ALA‐S γ‐3

LS

S‐EPA‐S β‐3

7.04

Tm

γ‐3

6.31

35.99

ΔHm

116.2

S‐DHA‐S γ‐3

7.16

7.35

40.1

32.5

36.2

138.7

116.7

130.2

ALA, α‐linolenic acid; ΔHm, enthalpy of melting (kJ/mol); DHA, docosagexanoic acid; EPA, eicosapentanoic acid; LS, long spacing (nm); S, stearic acid; Tm, melting temperature (° C).

microscopic mechanisms involved in the transformation of OSO polymorphs. A unique polymorphic transformation was observed for rac‐SSO because α is a triple‐chain‐ length structure and no β was detected. As a reference, the polymorphic behavior of optical enantiomers of 1‐oleoyl‐2, 3‐dipalmitoyl‐sn‐glycerol (S‐OPP) and 1, 2‐dipalmitoyl‐3‐oleoyl‐ sn‐glycerol (R‐PPO) was compared with that of rac‐PPO (Mizobe et al. 2013). The polymorphism of S‐OPP and R‐PPO was identical, both having α‐2 and β’‐3, whereas rac‐PPO revealed αrac‐3, β’rac‐2, and β. Although there are some differences in the structures of α among S‐OPP (R‐PPO), rac‐PPO, and rac‐SSO, the common result is the absence of β. Thus, a basic question may arise: why is the subcell of T// forming β not stabilized in SSO and PPO, whereas the subcell of O┴ of β’ is stabilized, despite the fact that SOS stabilized β with T//. The answer may lie in the interplay of the chair‐type glycerol conformation and the methyl end‐stacking, which may not stabilize the T// subcell in SSO. Further research may be needed on this problem because many asymmetric mixed‐acid TAGs have stable β’ polymorphic forms that exhibit high functionality in edible fat formulations. The transformation pathways of the five polymorphic forms of sn‐PPCa are quite complicated as summarized in Table 2.13 (Kodali et al. 1989). Two types of transformations occur: solid‐state transformation and melt‐mediated transformation (Fig. 2.31a). For example, α is formed by the quenching of neat liquid. The heating procedure causes successive melt‐mediated transformations of α–melt‐β’3 and β’3‐melt‐β’2‐2 as seen in the DSC heating patterns in Fig. 2.31(b). Another melt‐mediated transformation of α‐ melt‐β’1 occurs by rapid melting of α at 39 °C. The solid‐state transformations occur

Table 2.13  Polymorphism of 1, 2‐dipalmitoyl‐3‐caproyl‐sn‐glycerol (sn‐PPCa). α–2

LS Tm

4.04 23

β3’–2

3.70 34

β2’–2

3.71 39.5

LS, long spacing (nm); Tm, melting temperature (° C).

β1’–6

11.1 42.5

β–3

5.60 47.0

53

Crystallization of Lipids

(a)

(b) 39° C

β3′: cryst.

Liquid

Exo.

54

23° C 34° C 37° C solid β-3

β2′-2

β3′-2

21° C solid

β2′: cryst.

α-2

β1′-6

α: melt β3′: melt

β2′: melt

Fig. 2.31  (a) Thermodynamic stability and (b) polymorphic transformation pathways of 1,2‐ dipalmitoyl‐3‐caproyl‐sn‐glycerol (PPCa).

from α–2 to β’3‐2 and from β’2‐2 to β‐3. The mechanisms of these transformations seem to be open to question, particularly the occurrence of the hexa‐layer of β’1‐6 and its formation processes, the detailed structures of multiple β’ crystals, etc. It is worth noting that the TAG group of PPn with small n numbers represents high‐melting fractions of anhydrous milk fat and their polymorphs (see Chapters 3 and 10).

2.7 ­Conclusions Research on the polymorphism of lipid crystals has progressed substantially in the last decades. Today, however, emerging lipid technologies require more detailed knowledge so that blending, interesterification, and fractionation of different lipids can provide functional lipid materials to cope with the current needs for trans‐fat alternatives, reduction of saturated fats, and so on. The following points would be high on any priority list of needed information. a) The molecular mechanisms of polymorphic transformations of TAG crystals. b) The stabilization mechanisms of β’ polymorph. c) The occurrence and structures of multiple polymorphs of β’ and β.

­References Akita, C., Kawaguchi, T., & Kaneko, F. (2006) Structural study on polymorphism of cis‐unsaturated triacylglycerol: Triolein. Journal of Physical Chemistry B. 110, 4346–53. Arakawa, H., Kasai, M., Okumura, Y., & Maruzeni, S. (1998) Polymorphism of SOA triglerol prepared from sal fat. Japan Oil Chemists’ Society. 47,19–24 Arishima, T., Sagi, N., Mori, H., & Sato, K. (1991) Polymorphism of POS. I. Occurrence and polymorphic transformation. Journal of the American Oil Chemists’ Society. 68, 710–15.

Polymorphism of Lipid Crystals

Birker P. J. M. W. L., & Blonk, J. C. G. (1993) Alkyl chain packing in a β’ triacylglycerol measured by atomic force microscopy. Journal of the American Oil Chemists’ Society. 70, 319–21. Birker, P. J. M., S de Jong, W. L., Roijers, E. C., & van Soest, T. C. (1991) Structural investigation β’ triacylglycerols: An X‐ray diffraction and microscopic study of twinned β’ crystals. Journal of the American Oil Chemists’ Society. 68, 895–906. Boubekri, K., Yano, J., Ueno, S., & Sato, K. (1999) Polymorphic Transformations in SRS (sn‐1,3‐distearoyl‐2‐ricinoleyl glycerol). Journal of the American Oil Chemists’ Society. 76, 949–55. Bowes, C. L., Malek, A., & Ozin, G. A. (1996) Chemical vapor deposition topotaxy in porous hosts. Chemical Vapor Deposition. 2, 97–103. Chow, C. K. (1992) Fatty acids in foods and their health implications. New York, Marcel Dekker. Culot, C., Norberg, B., Evrard, G., & Durant, F. (2000) Molecular analysis of the b‐ polymorphic form of trielaidin: Crystal structure at low temperature. Acta Crystallographica B. 56, 317–21. de Man, J. M. (1999) Relationship among chemical, physical, and textural properties of fats. In: Widlak, N. (ed.) Physical properties of fats, oils, and emulsifiers. New York, Marcel Dekker, pp. 79–95. Di, L., & Small, D. M. (1993) Physical behavior of the mixed chain diacylglycerol, 1‐ stearoyl‐ 2‐oleoyl‐sn‐glycerol: Difficulties in chain packing produce marked polymorphism. Journal of Lipid Research. 34, 1611–23. Di, L., & Small, D. M. (1995) Physical behavior of the hydrophobic core of membranes: Properties of 1‐stearoyl‐2‐linoleoyl‐sn‐glycerol. Biochemistry. 35, 16672–77. Dorset, D. L. (2005) Crystallography of the polymethylene chain. An inquiry into the structure of waxes. New York, Oxford University Press. Dorset, D. L., & Pangborn, P. A. (1988) Polymorphic forms of 1, 2‐dipalmitoyl‐sn‐glycerol: A combined X‐ray and electron diffraction study. Chemistry and Physics of Lipids. 48, 19–28. Figlarz, M., Gérand, B., Delahaye‐Vidal, A., et al. (1990) Topotaxy, nucleation and growth. Solid State Ionics. 43, 143–70. Goto, M., & Asada, E. (1978) The crystal structure of the B‐form of stearic acid. Bulletin of the Chemical Society of Japan. 2456–59. Goto, M., & Takiguchi, T. (1985) The crystal structure of the β‐form of α‐monolaurin. Bulletin of the Chemical Society of Japan. 58, 1319–20. Goto, M., Honda, K., Di, L., & Small, D. M. (1995) Crystal structure of a mixed chain diacylglycerols, 1‐stearoyl‐3‐oleoyl‐glycerol. Journal of Lipid Research. 36, 2185–90. Goto, M., Kodali, D. R., Small, D. M., et al. (1992) Single crystal structure of a mixed‐chain triacylglycerol: 1,2‐dipalmitoyl‐3‐acetyl‐sn‐glycerol. Proceedings of the National Academy of Sciences of the United States of America. 89, 8083–86. Goto, M., Kozawa, K., & Uchida, T. (1988) The crystal structure of the β’1 form of optically active α‐monostearin. Bulletin of the Chemical Society of Japan. 61, 1434–36. Hagemann, J. W. (1988) Thermal behavior and polymorphism of acylglycerides. In: Garti, N., & Sato, K. (eds.) Crystallization and polymorphism of fats and fatty acids. New York, Marcel Dekker, pp. 9–95. Hernqvist, L. (1988) On the crystal structure of the β’1‐form of triglycerides and mechanism behind the β’1‐β transition of fats. European Journal of Lipid Science and Technology. 90, 451–54.

55

56

Crystallization of Lipids

Hernqvist, L., & Larsson, K. (1982) On the crystal structure of the β’‐forms of triglycerides and structural changes at the phase transitions liq‐α‐β’‐β. Fette, Seifen, Anstrichmittel. 84, 349–54 Hiramatsu, H., Sato, T., Inoue, T., Suzuki, M., & Sato, K. (1990). Pressure effect on transformation of cis‐unsaturated fatty acid polymorphs. 2. Palmitoleic acid (cis‐9‐ hexadecenoic acid). Chemistry and Physics of Lipids. 56, 59–63. Jensen, L. H., & Mabis, A. J. (1963) Crystal structure of β‐tricaprin. Nature (London). 197, 681–82 Jensen, L. H., & Mabis, A. J. (1966) Refinement of the structure of β‐tricaprin. Acta Crystallographica. 22, 770–81. Kaneko, F. (2001) Polymorphism and phase transitions of fatty acids and acylglycerols. In Garti, N. & Sato, K. (eds.) Crystallization processes in fats and lipid systems. New York, Marcel Dekker, pp. 53–97. Kaneko, F., & Kubota, H. (2011) Vibrational spectroscopic study on the polytypism of n‐alkanes and fatty acids. Current Opinion in Colloid and Interface Science. 16, 367–73. Kaneko, F., Kobayashi, M., Kitagawa, Y., & Matsuura, Y. (1990) Structure of stearic acid E form. Acta Crystallographica C. 46, 1490–92. Kaneko, F., Kobayashi, M., Sato, K., & Suzuki, M. (1997a) Martensitic phase transition of petroselinic acid: Influence of polytypic structure. Journal of Physical Chemistry B. 101, 285–92. Kaneko, F., Simofuku, T., Miyamoto, H., & Kobayashi, M. (1992) Vibrational spectroscopic study on the occurrence of stearic acid B and E forms: Heterogeneous nucleation of the B form on the surface of E crystals and the topotactic phase transition from E to B. J. The Journal of Physical Chemistry. 96, 10554–59 Kaneko, F., Sakashita, H., Kobayashi, M., et al (1994a) Double‐layered polytypic structure of the B form of octadecanoic acid, C18H36O2. Acta Crystallographica C. 50, 245–47. Kaneko, F., Sakashita, H., Kobayashi, M., et al. (1994b) Double‐layered polytypic structure of the E form of octadecanoic acid, C18H36O2. Acta Crystallographica C. 50, 247–50. Kaneko, F., Yamazaki, K., Kitagawa, K., et al. (1997b) Structure and crystallization behavior of the β1 phase of oleic acid. Journal of Physical Chemistry B. 101, 1803–9. Kaneko, F., Yamazaki, K., Kobayashi, M. (1996) Mechanism of the γ‐α and γ1‐α1 reversible solid‐state phase transitions of erucic acid. Journal of Physical Chemistry. 100, 9138–48. Kaneko, F., Yano, J., & Sato, K. (1998) Diversity in the fatty‐acid conformation and chain packing of cis‐unsaturated lipids. Current Opinion in Structural Biology. 8, 417–25. Kobayashi, M., Kaneko, F., Sato, K., & Suzuki, M. (1986) Vibrational spectroscopic study on polymorphism and order‐disorder phase transition in oleic acid. Journal of Physical Chemistry. 90, 6371–78. Kobayashi, M., Kobayashi, T., Ito, Y., & Sato, K. (1984) Polytypism in n‐fatty acids and low‐frequency Raman spectra: Stearic acid B form. Journal of Chemical Physics. 80, 2897–903. Kodali, D. R., Atkinson, D., Redgrave, T. G., Small, D. M. (1984) Synthesis and polymorphism of 1, 2‐dipalmitoyl‐3‐acyl‐sn‐glycerols. Journal of the American Oil Chemists’ Society. 61, 1078–84. Kodali, D. R., Atkinson, D., Redgrave, T. G., Small, D. M. (1987) Structure and polymorphism of 18‐carbon fatty acyl triacylglycerols: Effect of unsaturation and substitution in the 2‐position. Journal of Lipid Research. 28, 403–13.

Polymorphism of Lipid Crystals

Kodali, D. R., Atkinson, D., & Small, D. M. (1989) Molecular packing of 1,2‐dipalmitoyl‐ 3‐decanoyl‐sn‐glycerol (PP10): Bilayer, trilayer and hexalayer structures. Journal of Physical Chemistry. 93, 4683–91. Kodali, D. R., Atkinson, D., & Small, D. M. (1990b) Polymorphic behavior of 1,2‐ dipalmitoyl‐3‐lauroyl (PP12)‐ and 3‐myristoyl (PP14)‐sn‐glycerols. Journal of Lipid Research. 31, 1853–64. Kodali, D. R., Fahey, D. A., Small, D. M., & Atkinson, D. (1990a) Structure and polymorphism of saturated monoacid 1, 2‐diacyl‐sn‐glycerols. Biochemistry. 29, 10771–79. Kodali, D. R., Redgrave, T. G., Small, D. M., & Atkinson, D. (1985) Synthesis and polymorphism of 3‐acyl‐sn‐glycerols. Biochemistry. 24, 519–25. Koyano, T., Hachiya, I., Arishima, T., Sato, K., & Sagi, N. (1991) Polymorphism of POS. II. Kinetics of melt crystallization. Journal of the American Oil Chemists’ Society. 68, 716–18. Krog, N. (2001) Crystallization properties and lyotropic phase behavior of food emulsifiers. In: Garti, N., & Sato, K. (eds.) Crystallization processes in fats and lipid systems. New York, Marcel Dekker, pp. 505–26. Kubota, H., Kaneko, F., Kawaguchi, T. (2005) Study of thermodynamic stabilities of polytypes of n‐C36H74 by solubility measurements and incoherent inelastic neutron scattering. Journal of Chemical Physics. 122, 24903‐1‐6. Kulkarni, C. V. (2012) Lipid crystallization: From self‐assembly to hierarchical and biological ordering. Nanoscale. 4, 5779–91. Kuwahara, T., Nagase, H., Endo, T., Ueda, H., & Nakagaki, M. (1996) Crystal structure of DL‐12‐hydroxystearic acid. Chemistry Letters. 25, 435–36. Larsson, K. (1963) The crystal structure of the 1, 3‐diglyceride of 3‐thiadodecanoic acid. Acta Crystallographica. 741–48 Larsson, K. (1964) The crystal structure of the β‐form of trilaurin. Arkiv Kemi. 23, 1–15. Larsson, K., Quinn, P., Sato, K., & Tiberg, F. (2006) Lipids: Structure, physical properties and functionality. Bridgwater, UK: The Oily Press/Elsevier. Lovegren, N. V., & Gray, M. S. (1978) Polymorphism of saturated triglycerides: I. 1, 3‐distearo triglycerides. Journal of the American Oil Chemists’ Society. 55, 310–16. Low, J. N., Scrimgeour, C., & Horton, P. (2005) Elaidic acid (trans‐9‐octadecenoic acid). Acta Crystallographica E. 61, 3730–32. Lutton, E. S., & Fehl, A. J. (1970) The polymorphism of odd and even saturated single acid triglycerides, C8–C22. Lipids. 5, 90. Malta, V., Celotti, G., Zanetti, R., & Martelli, A. F. (1971) Crystal structure of the C form of stearic acid. Journal of Chemistry B. 548–53. McCusker, L. B., Von Dreele, R. B. Cox, D. E., Louër, D., & Scardi, P. (1999) Rietveld refinement guidelines. Journal of Applied Crystallography. 32, 36–50. Minato, A., Ueno, S., Smith, K, Amemiya, Y., & Sato, K. (1997) Thermodynamic and kinetic study on phase behavior of binary mixtures of POP and PPO forming molecular compound systems. Journal of Physical Chemistry B. 101, 3498 − 505. Mizobe, H., Tanaka, T., Hatakeyama, N., et al. (2013) Structures and binary mixing characteristics of enantiomers of 1‐oleoyl‐2, 3‐dipalmitoyl‐sn‐glycerol (S‐OPP) and 1,2‐dipalmitoyl‐3‐oleoyl‐sn‐glycerol (R‐PPO). Journal of the American Oil Chemists’ Society. 90, 1809–17.

57

58

Crystallization of Lipids

Moreno, E., Cordobilla, R., Calvet, T., et al. (2007) Polymorphism of even saturated carboxylic acids from n‐decanoic to n‐eicosanoic acid. New Journal of Chemistry. 31, 947–57. Mykhaylyk, O. O., & Hamley, I. W. (2004) The packing of triacylglycerols from SXAS measurements: Application to the structure of 1,3‐distearoyl‐2‐oleoyl‐sn‐glycerol crystal phases. Journal of Physical Chemistry B. 108, 8069–83. Mykhaylyk, O. O., Casteletto, V., Hamley, I. W., & Povey, M. J. W. (2004) Structure and transformation of low‐temperature phases of 1,3‐distearoyl‐2‐oleoyl‐sn‐glycerol. European Journal of Lipid Science and Technology. 106, 319–24. Pascher, I. (1996) The different conformations of the glycerol region of crystalline acylglycerols. Current Opinion in Structural Biology. 6, 439–48 Pascher, I., & Sundell, S. (1981) Glycerol conformation and molecular packing of membrane lipids: The crystal structure of 2,3‐dilauroyl‐D‐glycerol. Journal of Molecular Biology. 153, 791–806. Pascher, I., Lundmark, M., Nyholm, P‐G., & Sundell, S. (1992) Crystal structures of membrane lipids. Biochimica et Biophysica Acta. 1113, 339–73. Peschar, R., Pop, M. M., de Ridder, D. J. A., et al. (2004) Crystal structure of 1, 3‐distearoyl‐2‐ oleoylglycerol and cocoa butter in the β (V) phase reveal the driving force behind the occurrence of fat bloom on chocolate. Journal of Physical Chemistry B. 108, 15450–53. Pi, F., Kaneko, F., Iwahashi, M., Suzuki, M., Ozaki, Y. (2011) Solid‐state low temperature → middle temperature phase transition of linoleic acid studied by FTIR spectroscopy. Journal of Physical Chemistry B. 115, 6289–95. Helmholdt, R. B., Peschar, R., & Schenk, H. (2002) Structure of C15‐, C17‐ and C19‐mono‐ acid β‐triacylglycerols. Acta Crystallographica B. 58, 134–39. Sakurai, T., Masuda, Y., Sato, H., et al. (2010). A comparative study on chiral and racemic 12‐hydroxyoctadecanoic acids in the solutions and aggregation states: Does the racemic form really form a gel? Bulletin of the Chemical Society of Japan. 83, 145–50. Sala, S., Elizondo, E., Moreno, E., et al. (2010) Kinetically driven crystallization of a pure polymorphic phase of stearic acid from CO2‐expanded solutions. Crystal Growth & Design. 10, 1226–32. Sato, K., & Boistelle, R. (1984) Stability and occurrence of polymorphic modifications of stearic acid in polar and nonpolar solutions. Journal of Crystal Growth. 66, 441–50. Sato, K., & Koyano, T. (2001) Crystallization properties of cocoa butter. In: Garti, N., & Sato, K. (eds.), Crystallization processes in fats and lipid systems. New York, Marcel Dekker, pp. 429–56. Sato, K., Arishima, T., Wang, T. H., et al. (1989) Polymorphism of POP and SOS. I. Occurrence and polymorphic transformation. Journal of the American Oil Chemists’ Society. 66, 664–74. Sato, K., Goto, M., Yano, J., et al. (2001) Atomic resolution structure analysis of β’ polymorph crystal of a triacylglycerol: 1,2‐dipalmitoyl‐3‐ myristoyl‐sn‐glycerol. Journal of Lipid Research. 42, 338–45. Sato, K., Kigawa, T., Ueno, S., Gotoh, N., Wada, S. (2009) Polymorphic behavior of structured fats including stearic acid and ω‐3 polyunsaturated fatty acids. Journal of the American Oil Chemists’ Society. 86, 297–300. Sato, K., Morishita, H., & Kobayashi, M. (1988) Stability, occurrence and step morphology of polymorphs and polytypes of stearic acid. I. Stability and occurrence. Journal of Crystal Growth. 87, 236–42.

Polymorphism of Lipid Crystals

Sato, K., Yano, J., Kawada, I., & Kawano, M. (1997) Polymorphic behavior of gondoic acid and phase behavior of its binary mixtures. Journal of the American Oil Chemists’ Society. 74, 1153–59. Sato, K., Yoshimoto, N., Suzuki, M., Kobayashi, M., & Kaneko, F. (1990) Structure and transformation in polymorphism of petroselinic acid (cis‐ω‐12‐octadecenoic acid). Journal of Physical Chemistry. 94, 3180–85. Small, D. M. (1984) Lateral chain packing in lipids and membranes. Journal of Lipid Research. 25, 1490–1500. Sprunt, J. C., Jayasooriya, U. A., Wilson, H. H. (2000). A simultaneous FT‐Raman‐DSC (SRD) study of polymorphism in sn‐1,3‐distearoyl‐2‐oleoylglycerol (SOS). Physical Chemistry Chemical Physics. 2, 4299–4305. Suzuki, M., Ogaki, T., & Sato, K. (1985) Crystallization and transformation mechanisms of α, γ and β polymorphs of ultra‐pure oleic acid. Journal of American Oil Chemists’ Society. 62, 1600–4. Tachibana, T., & Kambara, H. (1969) Studies of helical aggregates of molecules. I. Enantiomorphism in the helical aggregates of optically active 12‐hydroxystearic acid and its lithium salt. Bulletin of the Chemical Society of Japan. 42, 3422–42. Takeuchi, M., Ueno, S., Yano, J., Floeter, E., & Sato, K. (2000) Polymorphic transformation of 1,3‐distearoyl‐sn‐ linoleoyl‐glycerol. Journal of the American Oil Chemists’ Society. 77, 1243–49. Takeuchi, M., Ueno, S., & Sato, K. (2002) Crystallization kinetics of polymorphic forms of a molecular compound constructed by SOS (1,3‐distearoyl‐2‐oleoyl‐sn‐glycerol) and SSO (1,2‐distearoyl‐3‐oleoyl‐rac‐glycerol). Food Research International. 35, 919–26. Uehara, H., Suganuma, T., Negishi, S., et al. (2008) Physical properties of two isomers of conjugated linoleic acid. Journal of the American Oil Chemists’ Society. 85, 29–36. Ueno, S., Miyazaki, A., Yano, J., et al. (2000) Polymorphism of linoleic acid (cis‐9, cis‐12‐ octadecadienoic acid) and α‐linolenic acid (cis‐9, cis‐12, cis‐15‐octadecatrienoic acid). Chemistry and Physics of Lipids. 107, 169–78. Uzu, Y., & Sugiura, T. (1975) Electron microscopic and thermal studies of optically active 12‐hydroxystearic acids in soap formation. Journal of Colloid and Interface Science. 51, 346–49. van de Streek, J., & Neumann, M. A. (2014) Validation of molecular crystal structures from powder diffraction data with dispersion‐corrected density functional theory (DFT‐D). Acta Crystallographica B. 70, 1020–32. van de Streek, J., Verwer, P., de Gelder, R., & Hollander, F. (1999) Structural analogy between β’ triacylglycerols and n‐alkanes: toward the crystal structure of β’‐2 p.p + 2.p triacylglycerols. Journal of the American Oil Chemists’ Society. 76, 1333–41. van Langevelde, A., Peschar, R., & Schenk, H. (2001a) Structure of β‐trimyristin and β‐tristearin from high‐resolution X‐ray powder diffraction data. Acta Crystallographica B. 57, 372–77. van Langevelde, A., Peschar, R., & Schenk, H. (2001b) Alternation of melting points in odd‐ and even‐numbered monoacid triacylglycerols. Chemistry of Materials. 13, 1089–94. van Langevelde, A., van Malssen, K., Driessen, R., et al. (2000) Structure of CnCn+2Cn‐type (n = even) β’‐triacylglycerols. Acta Crystallographica B. 56, 1103–11. van Langevelde, A., van Malssen, K., Hollander, F., Pechar, R., & Schenk, H. (1999a) Structure of mono‐acid even‐numbered β‐triacylglycerols. Acta Crystallographica B. 55, 114–22.

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van Langevelde, A., van Malssen, K., Sonneveld, E. D., Pechar, R., & Schenk, H. (1999b) Crystal packing of a homologous series β’‐stable triacylglycerols. Journal of the American Oil Chemists’ Society. 76, 603–9. van Mechelen, J. B., Goubitz, K., Pop, M., Peschar, R., & Schenk, H. (2008c) Structures of mono‐unsaturated triacylglycerols. V. The β’‐2, β’‐3 and β2‐3 polymorphs of 1, 3‐ dilauroyl‐2‐oleoylglycerol (LaOLa) from synchrotron and laboratory powder diffraction data. Acta Crystallographica B. 64, 771–779 van Mechelen, J. B., Peschar, R. & Schenk, H. (2006a) Structures of mono‐unsaturated triacylglycerols. I. The β1 polymorph. Acta Crystallographica B. 62, 1121–30. van Mechelen, J. B., Peschar, R. & Schenk, H. (2006b) Structures of mono‐unsaturated triacylglycerols. II. The β2 polymorph. Acta Crystallographica B. 62, 1131–38. van Mechelen, J. B., Peschar, R. & Schenk, H. (2007) The crystal structures of the β1 and β2 polymorphs of mono‐unsaturated triacylglycerols and cocoa butter determined from high resolution powder diffraction data. Zeitschrift für Kristallographie Suppl. 26, 599–604. van Mechelen, J. B., Peschar, R., & Schenk, H. (2008a) Structures of mono‐unsaturated triacylglycerols. III. The β‐2 polymorphs of trans‐monounsaturated triacylglycerols and related fully saturated triacylglycerols. Acta Crystallographica B. 64, 240–48. van Mechelen, J. B., Peschar, R., & Schenk, H. (2008b) Structures of mono‐unsaturated triacylglycerols. IV. The highest melting β’‐2 polymorphs of trans‐monounsaturated triacylglycerols and related saturated TAGs and their polymorphic stability. Acta Crystallographica B. 64, 249–59. van Mechelen, J. B., Peschar, R. & Schenk, H. (2009) Structure and polymorphism of trans mono‐unsaturated triacylglycerols. Zeitschrift für Kristallographie Suppl. 30, 491–95. Vereecken, J., Meeussen, W., Foubert, I., et al. (2009) Comparing the crystallization and polymorphic behaviour of saturated and unsaturated monoglycerides. Food Research International. 42, 1415–25. Wang, Z. H., Sato, K., Sagi, N., Izumi, T., & Mori, H. (1987) Polymorphism of 1, 3‐disaturated‐ acyl‐2‐oleoylglycerols: POP, SOS, AOA, BOB. Japan Oil Chemists’ Society.36, 671–79. Yano, J., Kaneko, F., Kobayashi, M., et al. (1997) Structural analyses of triacylglycerol polymorphs with FT‐IR techniques: II. β’1‐form of 1,2‐dipalmitoyl‐3‐myristoyl‐sn‐ glycerol. Journal of Physical Chemistry B. 101, 8120–28. Yano, J., Sato, K., Kaneko, F., Small, D. M., & Kodali, D. R. (1999) Structural analyses of polymorphic transitions of sn‐1,3‐distearoyl‐2‐oleoylglycerol (SOS) and sn‐1,3‐ dioleoyl‐2‐stearoylglycerol (OSO): Assessment on steric hindrance of unsaturated and saturated acyl chain interactions. Journal of Lipid Research. 40, 140–51. Yano, J., Ueno, S., Sato, K., et al. (1993) FT‐IR study of polymorphic transformations in SOS, POP and POS. Journal of Physical Chemistry. 97, 12967–73. Yoshimoto, N., Suzuki, M., & Sato, K. (1991) Polymorphic transformation in asclepic acid (cis‐ω7‐octadecenoic acid). Chemistry and Physics of Lipids. 57, 67–73. Young, R. A. (ed.) (1993) The Rietveld Method. New York: Oxford University Press/An International Union of Crystallography. Zhang, L., Ueno, S., Sato, K., Adlof, R. O., & List G. R. (2009) Thermal and structural properties of binary mixtures of 1,3‐distearoyl‐2‐oleoyl‐glycerol (SOS) and 1,2‐dioleoyl‐3‐ stearoyl‐sn‐glycerol (sn‐OOS). Journal of Thermal Analysis and Calorimetry. 98, 105–11.

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3 Molecular Interactions and Mixing Phase Behavior of Lipid Crystals Eckhard Floeter, Michaela Haeupler, and Kiyotaka Sato

3.1 ­Introduction The functionalities of fats in products are manifold. The natural and industrial lipids employed in manufacturing foods, cosmetics, and pharmaceuticals are mixtures of ­different types of lipid specimen of fatty acids and triacyglycerols (TAGs), in which fatty‐acid moieties and polar groups are largely varying from one specimen to the other. The nutritional delivery in food products is primarily based on the fatty‐acid moieties of the lipid molecules and only indirectly dependent on the physical state of the lipids in products. In contrast, the complicated behavior of melting, crystallization and transformation, morphology, and aggregation of lipid crystals into crystal networks depend on the physical properties of the crystals determined by the properties of the lipids and their mixing behavior. This implies that all properties related to structuring (e.g., macroscopic hardness, plasticity, Pickering emulsion stability, and organoleptic properties based primarily on deliberate product disintegration) are determined by the crystallization behavior of lipids. In a lot of fat‐based food products fat crystals are the major contributor to the macroscopic structure of the product. The functionality of the fat phase might simply be a sort of glue to bind numerous solid particles into a solid mass, as for example in bouillon cubes. Fat crystal networks in fat‐continuous shortenings and spreads essentially build a sponge‐like porous structure that prevents oil leakage from the soft‐solid matrix. However, also in water‐continuous emulsions and aerated products, such as whipping creams, the macroscopic properties of the product structure might be influenced by the physical state of the lipid phase inside the dispersed droplets. The ability of fat crystals to stabilize fat‐continuous emulsions via the so‐called Pickering stabilization (Pickering 1907) should be explicitly mentioned here. In spreads, the characteristics of the emulsion are strongly influenced by the physical properties of the fat crystals that form a kind of shell at the oil‐water interface of the droplets (Walstra et  al. 2001). Features influenced are temperature stability, initial and long‐term droplet size, and coalescence/ inversion kinetics of the emulsions during storage and usage. As mentioned, the presence of the lipid phases and their physical properties can have a profound effect on the Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, First Edition. Edited by Kiyotaka Sato. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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organoleptic properties of a product. First, the lubrication effect of fat during mastication and swallowing is important for the perceived quality of food products (De Bruijne et al. 1993; Prinz & Lucas, 1997; Lillford 2000; De Hoog et al. 2006). Second, the transition of fat from the solid to the liquid state is accompanied by a melting sensation. In emulsion products, the characteristic dissolution of the fat crystals is typically coupled with emulsion break‐up and thus flavor release. Because of the current pressure to innovate products that have to satisfy continuously changing, harder constraints (e.g., nutritional, raw material restrictions, costs), it is important to improve the understanding of the underlying principles that link final product properties to product formulations and manufacturing processes. A meaningful step into this direction is an improved knowledge of the possible states a system is likely to assume. This would benefit both the product design and the understanding of manufacturing processes. Currently, the consistent description of the physical chemistry of food matrices and the fat‐based food manufacturing processes appear impossible. A convincing theoretical description and comprehensive understanding are hampered by many factors. On one hand, the lipid phases present in food products are true multicomponent systems. Considering only the major 13 fatty acids (see also Chapter 1), a substantial—in excess of 1000—amount of possibly present triglycerides emerges. Due to the nature of the synthetic pathway some natural tropical fats such as cocoa butter are neat systems with only three major triglycerides accounting for the vast majority of the composition, above 80%. Mammalian fats in contrast contain a few hundred different fatty acids yielding a vast number of different triglycerides, by combination into triplets of fatty acids. On the other hand, numerous other species such as partial glycerides and other fat‐soluble components can be found in the fat compositions as well (see Chapter  10) and complicate matters even further. The crystallization behavior of the lipid compositions can be altered dramatically depending on the nature of these components (e.g., Smith et al. 2011). Fats are natural raw materials and hence subject to seasonal and regional variability, which introduces additional perturbations into the systems. The raw material nature of the food systems and the variety of science disciplines concerned make it difficult to advocate an engineering thermodynamics approach in this field. However, in understanding and improving processes observed, a sound understanding of the driving forces present is certainly beneficial. In this context, the appreciation of engineering thermodynamics and phase behavior considerations appear indispensable. In the first place this implies the knowledge of the bulk compositions of the phases, which would be coexisting in case one was ever able to produce a system that found itself in its equilibrium state. With manufacturing processes inducing rapid changes of external forces and gradients in temperature and pressure the fat systems are certainly far from equilibrium during processing. This application of significant driving forces increases the likelihood of the formation of “frozen,” metastable states. In combination with the monotropic polymorphism (see Chapter 2) of fats, it is consequently not surprising that many fat‐based systems are manufactured into metastable states that are preferentially persistent during product shelf life. This is possible because, different than in liquid or gaseous states, the relaxation of solid phases and hence the transformation toward the equilibrium state is slow despite the presence of the driving forces for transition. The possible recrystallization processes are typically slow once either the β or β ’ polymorphic forms are established and practically only occur during product shelf life once external stimuli such as temperature fluctuations are present.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Their presence is, however, certainly not limited to the prominent examples of chocolate bloom and the development of POP or tropical graininess in palm oil–based products. There is a long list of reasons to assume deviations from equilibrium. These are for example effects of interfaces on nucleation processes, inhomogeneous distribution of material, and significant temperature gradients. These ensure that crystallization actually takes place under rather ill‐defined local conditions. In polymorphic crystallization processes, the local liquid composition will significantly differ from the bulk’s because of a metastable polymorph disintegrating. It can be said that almost all lipid crystals contain long‐chain aliphatic moieties, which play an important role in defining the mixing behavior of the fatty acids and TAGs, together with polar groups of carboxyl and glycerol groups, respectively. Hence, this chapter is concerned with the description of the phase behavior of lipid mixtures. The hydrophobic interactions can be understood to a large extent by considering the mixing behavior of n‐alkanes and n‐alkenes in the solid state systematically. In addition to the description of this mixing behavior, this chapter covers the thermodynamic and molecular‐level understanding of the mixing behavior of fatty acids, mono‐ and diacylglycerols as well as TAGs The modeling of the solid and liquid phase states will be discussed briefly to facilitate its application for the purpose of interpretation of observations. The previous discussion points out, that the value of the application of engineering thermodynamics to fat mixtures is limited because of the complexity of the matter. However, when applied appropriately the understanding of the phase behavior helps to interpret experimental findings, supplies feedback with respect to data consistency and hence should guide the design of additional experiments. Also because of the complexity of multicomponent phase diagrams, this chapter will primarily use binary phase diagrams to illustrate how the interactions of different molecular species propagate into the solidification behavior of mixtures. The bulk thermodynamics is the basis to any driving force assessment and will thus indicate the tendency of a system to change. Furthermore, the sound generalization of experimental data allows to better benefit from the results of experimental efforts.

3.2 ­Thermodynamic Considerations 3.2.1  Framework for Engineering Calculations Before the discussion of the mixing behavior in solid phases, it is useful to consider first the phase behavior of highly asymmetric systems. Asymmetric in this context is meant to illustrate a significant difference in the pure component melting points of a binary system. For this type of systems, assuming one component to be permanently in the liquid state while the other component is at least in part solid in the temperature range considered, it seems justified to consider the terminology of solvent and solute. This is rightly used to describe fractionation processes based on organic solvents such as hexane. However, considering low temperatures where hexane might crystallize, the system is rather eutectic in its nature. This illustrates that the categorization of phase behavior will strongly depend on the perspective one takes with regard to the temperature and

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pressure ranges considered. Ignoring that, the simple case of a system with a high melting solute (A) and a low melting, thus permanent solvent (B), shall be considered here. Based on the condition that the chemical potential of the solute i is equal in the coexisting phases, the following equation, 3‐1, to compute solid‐liquid equilibria is derived. ln

xis

s i

T , p, x s

xiL

L i

T , p, x L

1 R T

hiFUS TiSL ,p T

hiFUS TiSL ,p TiSL

c P ,i TiSL T c p ,i ln

TiSL T

reshuffled ln

xis

s i

T , p, x s

hiFUS TiSL ,p

xiL

L i

T , p, x L

R T

1

T TiSL

c P ,i R T

TiSL T

T ln

TiSL T (3-1)

Here x si and x Li indicate the molar fraction of component i in the solid and liquid phase, respectively. The different activity coefficients are indicated as γ. Other symbols are ideal gas constant R, temperature T, heat of fusion Δh, melting point temperature of component i TiSL , and pressure, p. The difference of isobaric specific heat capacities between the liquid and the solid state ΔcP is however often ignored because of its marginal contribution. The way the activity, which describes the non-ideality of mixtures, is derived from commonly used GEXCESS models can be found in engineering thermodynamics textbooks (Poling et al. 2004; Sandler 2006). However, the solubility is often described based on the so‐called Hildebrandt equation (Eq. 3‐2). Different from Eq. 3‐1, an ideal mixture in the liquid phase and a pure solute i solid phase are assumed. Additionally, the temperature dependencies of the enthalpy and entropy changes on fusion are ignored. xiL

exp

hiFUS TiSL ,p R T

1

T TiSL



(3-2)

Introducing polymorphism of component i with the crystal structures P1 and P2, it is rather easy to establish the differences between monotropy, only one stable polymorph (Fig.  3.1a), and enantiotropy, more than one stable polymorph (Fig.  3.1b). Indicated in Fig. 3.1(a), the melting point temperature of the high temperature polymorph P1(TSL,P1) is higher than the melting point temperature of the polymorph 2(TSL,P2). In both figures the solubility curve of the high temperature stable polymorph P1 is identical and indicated as solid line. In Fig. 3.1(a), the polymorph P2 with lower melting point temperature has an enthalpy of fusion which is lower than the enthalpy of fusion of polymorph P1. The dashed curve represents the metastable solubility curve of polymorph P2. In contrast the solubility curve of polymorph P2* in Fig. 3.1(b; dashed dotted) indicates that the polymorph P2* is stable at lower temperatures (enantiotropic polymorphism). The difference in the solubility curves for P2 and P2* is a consequence of a significantly higher enthalpy of fusion for polymorph P2*, which is

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

(b)

300 L

290

T (K)

280

260

250

250 0

0,5

TSL, P1

240

1

TSL, P2, ms = TSL, P2*, s

270

260

240

L

280

TSL, P2

270

300 290

TSL, P1 T (K)

(a)

TSS, P1–P2

0

Xi

(c)

1

0,5 Xi

(d) L GSL,P1

G

G

P2→L GSL,P2

GSL,P2

P2→P1 P1→L

T

GSL,P1 L

T

Fig. 3.1  Solubility curves (top) and Gibbs free energy (bottom) for a system showing monotropy (a) and (c) or enantiotropy (b) and (d).

larger than the one of polymorph P1. The temperature at which the solubility curves of the different polymorphs cross each other is characterized by a discontinuity in the apparent solubility curve. This temperature is per definition the equilibrium solid‐ solid transition temperature (TSS) between the different polymorphic forms. Taking a look at the sketch of the Gibbs free energy as a function of temperature as shown in Fig. 3.1.(c and d) for monotropic and enantiotropic polymorphism, respectively, it is obvious that the enantiotropic polymorphism can only appear if the slopes of the energy curves differ. This is a necessary condition for the curves to cross. Additionally, the temperature at which the two different polymorphs are assumed to have the same energy level, the solid‐solid transition temperature, has to be lower than the melting point temperatures of both polymorphs. These are obviously the intercepts of the respective Gibbs energy curve per polymorph with the liquid Gibbs energy curve. Considering only Eq. 3‐2, the different curves result from a higher enthalpy of fusion for the low‐temperature stable polymorph P2*. This condition for enantiotropic polymorphism appears counterintuitive at the first sight but actually expresses that at zero Kelvin the polymorph P2* has a lower Gibbs free energy. Additionally, the entropy, which is the negative slope of the isobaric Gibbs energy over temperature curve, of the high melting polymorph (P1) is larger than for the low temperature stable polymorph (P2*). This condition can also be expressed in differences in the specific heat capacities being bigger for the high temperature stable polymorph P1 (Grunenberg et al. 1996).

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For this enantiotropic polymorphism, as displayed in Fig.  3.1 and found in for e­ xample  even numbered n‐alkanes with chain length in excess of 22 carbon atoms (Ksiazczak et al. 1994), we find that at the solid‐solid transition temperature TSS,P1 P2 polymorph 1 and polymorph 2 are in equilibrium. This solid‐solid equilibrium can be described analogue to the solid‐liquid equilibrium. For the condition of equal chemical potential of component i in the coexisting solid phases straightforwardly Eq. 3‐3 for mixed solid systems evolves. ln



xiP 2

P2 i

T , p, x P 2

xiP 1 , iP 1

P1

T , p, x

hiP 1 P 2 Ti P 1 P 2 , p R T Ti P 1 P 2 T

T ln

1 Ti

T

Ti

P 1P 2

P 1P 2

T

c P ,i R T (3-3)

In essence, compared to Eq. 3‐1, only the indication liquid (L) needs to be replaced by a second solid phase and the differences in enthalpy and specific heat capacity adjusted for the transitions considered. The transition from the high‐temperature stable polymorph 1 to the low‐temperature stable polymorph 2 involves a change in energy described in terms of the enthalpy of transition, ΔhP1P2. For a pure solute, the enthalpy of fusion of the low‐temperature stable polymorph 2 is consequently always exceeding the enthalpy of fusion of the high‐temperature stable polymorph 1. It should be noted here that the fact that the solubility of both polymorphs 1 and 2 are equal at the transition temperature, TP1P2, is not incidental but a necessary feature because in equilibrium the relative energies of the solid phases are independent of the solvent present. However, regularly conflicting observations are made which are the result of kinetic effects. 3.2.2  Phase Behavior of Co‐Crystallizing Components Attempts to systematically categorize the phase behavior of binary systems with limited asymmetry in melting point temperatures observed are actually scarce. Dating back a long time, the five types of phase behavior defined by Bakhuis‐Roozeboom and co‐ workers (Bakhuis‐Roozeboom 1901; Bakhuis‐Roozeboom et al. 1911) cover the main features observed. This is, for example, discussed by Kitaigorodsky (1973). In essence, the combination and repetition of a few main features allows us to describe the large number of different solid‐solid‐liquid phase behaviors, as for example found in the field of alloys. These building blocks are intermediate mixed solid phases, continuous solid‐ liquid two‐phase regions, and solid‐solid two‐phase regions. To simplify the discussion of the different combinations of the solid‐liquid phase behavior interactions in the liquid phase will be ignored. Figure 3.2 depicts some basic features for systems composed of components A and B. Insert I shows completely miscible solid and liquid phases separated by a neat two‐ phase region (solid solution liquid). The two‐phase region (S L ) separates the two one‐phase regions, liquid (L) and solid (S). Resulting from interactions in either the solid or the liquid phase a deformation of the two‐phase region (S L ) occurs. With increasing interaction energy, the limits of this deformation occur. The depicted hylotrops are characterized by congruent melting of the solid solution. Under isobaric

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals Ia

I

L

Ib L

L

T

S+L

T

T

S+L

S+L xB

xB

xB II

T

L

Sa + L

Sb + L

Sa + Sb xB

Fig. 3.2  Solid‐liquid phase transitions; complete miscibility of solid and liquid phase, hylotrop with a minimum (Ia), hylotrop with a maximum (Ib), and immiscibility of the solid phase (II).

I

II

III

L

L

L

S+L

Sβ Sα + Sβ xB

Sβ

L + Sβ

L + Sα

Sβ

T

Sα

L + Sα

T

T

L + Sβ

c.p. Sα

Sα+Sβ xB

Sα

Sα + Sβ xB

Fig. 3.3  Phase behavior of an eutecticum (insert I), a miscible system with a solid‐solid demixing two‐phase region (insert II), and a peritecticum (insert III).

conditions this must result in either a minimum (Ia) or maximum melting temperature (Ib; Schaum 1898; Smirnov & Kurnakov 1910). With stronger interactions in the solid phase partial or complete immiscibility in the solid phase emerges (Insert II of Fig. 3.2). Here, either pure solid A (S A L ) or pure solid B (SB L ) is in equilibrium with a mixed liquid phase at higher temperatures. According to the indication in insert II the two coexisting solid phases are crystalline pure A and pure B at temperatures below the three‐phase line. Anyhow, this is unrealistic and the coexisting solid phases are rather intermediate cases with limited mutual solubility of A and B. The coexisting solid phases are either rich in solute A (Sα) or solute B (Sβ). See insert I of Fig. 3.3.

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Crystallization of Lipids

The sequence of the inserts of Fig. 3.3 suggests that starting from insert II, insert I and III can evolve from the merger of the solid‐solid demixing two‐phase region (S S ) with the solid‐liquid two‐phase region shown in insert II. Insert II displays a low‐temperature solid‐solid two‐phase region with an upper critical point (c.p.) where both solid phases (S S ) become identical. When the solid‐liquid and solid‐solid two‐phase regions overlap a three‐phase equilibrium line emerges (see inserts I and III). Because of Gibbs’ phase rule, this solid‐solid‐liquid three‐phase line must be isothermal and separates three different two‐phase regions at any chosen pressure. In how far this overlapping of two‐phase regions results in an eutecticum (insert I) or a peritecticum (insert III) depends primarily on the shape of the solid‐liquid two‐phase region. By simple further geometric considerations it is easy to envisage a peritecticum or eutecticum adjacent to a hylotrop. This typically results in discontinuities in phase transition lines observed experimentally. For the description of the phase behavior of lipid systems, it often happens that very asymmetric eutectic systems are characterized as monotectic. In contrast to asymmetric eutectic systems, monotectic systems are characterized by the presence of a solid‐solid‐liquid three‐phase line involving a liquid which is rich in the lower melting component. Additionally, monotectic systems reveal a solid‐liquid‐ liquid three‐phase line at higher temperatures. Here, the solid phase rich in the high melting component is in equilibrium with a liquid phase rich in the low melting component and another one with an intermediate composition. Because the presence of two immiscible liquid phases in binary lipid systems composed of similar molecules is highly unlikely, to observe monotectic phase behavior is equally unlikely and likely often used inappropriately. Intermediate phases related to temperature maxima are mostly considered to be molecular compounds. This implies a mixed crystal composed of two or more molecular species being incorporated into the crystal lattice at a fixed ratio. The presence of molecular compounds has been reported for numerous TAG systems (e.g., Sato 2001; Sato & Ueno 2001; Engström 1992, see below). The occurrence of melting point maxima or minima is not strictly limited to fixed stoichiometric ratios, as can be seen in alloys where intermediate solid phases with extended composition ranges occur. However, depending on the quality of the experimental observation it remains often difficult to distinguish between a compound with compositional tolerance and a hylotrop. Careful interpretation of experimental observations should build on the fact that both phenomena relate to different phase boundaries, solidus lines (hylotrop), and three‐phase lines (compound), respectively. Combining the different features of phase behavior described a hypothetical phase diagram as complex as shown in Fig. 3.4 can be constructed. It should be noted here that additional solid‐solid transitions, thus polymorphism, as for example found for mixtures of long chain n‐alkanes (e.g., Coutinho & Stenby 1995) are not considered here. From left to right, with increasing concentration of component B, one finds the following regions; a solid solution, rich in A (Sα); a two‐phase region of Sα coexisting with a compound crystal (C) that has a stoichiometric ratio of 2 A : 1 B; the compound (C) itself; a two‐phase region where the compound (C) coexists with the mixed intermediate solid phase Sγ; a one‐phase region of mixed solid phase Sγ; a two‐ phase region of solid γ coexisting with a mixed solid phase rich in solute B (Sβ); and the solid phase rich in B (Sβ). All three‐phase lines separate a set of three different two‐ phase regions. Both, the hylotrop and the compound cause an intermediate local

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals L C L + Sγ L + Sα

L+C

L + Sγ

T

L+C

L + Sβ

Sγ Sα

Sγ+ C

Sγ+ Sβ

Sα + C

Sβ xB

Fig. 3.4  Hypothetical binary phase diagram of a binary system not showing polymorphism. Phases shown: three mixed solid phases (plain); three solid‐solid two‐phase regions (vertically hatched); and six solid‐liquid two‐phase regions (horizontally hatched). Temperature maxima relate to a compound crystal (C) and a hylotrop.

temperature maximum. Identification of the causes of these two temperature maxima, either hylotrop or compound, is impossible by analysis of the liquidus line only. However, depending on the type of phenomenon the signals from either thermal (differential scanning calorimetry [DSC]) or structural analysis (X‐ray diffraction [XRD]) will display different patterns (solidus or three‐phase line) in the vicinity of the temperature maximum. In detail, the properties of the mixed solid phase γ change continuously with composition in the solid one‐phase region adjacent to the maximum. In contrast to this, the solid‐solid two‐phase regions (S C ) and (C S ) are characterized by ­linear combination of each of the two characteristic signals of S(C) and respectively (Sα) or (Sγ) that will gradually change in relative strength. Figure 3.1 already illustrates the effect of a solvent on the solid phase behavior. As shown, the relative stability of different solid phases is practically independent of the presence of the liquid phase. For the phase behavior shown in Fig. 3.4, solvent addition would hence primarily cause a shift to lower dissolution temperatures. This is because the system does not show any temperature‐induced solid‐solid phase transitions. However, if solid‐solid phase transition occurs, which is likely for the chainlike molecules considered here, different solid phases could be present at lower dissolution temperatures. For example, the discontinuities of the liquidus line found in the ternary n‐alkane system methane docosane tetracosane are explained this way (Floeter et al. 1998). These phenomena can properly be described based on Eqs. 3‐1 and 3‐2, but depending on the system at hand, this is often hampered by the lack of knowledge of physical properties and interaction parameters for the different solid phases. This challenge is further complicated through possible co‐crystallization of the solvent component with either component A or B. On the other hand, the rules as described for simpler systems are also valid for complex systems and the underlying binary phase diagrams rarely look as complex as shown in Fig. 3.4. Beyond these considerations concerning equilibrium states only, it has to be acknowledged that the effect of solvents on

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the appearance of different phases can be substantial but is primarily of kinetic nature. Consequently, in systems with monotropic polymorphism, which is characteristic for lipid systems, the transition from kinetically induced metastable states to more stable states does not strictly follow the framework outlined here. This is easily understood by considering the lack of reversibility of the transitions from a metastable to more stable polymorphic forms. However, one should note that considering a single polymorphic form the application of the set of rules presented appears appropriate. 3.2.3  Governing Principles for Phase Boundaries The configuration and composition of phase boundaries are guided by a theoretical framework, basic thermodynamic principles, formulated in part already in the 19th century. The Gibbs’ phase rule should by all means be obeyed. It postulates that for a given system, the degree of freedom is equal to the number of components present minus the number of coexisting phases plus two. The rule hence defines the dimensionality of a phase region. Consequently, a three‐phase (solid‐solid‐liquid) equilibrium in a binary system yields one degree of freedom. With the pressure fixed only isothermal three‐phase lines are possible. Goodman et al. (1981) reviewed the Gibbs‐Konovalov rule and reiterated a few important constraints on the slopes of coexistence curves. In the case of congruent melting, melting of a compound crystal or melting at a hylotrop, the slope of the liquidus line must be zero. This is valid for any liquidus line ending at this point. Consequently, intermediate temperature maxima in phase diagrams should never have the shape of a cusp. Furthermore, the solidus lines in a hylotrop are similarly constrained necessitating it to be tangential to the horizontal liquidus line (dT/dx 0) under isobaric conditions. It is tempting to treat a compound crystal as an additional pure component that practically splits a phase diagram in essentially two separate phase diagrams. However, the liquidus lines at the compound are subject to the constraint described previously. Hillert (1982) formulated another set of constraints for the slopes of phase boundaries by arguing based on the Gibbs‐Konovalov rule, the Gibbs‐Duhem relation and Schreinemakers’ rule (Schreinemakers 1911). In simple terms it is stated that for two univariant lines crossing, a higher‐order phase equilibrium line (e.g., two binary liquidus lines crossing on a solid‐solid‐liquid line) appears. The two‐phase lines metastably extend into another phase region. These extensions are c­ onstraint additionally to point into phase regions of identical order. Even though this appears almost trivial for binary systems, this becomes more relevant once three‐phase regions in ternary systems are considered. It should be noted that the framework ­outlined gives guidance to the construction of phase diagrams in systems based on a defined number of pure components. Admittedly, this is in fact rarely the case for ­practical multicomponent fat compositions.

3.3 ­Effects of Molecular Structures on the Phase Behavior The molecular interactions of the lipid molecules in the solid phases are most clearly expressed in the phase behavior displayed. In the following, the comparison of the phase behavior of similar systems illustrates how changes in the molecular structure influence the phase behavior in mixed systems. Application of the framework outlined

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

previously causes in some cases changes with respect to the translation of the experimental evidence into a phase diagram compared to the original publications. These modified interpretations are, however, not based on any new experimental evidence but just based on interpretation of the originally published data. One of the distinguishing elements is that a first‐order equilibrium polymorphic transition should always relate to a two‐phase region. This interpretation can however not be applied strictly because a lot of the phase transitions observed in lipids are not subject to equilibrium but rather as a result of kinetics, hence relating to metastable states. Furthermore, it appears that in rather asymmetric systems it is necessary to consider a so‐called degenerate eutectic, which means that even though the eutectic or eutectoid point is very close to the pure lower melting component, there should be a three‐phase line and a two‐phase region representing the solid lower melting component in equilibrium with a liquid solution. 3.3.1  Aliphatic Chain‐Chain Interactions: n‐Alkanes To illustrate the effects of chain‐chain interactions in the solid phase n‐alkane systems are most appropriate. For pure n‐alkanes, the increase of the melting point of pure components increases systematically with chain length, taken the same crystal structure is considered. For reasons of simplicity and lack of relevance of aliphatic chains with odd number of carbons, the differences between odd‐ and even‐numbered carbon chains will not be discussed here. However, taking a closer look at the phase behavior of the pure n‐alkanes one finds that starting at carbon numbers equal or greater than 22, enantiotropic polymorphism occurs and a stable rotator phase is present as high‐temperature solid phase. Considering now binary n‐alkane mixtures with chain length differences of two carbon atoms, the systematic study by Mondieig et al. (2004) impressively illustrates the complexity of phase diagrams evolving for these simple mixtures. In Fig. 3.5 the phase diagram for the octane (C8) decane (C10) binary system is depicted (Mondieig et al. 2004). The display shows a clear eutectic system with limited miscibility close to the pure components. In contrast, the mixtures of docosane (C22) and tetracosane (C24) reveal a much more complex picture (see Fig. 3.6). At high temperatures, Fig. 3.5  Phase behavior of two n‐alkanes, octane (C8) and decane (C10), differing by two carbon atoms. Adapted by Mondieig et al. (2004).

–30

L

Temperature (° C)

–40

–50

–60

0 C8

0.2

0.4

0.6 x

0.8

1 C10

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Crystallization of Lipids

Fig. 3.6  Phase behavior of two n‐alkanes, docosane (C22) and tetracosane (C24), differing by two carbon atoms. Data according to Mondieig et al. (2004).

333 L 323 Temperature (K)

72

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293

0

C22

0.2

0.4

0.6

0.8

x

1 C24

the aforementioned rotator phase occurs spanning the whole composition range. The necessary two‐phase regions separate this solid phase from the one‐phase liquid and the intermediate lower rotator phase. At lower temperatures, the limited solubility regions are found close to the pure components similar to the C8‐C10 system. However, in the intermediate composition range three distinct (orthorhombic, monoclinic, and orthorhombic) mixed crystal phases occur. Consequently, four three‐phase lines and numerous two‐phase regions are present. Because the rotator phase also occurs in the intermediate composition range of systems constituted of shorter chain lengths n‐ alkanes, the conclusion that the occurrence of the rotator phase is driven by entropy (Mondieig et al. 2004) is certainly justified. What Mondieig et al. (2004) further demonstrated is that with decreasing chain lengths of the constituting n‐alkanes, the low temperature solid phase behavior becomes less complex. 3.3.2  Mixtures of Fatty Acids This section discusses the phase behavior of the binary mixtures of principal fatty acids. Because of the presence of unsaturation in hydrocarbon chains in naturally occurring fatty acids, their mixing behavior becomes more complex compared to that of n‐alkanes having a fully extended chain conformation (Dorset 2005). However, this chapter aims to clarify the systematic sequence behind the phase behavior of fatty acids following the rules stated in the previous sections. 3.3.2.1  Saturated/Saturated Fatty‐Acid Systems

Taking the nature of fatty acids into account it is not surprising that also for their binary mixtures a systematic evolution of the solidification behavior emerges with increasing chain length of the fatty‐acid moieties. In 1986, Small provided a brief summary of the binary mixtures of saturated fatty acids. Between 2007 and 2009 Costa reported rather comprehensive data sets for binary mixtures of saturated fatty acids (Costa et al. 2009a). Subsequent to the original study of two series of binary mixtures (Costa et al. 2007),

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Fig. 3.7  Phase behavior of saturated fatty acids (FAn) differing by two carbon atoms (FAn=2), with n = 8, 10, 12, 14, and 16. Modified Costa et al. (2009a, 2009b, 2009c).

350

Temperature (K)

330

310

290

270

0 FAn+2

0,25

0,5 x

0,75

1 FAn

data on three series of the binary mixtures each composed of fatty‐acid pairs with constant differences in carbon numbers were published. Systems with differences of two (Costa et al. 2009a), of four (Costa et al. 2009b), and of six carbon atoms were studied (Costa et al. 2009c). Figure 3.7 is based on the experimental data reported for the systems with fatty‐acid chains n and n + 2 with n = 8, 10, 12, 14, and 16. Even though Fig. 3.7 is based on the data reported, it was necessary to marginally modify the phase boundaries given (Costa et al. 2009b) to obey the rules for phase boundaries as outlined. The introduction of an additional three‐phase line was necessary because the original paper suggested unrealistically the existence of a four‐phase line. Starting from the caprylic and capric acid system at lowest temperatures one finds that the phase behavior is characterized by four three‐ phase lines and five mixed solid phases (hatched areas). Four of these mixed solid phases are adjacent to the pure components. This general pattern of the phase behavior is repeated throughout this series of binary systems. It appears remarkable in the first place that the extension of the temperature range in which the solid‐liquid transitions take place is shrinking with increasing chain lengths of the fatty acids in the system. However, this is less surprising considering that increasing length of the aliphatic chain relates to larger enthalpies of fusion of the pure components and hence less steep solubility curves. Additionally, the relative effect of extending the aliphatic chain by two carbon atoms is decreasing with increasing chain length, and consequently differences in pure component melting points shrink as well. The pattern found for the systems with chain lengths differing by two carbons is actually also found for the systems with chain length differences of four or six carbons. Compared to the evolution of phase behavior in n‐alkanes, it is thus found that the presence of the carboxyl group is

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dramatically damping the effect of chain length elongation or length differences because a wide range of saturated fatty‐acid systems shows qualitatively the same phase behavior. 3.3.2.2 Saturated/cis‐Monounsaturated Fatty‐Acid Systems

By introduction of aliphatic chains with unsaturated bonds it is expected that the regularity in the phase behavior of binary systems becomes less obvious. In the systems considered previously, the systematical order relates to simple geometric arguments of overlapping elements of the aliphatic chains. In general, binary mixtures made of saturated and cis‐monounsaturated fatty acids exhibit practically no miscibility nor do they form molecular compounds. Inoue’s group conducted systematic studies of the binary mixtures of oleic acid (OA) with different saturated fatty acids having different chain lengths, Cn. Both, DSC and Fourier transformed infrared spectroscopy (FT‐IR) were applied to binary systems composed of oleic acid with either C8 (caprylic), C10 (capric), C12 (lauric), C14 (myristic), C16 (palmitic), C18 (stearic), and C22 (behenic) (Inoue et al. 2004a, 2004b, 2004c). The phase behavior found by DSC melting studies for long‐chain saturated fatty acids, respectively stearic and behenic acid, mixed with oleic acid is characterized as monotectic in the original paper. However, seeing the evolution of the systematic phase behavior, it was found that the type of mixtures involving shorter saturated fatty‐acid chains reveal eutectic behavior. This is shown for the oleic/lauric acid system in Fig. 3.8 (Inoue et al. 2004a). This leads, in agreement with the comments on monotectic systems made previously, to the conclusion that the mixtures of saturated fatty acids and oleic acid show eutectic behavior. With increasing chain length of the saturated fatty acid, the system becomes however increasingly asymmetric such that the crossing point of the solubility curves, the eutectic point, approaches the composition and melting temperature of pure oleic acid. The derived eutectic behavior is additionally confirmed by the FT‐IR spectra obtained for the mixtures that correspond to the superposition of the spectra characteristic for the respective pure components. At lower temperatures, these eutectic systems are characterized in that the oleic acid crystals assume the γ polymorph, whose presence is expected from the pure component Fig. 3.8  Eutectic behavior of oleic acid and lauric acid. filled symbols, stabilized samples; L, liquid; open symbols, nonstabilized, transient samples. Inoue, Hisatsugu, Ishikawa, et al. (2004a, 2004b, 2004c).

50 L 40 30 T (° C)

74

20

L + LA

L + OA (α)

10 OA (α) + LA

0 –10

OA (γ) + LA –20 100

80

60

40

coleic acid (mol%)

20

0

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

behavior. Since the γ to α polymorphic transition is reversible, this order‐disorder conformational change should be considered as an equilibrium transition (Kobayashi et al. 1986). Hence, the isothermal three‐phase line in this case is defined by two two‐phase regions, crystalline lauric acid in equilibrium with the γ or the polymorph of oleic acid, respectively, and a transition point (see Fig.  3.8). The open symbols in Fig.  3.8. indicate the transition temperatures determined for nonstabilized samples. These data  indicate effects of possible kinetically induced pseudo‐miscibilities and will be ­discussed later. In summary, the results show that all saturated fatty acids are immiscible with oleic acid in the crystalline phase. According to a thermodynamic analysis of the liquidus lines based on the regular solution model for the melt, the liquid mixtures composed of oleic acid and stearic acid behave almost ideally. Increased non‐ideality in the liquid phase, even though small in these mixtures, emerges with increased disparity of the lengths of the chains irrespective of the saturated aliphatic chain becoming shorter or longer than the 18 carbon atoms of oleic acid (Inoue et al. 2004a, 2004c). Different from the liquid mixtures, it is justified to assume that for solid mixtures steric hindrance due to chain conformation is important. The all trans straight aliphatic chains of the saturated fatty acids make it difficult to accommodate the bend oleic acid chains in the crystal lattice, and hence cause eutectic behavior. This interaction is most certainly also present in TAG crystals accommodating both saturated and unsaturated fatty‐acid moieties. 3.3.2.3  cis‐Monounsaturated/cis‐Monounsaturated Fatty Acid Systems

Next to the length of the aliphatic chain and the level of saturation of the carbon‐carbon bonds, the position of the unsaturated bond in the aliphatic chain determines the interaction of aliphatic chains. Table 3.1 summarizes the solid‐liquid phase behavior of the binary mixtures composed of different pairs of cis‐monounsaturated fatty acids. Figure 3.9 visually characterizes the different fatty acids and illustrates the differences in Δ‐ and ω‐chain expression of the position of unsaturated bonds. The complicated mixing behavior mentioned in Table 3.1 is attributed to the combined effects of the chain length disparities, the position of the double bonds and the  polymorphism of the pure components. To this end, the characterization of cis‐ monounsaturated fatty acids using ( x y ), in which x is the number of carbon atoms Table 3.1  Phase behavior of binary mixtures of cis‐mono‐unsaturated fatty acids. Combinations

Mixing Behavior

gondoic acid (GOA, C20:1 Δ11ω9)/asclepic acid (APA, Δ11ω7)

miscible

GOA (C20:1Δ11ω9)/oleic acid (OA, C18:1Δ9ω9) OA (C18:1Δ9ω9)/petroselinic acid (PSA, C18:1Δ6ω12) OA (C18:1Δ9ω9)/ APA (C18:1Δ11ω7) palmitoleic acid (POA, C16:1’Δ9ω7)/APA (C18:1Δ11ω7) OA (C18:1Δ9ω9)/POA (C16:1’Δ9ω7) OA (C18:1Δ9ω9)/myristoleic acid (MOA, C14:1’Δ9ω5)

eutectic eutectic eutectic eutectic peritectic/hylotrop peritectic/hylotrop

Cn, number of carbon atoms.

75

Crystallization of Lipids Δ

ω

O CH3 GOA, C20:1 Δ11ω9

HO O HO

CH3

APA, C18:1 Δ11ω7

CH3

OA, C18:1 Δ9ω9

CH3

PSA, C18:1 Δ6ω12

O HO O HO O CH3

HO

POA, C16:1 Δ9ω7

O CH3

HO

MOA, C15:1 Δ9ω5

Fig. 3.9  Fatty acids with different Δ‐ and ω‐chain lengths: gondoic acid (GOA), asclepic acid (APA), oleic acid (OA), petroselinic acid (PSA), palmitoleic acid (POA), and myristoleic acid (MOA). Fig. 3.10  Mixtures of oleic acid (black solid curve, liquidus line) with stearic acid (dashed double dotted line), lauric acid (dotted line), petroselinic acid (dashed line), gondoic acid (dashed dotted line), or asclepic acid (grey solid line). Arrows indicate eutectic mixture. Adapted from Inoue et al. 2004a, 2004c; Sato et al. 1997; and Yoshimoto et al. 1991; individual data points omitted for clarity of presentation.

80

60

T (° C)

76

40

20

0 –20 100

80

60

40

20

0

coleic acid (mol%)

of the chain segments between the COOH‐group and the double bond (Δ chain), and y is the number of carbon atoms between the double bond and CH3 end group (ω chain), respectively appear quite useful. As Fig.  3.10 illustrates, the liquidus lines of systems containing oleic acid show a remarkable systematic order. The mixtures with petroselinic acid (PSA), gondoic acid (GOA), and asclepic acid (APA) show eutectic behavior like the systems with saturated fatty acids. Consequently, eutectic compositions and temperatures of the solid‐solid‐ liquid three‐phase lines are defined by the respective solubility curves and their

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Fig. 3.11  High‐temperature phase behavior of palmitoleic acid mixed with oleic acid (circles, solid line) and myristoleic acid mixed with oleic acid (squares, dashed line). Adapted from Inoue et al. 1992).

20

T (° C)

10

0

–10

–20 100

80

60

40

20

0

coleic acid (mol%)

intercept with the oleic acid solubility curve (full black line). The eutectic behavior is hence found when the Δ‐ and ω‐chain lengths are different, even if the polymorphic behavior of the pure components constituting the mixtures are similar. However, when the mixed monounsaturated fatty acids have the same Δ‐chain length, differences in the ω‐chain length appear to be less disturbing. This is found in the gondoic (Δ11, ω9)/asclepic (Δ11, ω7) acid system where it becomes increasingly difficult to identify eutectic behavior (Sato et al. 1997). However, the dissolution temperatures identified for gondoic acid rich mixtures coincide with those found in the system of gondoic/oleic acid, which shows eutectic behavior. For shorter chain lengths, the effect of differences in Δ‐ and ω‐chain lengths becomes more apparent because the binary systems composed of oleic acid (Δ9, ω9) with either palmitoleic acid (Δ9, ω7) or myristoleic acid (Δ9, ω5) show at least partial miscibility, see Fig. 3.11. This indicates that an identical Δ‐chain length supports the miscibility of different fatty acids. In essence, both systems reveal the same type of phase behavior that resembles that of n-alkanes with different chain lengths (Fig.  3.7). At high oleic acid concentrations, a limited miscibility is found. A peritectic three‐phase line with a low‐temperature solid‐solid two‐phase region marks the border of miscibility. The solubility of palmitoleic acid in the oleic acid dominated crystal lattice is expectedly higher than the one of myristoleic acid. At higher concentrations of the shorter chain fatty acids, palmitoleic or myristoleic, the experimental data suggest miscibility in the solid phase with a low‐temperature hylotrop at approximately 25% and 17% (molar) of oleic acid, respectively (Fig. 3.11). Not surprisingly, the liquidus lines of the system containing myristoleic acid cover a wider temperature range. It should be recognized that the phase behavior displayed here is derived from DSC data only. Regarding the solid‐solid transitions in the systems considered in this section that have been identified at lower temperatures, a similar systematic order emerges. A three‐phase line as mentioned previously in the systems composed of saturated fatty acids and oleic acid is present at the transformation temperature of pure oleic acid,  see Fig.  3.8. This line separates two two‐phase regions with oleic acid being present in  the conformation at lower temperatures and in the conformation at higher  

77

Crystallization of Lipids

Fig. 3.12  Low‐temperature phase behavior of different binary fatty‐acid systems. Kinetically induced (high cooling rates, no annealing) solid‐solid phase transitions of different fatty acid mixtures: stearic/oleic acid (circles, dotted curve), lauric/oleic acid (squares, dashed curve), gondoic/oleic acid (triangles, solid curve), asclepic/oleic acid (hexagons, dashed double dotted curve), petroselinic/oleic acid (diamonds, dashed dotted curve). Open symbols relate to γ–α transformation of pure oleic acid.

0

–10

T (° C)

78

–20

–30

–40 –50 100

80

60

40

20

0

coleic acid (mol%)

temperatures; see Fig. 3.12 horizontal line at the top. This is most obvious in the systems with saturated fatty acids. In other systems (e.g., oleic acid mixed with petroselinic or asclepic acid,) this transition is much less prominent. In the petroselinic containing system this three‐phase line is only detected once cooling is done at limited rates (Yoshimoto et al. 1991). For mixtures of oleic acid and asclepic acid, transitions found at lower cooling rates are less clearly identifiable. However, one could argue that the indication of the three‐phase line as to ’ transition of asclepic acid is doubtful because  asclepic  this transition does not occur in pure acid and its temperature coincides with the γ–α transformation of pure oleic acid. Looking at Fig.  3.12. it appears that at high cooling rates (50 K/min) significantly reduced temperatures of the γ–α transformation for oleic acid rich mixtures are observed. This is most likely because of the metastable inclusion of other fatty‐acid molecules in the oleic acid γ–crystal structure because of the high cooling rates applied. In contrast, for stabilized samples, assumed to be in equilibrium, this line practically coincides with the γ–α transition of pure oleic acid, see open symbols in Fig. 3.12. The fact that the slope of the metastable transition curve is very similar for the different systems indicates the disturbance of the quite ordered polymorph by kinetic inclusion  of alien fatty acids. For combinations of oleic acid with either saturated fatty acids, asclepic or petroselinic acid, the data suggests a metastable eutectic behavior. For mixtures of oleic with gondoic acid the low temperature transition curves give less clear clues. For this system, the Δ‐chain length differs by two carbons, while both ω‐chain lengths are equal to nine. Different than for the other systems also for stabilized systems and slow cooling rates the three‐phase line, which is related to the γ–α transition of pure oleic acid has not been found experimentally. In the original publication, this course of the solid‐solid transition is related to immiscible and phases. Such a four‐   impossible—so phase transition is however unlikely—theoretically actually that the phenomena encountered are certainly related to kinetics. The mixtures of oleic acid with either palmitoleic or myristoleic acid also show a similar course of the γ–α transformation temperatures for oleic acid rich mixtures. Overall,

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

these systems, composed of fatty‐acid pairs with identical Δ‐chain length, reveal quite a different phase behavior compared to the other systems. This is as a result of the higher compatibility of the different fatty acids in these systems. Also here, no three‐ phase line relating to the γ–α transformation of pure oleic acid is found because of the miscibility found for these systems. In the intermediate composition range a maximum transition temperature for the γ–α transformation was found. In the original publication, this maximum was related to a compound crystal in the polymorph (Inoue et  al. 1992). Compared to the original paper, Fig. 3.12 suggests a somewhat different low‐temperature phase behavior. It seems fair to relate the solid‐solid transition temperature maximum to the solid‐liquid phase behavior depicted in Fig.  3.11, which is  characterized by a low‐temperature hylotrop. This indicates an energetically less favorable mixing state in the polymorph for concentrations around 30% of oleic acid. Consequently, this maximum appears to be induced by the polymorph. Respecting  speculative. However, this, the detailed phase behavior shown in Fig. 3.13 remains rather taking the miscibility in the polymorph into account proposing an intermediate  one‐phase region in the polymorph appears justified. Furthermore, it remains ­speculative but likely that mixtures of oleic acid and myristoleic acid will show a ­similar phase behavior. Compared to systems composed of saturated fatty acids or alkanes, systems containing unsaturated fatty acids offer more parameters to consider. Even though the discussion was limited to fatty acids with only a single double bond, chain length and relative position of the double bond (Δ, ω) need to be considered. It appears that both matter for the stability of mixed solid phases with a dominance for the distance between the carboxyl group and the double bond (Δ chain). Analysis of the DSC data reveal that the interaction in the liquid phase is primarily depending on chain‐length disparities. The contribution of non‐idealities in the liquid phase remains negligible for practical considerations. The phase behavior found for the different systems follows rather systematic patterns. In particular, for the low temperature solid‐solid transitions, ­ it needs to be acknowledged that metastable states induced by high cooling rates tend to overshadow the systematic order identified. Fig. 3.13  Phase behavior of mixtures containing palmitoleic/oleic acid (circles, solid line) and myristoleic/oleic acid (squares, dashed line).

0

T (° C)

–20

–40

–60

–80 –100 100

80

60

40

coleic acid (mol%)

20

0

79

Crystallization of Lipids

3.3.2.4  Other Mixed Fatty‐Acid Systems

The physiological functions of conjugated linoleic acids (CLAs) vary between different isomers. Thus, the separation of CLA isomers has a high significance in the industrial uses of the CLAs. Uehara et al. (2008) found that highly efficient separation of the two CLA isomers was achievable by crystallization from acetone solutions applying medium‐chain fatty acids as additive. For this purpose, the physical properties of binary mixtures of two isomers of CLAs, 18 carbons, 9‐cis, 11‐trans‐CLA (9c,11 t) and 10‐trans, 12‐cis‐CLA (10 t,12c) and a medium‐chain fatty acid, capric or decanoic acid (C10A; see Fig. 3.14) were examined (unpublished). The phase diagrams reveal clear differences between the two systems. The mixtures of capric acid (CA) with CLA‐9c,11 t indicate eutectic behavior. Close to the pure components both systems show comparable limited miscibility. However, for mixtures of capric acid and CLA‐10 t,12c at the molar concentration ratio of 1:1, the formation of a molecular compound (MC) was observed. The existence of this compound crystal is prominently demonstrated by small‐angle XRD patterns. These indicate that the chain‐length structure of the molecular compound is quite different from the  simple double chain‐length structure of the constituting pure components, CLA‐10t,12c or capric acid (CA). For the molecular compound an interdigitated double chain‐length structure is assumed. This is driven by acyl chain interactions between the chains of CA and the chain segments of the CLA‐18:2, 10t,12c chain between the carboxyl group and the first double bond. This indicates a good geometrical fit. Looking at Fig. 3.15 on the left, such acyl chain interactions appear less beneficial between CLA‐9c,11t and CA because of a mismatch of the length of the straight saturated chain segments. This is further supported by the suggested interdigitated packing shown on the right.

(a)

(b)

35 L

30

35

25

25

20

20

15 10

MC + L

15 10t12c + MC

5

CA + MC

0

0 –5

CA + L 10t12c + L

10

CA + L

9c11t + L

5

L

30

T (° C)

T (° C)

80

CA + 9c11t 0

20

40

60

cCA (mol%)

80

100

–5

0

20

40

60

80

100

cCA (mol%)

Fig. 3.14  Phase behavior of capric acid (CA) mixed with different conjugated linoleic acids: 18:2, 9c,11t (a) and 18:2, 10t,12c (b).

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals C10A/10t12c = 50/50 C10A

9 10

10

12 13

10t12c

9 9 11 10

9c11t

C10A/9c11t = 50/50.

11 12

Fig. 3.15  Sketches of molecular structure of capric acid (CA), CLA‐10trans, 12cis, and CLA‐9cis, 11trans. Sketch of molecular packing in the crystal structure for equimolar mixtures of CA and the respective CLA.

3.3.3  Mixtures of Partial Glyceride Fatty‐Acid Esters A logical intermediate class of molecules between fatty acid systems and TAGs are monoacylglycerols (MAGs) and diacylglcerols (DAGs). Because these molecules are surface active, most of the respective research concerns membranes and interfaces and straight solidification behavior has much less been subject of detailed studies. Because these molecules are often present in natural fats, predominantly in palm oil and its derivatives, and deliberately added to food compositions, crystallization studies mainly concern the influence of partial glycerides on TAG crystallization. The reason for this is that the hydroxyl groups introduce polarity and complicate the molecular interaction in the solid phases significantly. The studies on the solidification behavior of pure MAGs (Vereecken et al. 2009) reveal a clear systematic development of melting point temperatures and heats of fusion with chain length within homologous series. Also for pure DAGs, the typical evolution of physical properties with chain length is found, taken polymorphic forms remain constant within a homologous series (e.g., Abes & Narine 2007). Some binary mixtures of monoacid saturated DAGs have been studied as well (e.g., Abes et  al. 2007, 2008; Craven & Lencki 2011). At high temperatures, eutectic behavior is found already for smallest chain‐length differences, two carbon atoms, respectively. The low‐temperature phase behavior appears to be too complicated to quantify because it is sensitive to different cooling rates and hence dominated by the superimposing of metastable states. Despite of the recent interests to edible applications of liquid and solid DAGs (see Chapter 2), the phase behavior of partial glycerol fatty acid esters is not further elaborated in this section.

81

82

Crystallization of Lipids

3.3.4  Mixtures of TAGs The mixing behavior of TAGs in the metastable and stable states becomes extremely complicated, compared to that of the fatty acids or alkanes. This is because of the effects of the different chemical nature of the fatty‐acid moieties which are combined into a single molecule, the sn‐positions of the fatty‐acid moieties at the glycerol backbone, respectively. This causes already complicated polymorphic behavior of the pure TAGs. Another factor, that makes the mixing behavior of all the systems so complex, is the effect of cooling and heating rates on the mixing and polymorphic behavior of the mixtures during crystallization and melting processes. Despite these complications, being driven by the facts that naturally occurring fat resources contain different types of acylglycerol specimen, in particular TAGs, and that end products can reveal functionality partly because of the blending of the different TAGs, many researchers have worked on the mixing behavior of TAGs and its consequences for product functionality. This section discusses the phase behavior of binary (Table 3.2) and ternary mixtures of major TAGs. As mentioned previously, the phase transitions in TAG mixtures are sensitive to cooling and heating rates and hence prone to observations being obscured by kinetics. Anyhow, the presence of monotropic polymorphism, which is the basis for many applications of fats in foods, is also basically a kinetic phenomenon. With respect to applying the framework outlined at the beginning of this chapter this poses a problem. Transition from a metastable to a stable state could occur spontaneously and hence do not need to obey the rules outlined for reversible equilibrium phase transitions. This is reflected in the fact that often simple lines connecting experimental data points are found when phase behavior is discussed. In most cases, the quality and quantity of the experimental evidence is not sufficient to judge in how far this is realistic or if the equilibrium or pseudo‐equilibrium considerations made above yield more appropriate interpretations. This dilemma is already apparent in the interpretation of the low‐temperature solid‐ solid transitions of the mixed fatty‐acid systems when initial states after cooling at high Table 3.2  Typical phase behavior of binary mixtures of major triacylglycerols. Mixtures*

Phase behavior

References

PPP/SSS LLL/MMM LLL/PPP PSP/sn‐PPS PPP/POP POP/PPO POP/OPO SOS/sn‐OOS SOS/SLiS SOS/POS SOS/POP

miscible (α and β’), eutectic (β) miscible (α and β’), eutectic (β) eutectic eutectic eutectic MC forming MC forming eutectic miscible miscible eutectic

Kellens et al. 1991 Takeuchi et al. 2003 Takeuchi et al. 2003 Boodhoo et al. 2009a, 2009b Minato et al. 1996 Minato et al. 1997a Minato et al. 1997b Zhang et al. 2009 Takeuchi et al. 2002 Rousset et al. 1998 Sasaki et al. 2012

* Abbreviations of fatty‐acid moieties: L, lauroyl; Li, linoleoyl; M, myristoyl; O, oleoyl; P, palmitoyl; S, stearoyl.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

rates are metastable. Specifically, the low‐temperature phase behavior as shown in Fig.3.13 might be more complex than reality is. On the other hand, it properly explains the experimental observations. A practical guidance for the assessment of likely phase behavior is rather the time dependence of transitions than the question if metastabile states are involved. The melting behavior of a metastable state is a transition involving the change of the relative stabilities of the two phases considered. Hence, well defined two‐phase regions should be present in binary systems. The same is true for polymorphic transitions relating to enantiotropic polymorphism. In contrast, transitions relating to monotropic polymorphism are “spontaneous” and only driven by kinetics. In these cases, the line of transition temperatures essentially marks the activation of an overdue transition. In monotropic systems, this difference is clearly illustrated by the fact that melting of metastable states in binary systems proceeds analogue to melting of stable states over a solid‐liquid two‐phase region, while the solid‐solid transition appears spontaneous. The later process being primarily characterized by the time a system needs to transform. These considerations are additionally complicated by the lack of clarity of relative polymorphic stability of mixed TAG solid phases. For numerous compositions, the β’ appears to be stable (e.g., spreads), which could support the proposition that instead of being strictly monotropic, the polymorphism of fats could also be enantiotropic in mixed systems. 3.3.4.1  Binary Mixtures of Monosaturated Acid TAGs

Kellens et al. (1991) analyzed the phase behavior of the binary mixtures of saturated monoacid TAGs of palmitic and stearic acid (PPP/SSS), by using synchrotron radiation (SR) XRD technique. It was found that miscible mixtures were formed for α and β’ polymorphs in the whole concentration range of the PPP/SSS mixtures, whereas immiscible phases indicating eutectic behavior were found when α and β’ transformed to the most stable β form. Later, the same system was studied under isothermal and non-isothermal conditions and kinetic phase diagrams for the occurrence of the metastable and stable polymorphic forms were constructed (Himawan et al. 2006, 2007; MacNaughtan et al. 2006). Figure 3.16 shows the phase diagram described. The α to β’ and β’ to β transitions expand over the whole composition range connecting the respective pure component transition points. Looking carefully at the original DSC data (MacNaughtan et al. 2006), one could argue that these transitions are melt‐mediated and hence, instead of the line of transition temperatures displayed for spontaneous processes, a two‐phase region should separate the α, β’, and β regions. Takeuchi et al. (2003) studied the phase ­diagrams of different mixtures of monoacid TAGs, based either on lauric, myristic, palmitic, or stearic fatty acid moieties (PPP‐LLL, MMM‐LLL, and SSS‐LLL). Figure 3.17 depicts the phase diagram for the binary system of trimyristin and trilaurin (MMM/LLL), and Fig. 3.18 that for tripalmitin and trilaurin (PPP/LLL). Both were derived from DSC and SR XRD experiments. Analogue to Fig. 3.16, Fig. 3.17 illustrates complete miscibility in the α‐ and β’‐polymorph over the whole composition range. Also for the system MMM/LLL, it is found that the β‐polymorph is characterized by eutectic behavior. Different than shown for the PPP/SSS system (Fig. 3.16) it is found that for the pure components, α directly transforms to β under the experimental conditions studied. This indicates that β ’ is preferentially formed in mixed solid phases. These findings are basically in line with the original study of the PPP/SSS system by

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Crystallization of Lipids

Fig. 3.16  Phase behavior of tripalmitin and tristearin. Compared to original publication (MacNaughtan, Farhat, & Himawan 2006); data points are omitted for reasons of clarity. Dotted line, boundary of solid‐solid two‐phase region, not supported by original data.

75 L

70

Temperature (° C)

βPPP + L

βSSS+ L

65

βSSS+ βPPP

60 55

β′PPP/SSS 50 45

αPPP/SSS

40 35

0

0.2

0.4

0.6

0.8

1

xPPP

Fig. 3.17  Phase behavior of the trimyristin‐trilaurin system, data points according to Takeuchi et al. (2003).

90 80 L 70 Temperatrue (° C)

84

60

βLLL+ L

βMMM + L

50 βMMM + βLLL

40 30

β′MMM/LLL

20 10

αMMM/LLL 0

0.2

0.4

0.6

0.8

1

xLLL

Kellens et al. (1991), indicating that a chain‐length difference of two carbons in saturated mono‐acid TAGs is tolerable in the α‐ and β’‐polymorphs. In contrast, the phase diagram shown for the system composed of tripalmitin and trilaurin as shown in Fig. 3.18 appears more complicated. The data shown in this figure originate from the same lab as the one for the MMM/LLL system (Takeuchi et al. 2003). Different than in the Figs 3.16 and 3.17, the lines indicating phase boundaries have been derived based on the equilibrium rules outline in this chapter. In the original manuscript, the phase diagram is subdivided into three regions: Miscibility of PPP in LLL up to 10%; miscibility of LLL in PPP up to 50%; and complex phase transition with limited

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals 90 80

L

Temperature (° C)

70

βLLL + L

60

βPPP + L

50

βPPP

40

βPPP + βLLL

αPPP + βPPP

30

αPPP + βLLL

αPPP 20 10

αPPP + β′LLL 0

0.2

0.4

0.6 xLLL

βLLL

β′LLL 0.8

β′LLL + βLLL 1

αLLL

αLLL + β′LLL

Fig. 3.18  Phase behavior of the tripalmitin‐trilaurin system. Data according to Takeuchi et al. (2003) with phase boundaries drawn according to the rules of equilibrium phase transitions instead of the original interpretation.

miscibility in the intermediate composition range. Analogue to the phase behavior shown in Fig. 3.17 for the MMM/LLL system, also here the pure components directly convert from the α‐ to the β‐polymorph. The newly introduced phase boundaries as shown in Fig. 3.18 are not in conflict with this assessment but add much more detail to the interpretation of the experimental data. The system composed of tristearate and trilaurin actually shows qualitatively the same phase behavior. From these results, we may draw the following conclusions. The metastable α‐ and β’‐ polymorphic structures in binary mixed systems can tolerate differences in the chain length of fatty acids (Cn) of monoacid TAGs of two without being immiscible. In contrast, the most stable polymorph β exhibits eutectic behavior for these systems. Once the chain‐length difference between the fatty acids of the monoacid TAGs (ΔCn) is increased to four or six carbons, partial immiscibility in the α‐ and β’‐polymorphs also emerges. In short, the binary mixing behavior between the saturated monoacid TAGs is hence driven by both the disparities of chain length of the fatty acid moieties and the polymorphic structure considered. 3.3.4.2  Binary Mixtures of Saturated Mixed‐Acid TAGs

Because the phase behavior of TAG mixtures is dramatically influenced by the conditions of the crystallization process, the discussion on the mixtures concerned in this paragraph are limited to one origin of experimental data. Systematic studies of binary mixtures of symmetric and asymmetric mixed‐acid TAGs were reported out of the University of Alberta. The stearic acid moiety (S) was placed at the sn‐2 and sn‐3 positions of the glycerol backbone to fabricate symmetric and asymmetric TAGs, respectively. Capric (C), lauric (L), myristic (M), and palmitic (P) acids were placed at the other

85

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Crystallization of Lipids

positions of the TAGs, resulting in, for example, CSC/CCS (Boodhoo et al. 2009b), LSL/ LLS (Bouzidi et al. 2010), MSM/MMS (Boodhoo et al. 2008), and PSP/PPS (Boodhoo et  al. 2009a) systems. The authors summarized the overall study in a book chapter (Bouzidi & Narine 2012). As a characteristic property of the binary systems, the chain‐length mismatch (CLM) is defined, referring to the differences in the carbon numbers between stearic acid (C18) and the other fatty‐acid moiety of the component TAGs. Namely, CLM is two for the PSP/PPS system, four for MSM/MMS, six for LSL/LLS, and eight for CSC/CCS. Data were gathered without long term stabilization. The phase behavior was studied by determining the melting temperatures with DSC of mixtures crystallized at slow (0.1 ° C/min) and rapid (3 °C/min) cooling rates. In broad terms, the results show that the aforementioned mixtures are immiscible. The details differ from one system to another because of different levels of disparity. For mixtures of PSP/PPS (CLM = 2), a singularity in the liquidus line at the 1:1 concentration ratio is found indicating the presence of a molecular compound. Typical for compound forming systems, the remaining mixed phases, above and below 50%, respectively, show eutectic behavior, which is often for reasons of asymmetry wrongly described as monotectic. Increasing the chain‐length mismatch to four in the mixtures of MSM/ MMS again rather asymmetric eutectic behavior is found. Surprisingly, increasing the mismatch to six carbons (CLM = 6) in the LSL/LLS system leads to the formation of a molecular compound similarly to the PSP/PPS system. On further increase of the mismatch to eight (CLM = 8) for mixtures of symmetric and asymmetric TAGs containing two capric acid and one stearic acid moiety, eutectic phase behavior was observed. This indicates that the formation of molecular compounds is certainly promoted by the presence of symmetric and asymmetric TAGs but also depends on the chain‐length mismatch present. This synergistic compatibility, which is necessary to form a molecular compound is most apparent for systems without chain‐length differences but differences in saturation (see next section). To further confirm this, additional structural analysis at the molecular level is needed to understand the immiscibility and formation of molecular compounds in mixtures of symmetric and asymmetric mixed‐acid fully saturated TAGs. In conclusion, the findings on the phase behavior of systems composed of symmetric and asymmetric TAGs being composed of the same saturated fatty‐acid moieties appears surprising. Taking the data of fatty acids and n‐alkanes into account, the limited affinity to mix in the solid phase is less surprising. Additionally, it has to be kept in mind that to optimize the chain‐chain packing in these systems, as a main contributor to the molecular interaction, the conformation of the glycerol backbone has to be considered as a contribution to the energetic state. With regard to the occurrence of molecular compounds, it can be concluded that the combination of symmetric and asymmetric TAGs is certainly promoting but not generally sufficient to overcome interactions from chain‐chain origin or glycerol conformation. 3.3.4.3  Binary Mixtures of Saturated‐Unsaturated Mono‐Acid and Mixed‐Acid TAGs

Mixtures of saturated monoacid and unsaturated monoacid TAGs, such as tripalmitin and triolein (PPP/OOO) and tristearin and triolein (SSS/OOO), show immiscible behavior, which can be characterized as asymmetric eutectic. Similarly, the binary mixtures of saturated monoacid TAGs and saturated‐unsaturated mixed‐acid TAGs are

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

T (° C)

immiscible, as for example revealed in the tripalmitin and di‐1,3‐palmitoyl‐2-oleoyl‐ glycerol (PPP/POP; Minato et al. 1996). However, quite different mixing behavior was observed for systems composed of different TAGs composed of saturated and unsaturated mixed acids. Limited miscibility was observed for binary mixtures of symmetric TAGs composed of oleic acid and stearic and palmitic acid (POS/SOS), solid solution in the β structure and ideal mixing in the α‐ polymorph (Rousset et  al. 1998). Another system with different levels of unsaturation of the fatty‐acid moiety (oleic and linoleic) on the sn‐2 and stearic acid on all terminal positions was studied (SOS/SLiS; Takeuchi et al. 2002). Concerning the mixtures of SOS and SLiS, the component TAGs of SOS are known to appear in α, γ, β’, β2, and β1 structures and SLiS appears as α‐ and γ‐polymorph, for molecular structures see Chapter 2. Figure 3.19 shows the miscible solid‐solutions that were observed in the metastable α and γ forms in all concentration ranges of SOS/SLiS. Consequently, the melting in this system would typically involve the transition of the miscible γ form into a liquid. This  transition is considered not spontaneous but is for obvious reasons related to a solid‐liquid two‐phase region. However, because of the similar melting points of the two pure components this two‐phase region is of limited extension. Most likely because of the proximity of the pure SOS melting temperatures for γ and β′ polymorphic forms, the miscible γ form did not transform into the more stable β′ form, which is found for pure SOS on different temperature programs. However, an α‐melt‐mediated transformation into β′ and β2 resulted in an immiscible system (Takeuchi et al. 2002). This way a solid (pure β2 SOS)‐solid (mixed γ, 30/70 SOS/SLiS) two‐phase region emerges; see dashed line in Fig. 3.19. This is because at SLiS concentrations above 30%, the α‐melt mediated transformation results only in the γ‐polymorph. Consequently, a three‐phase line as show in Fig. 3.19 would emerge. For this system, the discussion of the phase behavior is restricted to the melting behavior despite the fact, that the transition from the α‐ to 40 β′+ L the γ‐polymorph remains interesting as L + γSOS/SLiS L well (see original publication). Anyhow, 35 these results impressively document how the specific interactions between SOS and 30 SLiS, governed by the oleic and linoleic γSOS/SLiS β′+ γSOS/SLiS fatty acid moieties, result in a quite complicated phase behavior. 25 The formation of molecular compounds  was repeatedly reported for sev20 eral ­systems, in SOS/SSO (Engström 1992; αSOS/SLiS Takeuchi et al. 2002), SOS/OSO (Engström 15 1992; Koyano et  al. 1992), POP/PPO 100 0 20 40 60 80 (Ikeda‐Naito et  al. 2014; Minato et  al. cSLiS (mol%) 1997a), and POP/OPO (Ikeda et al. 2010; Minato et  al. 1997b), and PPO/OOP and Fig. 3.19  Phase behavior of the SOS/SLiS system. OPO/OOP (Bayés‐Garcia et  al. 2015). It Data points from Takeuchi et al. (2002). dashed must be mentioned that the molecular lines: stable states that do not appear on melting after cooling process applied, but are mentioned compounds of POP/OPO and POP/PPO in original source; solid lines, interpretation of occurred on crystallization from diluted measured data, metastable; miscibility up to alkane solutions (Ikeda‐Naito et al., 2014; 70% SLiS, melting of β’ polymorph SOS.

87

Crystallization of Lipids 30

40 L

35

L

βPOP + L 20

30 βMC+ L

25

βMC + βPOP

20 15

βPOP + L

T (° C)

T (° C)

88

10

βC + L βC + βPOP

βOPO + βMC

0

20

40

60

cPOP (mol%)

80

100

βOPO + βC 0 0 20

40

60

80

100

cPOP (mol%)

Fig. 3.20  POP/OPO phase behavior crystallized from n‐dodecane solution. Left, 50% solution; right, 20% solution. Adapted from Ikeda et al. (2010).

Ikeda et al., 2010), proving evidence that these molecular compounds are thermodynamically stable. Differently, the molecular compounds found in PPO/OOP and OPO/ OOP mixtures appeared to be metastable because quite slow transitions to eutectic states were observed during long‐term storage (Bayés‐Garcia et al. 2015). Figure 3.20 shows the solid phase behavior for mixtures of POP/OPO. The two different graphs represent different crystallizations from n‐dodecane solution at either 50% or 20%. Similar to the left‐phase diagram observations were made for the crystallization without solvent. The compound in the β‐polymorph (βC) has a long‐spacing value of 4.2 nm with a double chain‐length structure and a melting point of 31.9 ° C. In the polymorphic occurrence, the POP fraction transformed from α to β′, and then to β. The presence of OPO in POP promoted the β′ ‐ β transformation of POP during the melt‐ mediated crystallization (Minato et al. 1997b). Crystallized from n‐dodecane solutions, the molecular compound formed at a ratio of POP/OPO = 1:1 for stable and metastable polymorphic forms. The assumption that aliphatic chain‐chain interactions through palmitic‐ and oleic‐acid moieties are the main reason for the occurrence of the molecular compound and not solute‐solvent interactions is confirmed by the fact that the molecular compound was observed on crystallization from different solvents (Minato et al.1997a; Ikeda‐Naito et al. 2014). The mixtures of POP/rac‐OOP (Zhang et al. 2007), SOS/sn‐OOS (Zhang et al. 2009), and rac‐PPO/OPO (Bayés‐Garcia et al. 2015) all reveal eutectic behavior. As shown in Fig. 3.21, the POP/rac‐OOP mixtures exhibited immiscible eutectic behavior, both in metastable and most stable states. A time‐resolved synchrotron radiation X‐ray diffraction (SR‐XRD) study was undertaken during the cooling and heating processes and indicated that the α and β ’ forms of the POP and OOP fractions crystallized and melted in separate manners. In addition, the study showed that the crystallization of the β ’ form and the polymorphic transformation from α to β ’ of POP and OOP were promoted by the presence of another component. Basically, the same results were observed for the mixtures of SOS/sn‐OOS and rac‐PPO/OPO.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals 50 L

T (° C)

40

30

β′OOP + L

βPOP + L

20 βPOP β′OOP + βPOP

10 β′OOP 0

0

20

40

60

80

100

cPOP (mol%)

Fig. 3.21  Phase behavior of the POP/OOP system. Data from Zhang et al. (2007).

PPP/POP

POP/OPO

Fig. 3.22  Possible binary mixing behavior of TAGs consisting of palmitic (P) and oleic (O) acid.

Thermal, X‐ray diffraction, and FT‐IR absorption studies showed that steric hindrance as a result of repulsive interactions between saturated and oleic acid moieties are critical for the formation of molecular compounds (Fig. 3.22). Figure  3.22 depicts the basic structure of fatty acid orientation for different pure TAGs. Three main points emerge in considering the ability of combinations of TAGs to form molecular compounds. 1) The mixing behavior is governed by chain‐length structures and lateral molecular interactions in the fatty‐acid chains of saturated‐ and oleic‐acid moieties. 2) The eutectic mixture is formed in POP/PPP because the stable β forms of PPP and POP construct double and triple chain‐length structures, respectively. The mixture of SOS/SSS also shows the same result. 3) The molecular compound forming mixture is formed in POP/OPO, since the lateral interactions between palmitic‐ and oleic‐acid moieties are stabilized by forming double chain‐length MC crystals, in which oleic‐ and palmitic‐acid moieties are

89

90

Crystallization of Lipids POP

OPO

PPO

OOP

Molecular compound (metastable and stable states) Molecular compound (metastable) Eutectic (metastable and stable states)

Fig. 3.23  Map of molecular compound formation for binary mixtures of TAGs containing palmitic and oleic acid.

packed in separate leaflets to avoid steric hindrance. The mixtures of POP/PPO, SOS/OSO, and SOS/SSO also show the same result. Figure 3.23 illustrates the tendency to form molecular compounds for binary combinations of four typical mixed‐acid TAGs composed of palmitic‐ and oleic‐acid moieties (Bayés‐Garcia et al. 2015). Stable molecular compounds are formed in POP/PPO and POP/OPO mixtures, whereas metastable molecular compounds are found in OPO/ OOP and PPO/OOP. In contrast, the binary combinations of POP/OOP and PPO/OPO show eutectic phase behavior illustrating how delicate the molecular interactions of TAGs are. For the interpretation of the underlying interactions the following factors have to be considered: the conformation of the glycerol backbone, the mutual interactions of neighboring fatty‐acid chains, and the polymorphism of the pure components. The main molecular interactions that influence the stabilization of the crystal structure of TAGs containing saturated and unsaturated fatty acids are listed as follows and discussed in more detail in Chapter 2. 1) Aliphatic chain packing, caused by molecular interactions between saturated and unsaturated fatty‐acid chains. 2) Glycerol conformation, which determines the configuration of all TAG molecules. 3) Methyl end stacking, which determines the chain inclination and the chain‐length structure. The ability to form molecular compounds with a double‐chain length structure may be explained by the combined effects of these interactions. Figure  3.24(a) illustrates different glycerol conformations of two asymmetric units of TAG molecules in their most stable form. The glycerol conformation is symbolized using the direction of the glycerol group, indicated by an arrow pointing at the sn‐2 carbon atom, and defines the direction between the middle point of the two glycerol carbon atoms at the first and third positions, and the glycerol carbon atom at the ­second position. The glycerol conformation determines the lateral chain packing of the palmitic‐ and oleic‐acid moieties. In more stable conformations, the palmitic and oleic acid chains should be packed in separated leaflets. In contrast, the coexistence of palmitic and oleic acid in the same leaflet may

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

(a)

(b)

Glycerol conformation

POP/OPO

POP/PPO

OPO/OOP

PPO/OOP

Tuning fork POP

stable Chair PPO

stable

POP/OOP (case 1) (case 2)

OPO

stable OOP

PPO/OPO (case 1)

(case 2)

stable

Fig. 3.24  Sketches of different structural arrangements for binary pairs of TAGs composed of palmitic and oleic acid.

decrease the packing coefficient in aliphatic chain packing. Thus, the conformation obtained should become metastable. Typically, chiral TAGs adopt the chair conformation while achiral TAGs adopt the tuning fork (Craven & Lencki 2013). Therefore, the most stable conformation for symmetric (achiral) POP and OPO is tuning fork (van Mechelen et  al. 2006), but asymmetric (chiral) PPO and OOP may adopt a chair conformation. Figure 3.24(b) depicts possible structure models of the molecular compound of TAGs containing palmitic and oleic fatty acids. The adjacent glycerol groups found in parallel arrangement are directed along the chain axis with an opposite turn in the POP‐OPO mixture. The tuning fork conformation is the most stable for both POP and OPO, and it enables the oleoyl‐ and palmitoyl‐chains to form separate leaflets with stabilized aliphatic interactions. This gives rise to the assumption that POP and OPO are capable to form a molecular compound. For the combination of POP and PPO, it can be assumed that both TAGs may adopt the tuning fork conformation. With this structure, the adjacent glycerol groups should be directed along the chain axis with an opposite turn, similar to the arrangement in the POP/OPO combination. However, here the palmitoyl‐ and oleoyl‐chains are inevitably located in one leaflet, and the other leaflet may be fully occupied by ­palmitoyl chains. Chain packing of the palmitoyl‐palmitoyl leaflet may be stabilized, but steric hindrance may occur in the leaflet with mixed palmitoyl and oleoyl ­occupation. However, this hindrance does not seem to completely destabilize this molecular compound structure. For the combination of POP and OOP, which does not form molecular compounds, two possible structural models may be assumed. Case 1 assumes a parallel direction for the glycerol groups, which may be more stabilized than the nonparallel direction

91

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Crystallization of Lipids

depicted for case 2. However, the structural model of case 1 may destabilize the acyl chain packing because of the coexistence of oleoyl‐ and palmitoyl‐chains in both leaflets. In case 2, the oleoyl‐ and palmitoyl‐chains are separately located in their own leaflets. But in this case the glycerol conformation appears less favorable. Consequently, neither of the two possible models of the POP/OOP mixture may alleviate destabilization of the acyl chain packing, and hence the fact that no molecular compound was observed appears likely. Similar considerations lead to the reconfirmation that mixtures of PPO and OPO do not form molecular compounds. In summary, the following properties have been observed in mixtures of saturated‐ unsaturated TAGs: 1) Eutectic mixtures are formed between saturated monoacid TAGs and saturated‐ unsaturated mixed‐acid TAGs. 2) Miscible mixtures are formed between TAGs revealing isomorphic polymorphism (e.g., SOS/SLiS.) 3) The molecular compound crystals are formed between specific TAGs through aliphatic chain interactions (e.g., POP/OPO). The discussion on the mixing behavior of simple binary TAG mixtures as described illustrates that basically the systematic order found for the evolution of phase behavior in n‐alkane and fatty‐acid systems is also present in TAG systems. However, the fact that the aliphatic chains are restricted in their spatial distribution because of the bonds to the glycerol backbone complicates matters even more. Again, disparity of the chain lengths or level of unsaturation of neighboring fatty acid moieties drives demixing. Depending on the conformation of the glycerol back bone systems might arrange however in such a way that favorable homogeneous leaflets emerge. These are the basis for the occurrence of hylotrops and molecular compounds. In general, the β‐polymorph shows little tolerance to accommodating significant amounts of alien TAGs as most systems behave eutectic with limited miscibility. The α–,γ–, and β’‐polymorphs on the other hand show, depending on the TAG mismatch, complete miscibility in a lot of cases. Furthermore, it should be emphasized that the experimental observations made on solid‐solid phase transitions and melting are extremely sensitive to the history of the sample, crystallization, and storage/annealing, so that great care needs to be taken during the interpretation of spontaneous, equilibrium, and pseudo‐equilibrium phase transitions.

3.4 ­Mixing Behavior of TAGs in Natural and Interesterified Fats Almost all natural fats and oils contain a vast amount of different TAGs and a raft of minor components such as free fatty acids, MAGs, and DAGs. Just considering the number of main fatty‐acid chains present in most natural fats, the possible number of TAGs derived by simple statistics is discomforting. Because TAGs are the main components of fats, many physical properties are directly related to the mixing behavior of the major TAGs present as outlined in detail in the previous section. For example, the fractionation of a fat, separating TAGs differing in melting temperatures poses different levels of difficulty depending on whether one deals with a system that is eutectic, shows

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Table 3.3  Major relatively high‐melting triacylglycerols* in vegetable oils and fats. Palm oil and its fractions†

Cocoa butter†

TAG

Palm oil (IV 52.3)

Palm stearin (IV 34.3)

Palm olein (IV 56.7)

Coconut oil†

POO (1.9) SOO (3.9) POP (19.0) POS (39.6) SOS (28.5)

POO SOO POP/PPO/PLS POS PPP PPS

22.7 2.5 30.3 5.5 6.1 1.2

12.9 1.5 27.5 4.8 26.5 5.3

24.5 3.1 30.2 6.0 1.7 0.2

CCL (15.08) CLL (19.45) LLL (22.59) LLM (16.48) LMM (9.74) LMP (5.08)

* TAGs, concentration in %. †  Abbreviation of fatty‐acid moiety: C, capric acid (C10:0); L, lauric acid (C12:0); M, myristic acid (C14:0); O, oleic acid (C18:1); P, palmitic acid (C16:0); S, stearic acid (C18:0).

miscible solid solutions, or tends to form molecular compounds. In addition, inhomogeneous texture of products may occur if the TAGs reveal immiscible eutectics or if molecular compounds with elevated melting points are present in the fats which form the lipid crystal network. In the following section, the relevance of the mixing behavior described previously for industrially relevant systems containing high‐melting TAGs will be illustrated briefly. Systems include natural vegetable fats (Table  3.3), milk fat, and interesterified fats. Table 3.3 lists the TAG composition of three major tropical oils. The compositions are reduced to the main TAGs. It should be noted that, in particular, palm oil might contain amounts of partial glycerides in the order of several percent such that these should not be disregarded considering crystallization behavior. Cocoa butter, coconut oil, and palm oil and its fractions are the main vegetable sources of natural saturated fatty acids. As the table indicates, these fats comprise binary mixtures as described in the previous sections for their specific phase behavior. 3.4.1  Cocoa Butter The TAG composition of cocoa butters varies between the geographic regions of ­production. However, the main TAGs present are combinations of oleic‐, palmitic‐, and stearic‐acid moieties, namely POS, SOS, and POP. The major high‐melting TAGs in cocoa butter grown in Ivory Coast are shown in Table 3.3 (Chaiseri & Dimick 1989). Previous studies showed that the binary mixtures of SOS/POS are miscible (Rousset et al. 1998) or widely miscible with a partial eutectic region near POS 85% (Smith et  al. 2013). The other subsystems POP/POS (Smith et  al. 2013) and POP/ SOS (Sasaki et al. 2012; Smith et al. 2013) are eutectic. However, the composition of cocoa butter is approximately a ternary mixture of POP/POS/SOS with a 22/46/32 mixing ratio. The phase behavior of the POP/POS/SOS system has been studied by many researchers (e.g., Timms 2003). Two recent studies reported the phase behavior of the ternary mixture system of POP/ POS/SOS after long incubation times aiming at most stable states. Using pure component

93

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Crystallization of Lipids POP Solid solution Eutectic Concentration ratio of cocoa butter

POS

SOS

Fig. 3.25  Solid state for selected ternary mixtures of POP, SOS, and SOS. Smith et al. 2013

TAGs, the compositional grid had 5–10% intervals. Smith et  al. (2013) observed that, based on iso‐solid lines and melting measurements, a possible ternary eutectic mixture was present at about POP : POS 1 : 1 ratio combined with less than 10% SOS. Sasaki et  al. (2012) observed that based on DSC melting and SR‐XRD long‐spacing patterns, eutectic mixtures occurred when more than 50% POP or more than 65% SOS were present. Once high amounts of POS were present, miscible solid‐solutions occurred, which is in line with the behavior of cocoa butter (Fig. 3.25). The data illustrated the compositional proximity of the natural cocoa butter composition to compositions that might show eutectic behavior, which poses a major threat to product quality. The differences found between the two studies may stem from kinetic effects induced by the crystallization process and the thermal incubation, apart from general differences in the experimental techniques employed. This risk indication should certainly be taken into account once other TAG —CBE, CBI and CBR—are added to cocoa butter. However, further studies, also conducted in the presence of minor components and other chocolate ingredients, may give an even more detailed but less generic insight into the cocoa butter crystallization and related mixing behavior as a function of crystallization process and storage conditions. 3.4.2  Palm Oil The main TAGs in palm oil and its two predominantly traded fractions are shown in Table 3.3 (Kellens et al. 2007). Obviously, the composition entangles TAG combinations, and therefore, subsystems that show specific mixing behavior. In particular, looking at PPP, POP/PPO/PLS, and POO, eutectic mixtures are formed in PPP/POP, POP/POO, and most probably in PPP/POO. The increased levels of PPP PPS and POO in palm stearin and palm olein, respectively, support this interpretation. Taking the POP/PPO/ PLS combination into account, the formation of a molecular compound of POP and PPO may make it difficult to separate POP and PPO during the fractionation processes.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

The  tendency to demix is also found in palm oil crystallization because the separate crystallization of subsystems causes the presence of multiple mixed solid phases coexisting next to each other. In line with the previous section the level of co‐crystallization and demixing can be manipulated by variation of the crystallization process. 3.4.3  Coconut Oil The major TAGs in coconut oil are also shown in Table  3.3 (Marikkar et  al. 2013). Building on the discussion in Section 3.3.2. it may be assumed that a miscible mixture is formed among CCL, CLL, and LLL, where a chain‐length mismatch of two is present but no symmetry mismatch. This structure most likely cannot accommodate TAGs which increase the mismatch to four and hence another coexisting miscible mixture is formed between LLM and LMM, that might also contain LLL. This demixing hypothesis is supported by the results from solvent fractionation using acetone. Here CCL, CLL and LLL, and LLM and LMM are found in different fractions (Marikkar et al. 2013). 3.4.4  Milk Fat The crystallization properties of milk fat are explicitly discussed in Chapter  10 and hence the mixing properties of the major TAGs, see Table 3.4 (Zou et al. 2013), will only briefly be covered. Figure 3.26 shows three postulated mixed crystal configurations of the major TAGs in cow milk fat, in which butyric acid is represented with “4.” In detail, Fig.  3.26 suggests the following. One crystalline structure is the miscible mixture of the TAGs made up of long‐chain and short‐chain saturated fatty‐acid moieties such as PP4, PM4, and PM6, whose crystal structures may be similar to PP2 (see Chapter  2). The next crystalline structure is a molecular compound consisting of Table 3.4  Major triacylglycerols (TAGs) in cow milk fat. TAG*

Concentration (%)

MOBu + MMCp + PBuL PMBu MOCp PMCp + POBu PPBu + SMBu POCp + PCaL SOBu PSBu + PoLL POM POO PPO

5.39 6.19 4.19 11.61 10.63 4.29 4.42 3.29 5.04 4.16 5.43

* Abbreviation of fatty‐acid moiety: Bu, butyric acid (C4:0); Ca, caric acid (C10:0); Cp, caproic acid (C6:0); L, lauric acid (C12:0); Li, linoleic acid (C18:2); M, myristic acid (C14:0), O, oleic acid (C18:1); P, palmitic acid (C16:0); Po, palmitoleic acid (C16:1); S, stearic acid (C18:0).

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P

P

M

P

Molecular compound PPO, POO, POM M

P

P

P

P

Miscible PO6+PO4+MO4 O O

M 4

6 4

4

P

6

P

M

4 O O

Fig. 3.26  Sketch of the molecular packing of major TAGs in milk fat.

saturated‐unsaturated mixed‐acid TAGs such as PPO, POO, and POM, with a structure similar to the POP/PPO and POP/OPO molecular compounds. The third crystalline structure are miscible mixtures of the TAGs made up of long‐chain and short‐chain saturated and oleic acid moieties such as PO6, PO4, and MO4, which may be interdigitated. It seems fair to assume that these three different crystalline structures are immiscible with each other because of their significant dissimilarities. However, keeping the vast amount of TAGs present in milk fat in mind (see Table 3.4), the most likely numerous mixed solid phases existing next to each other make it d ­ ifficult to unravel the detailed crystallization behavior. Hence, the often applied ­subdivision into three melting fractions is practical but of limited value to create an in‐depth understanding. 3.4.5  Interesterified Fats Combining interesterification and fractionation is a useful method to design functional fat compositions. This approach has been the major solution to develop trans‐fat alternatives (see Chapter 1). In essence, esterification causes a random distribution of the fatty‐acid moieties present in the system over all TAG molecules. The differences between the enzymatically and chemically catalyzed processes are mainly in the reaction rate and the fact that sn‐1,3 specific lipases cleave only terminal fatty‐acid moieties from the TAG molecules. Consequently, the resulting TAG mixtures will depend on the  fatty‐acid composition, plus the symmetric distribution of the fatty acids on the TAGs in chemical and enzymatic interesterification, respectively. The resulting composition can thus be derived statistically and basic design rules can be found elsewhere (Kellens & Calliauw 2013). Taking this and the respective crystallization process of a specific product into account one should, building on the information given in this chapter, consider the subsystems that are present in a fat composition. High levels of

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Table 3.5  Major TAGs (concentration in %) in FHCO, HOSO, CI, and EI of FHCO / HOSO = 1/1.* FHCO†

HOSO†

CI†

EI†

SSS (79.0) SSP (9.4) SOS (5.1)

OOO (49.4) OOLi (27.0) OPO (5.5) PLiO (4.4)

OOS (30.4) SOS (27.3) OOO (12.0) SSS (9.8) POS (4.4) PPS (4.2) POP (3.5)

OOS (31.0) SOS (28.3) OOO (11.9) SSS (5.6) POS (3.9) PPS (4.2) POP (3.2)

* CI, chemical interestification; EI, enzymatic interesterifcation; FHCO, fully hydrogenated canola oil; HOSO, high‐oleic sunflower oil; TAGs, triacylglycerols. †  Abbreviations of fatty acid moiety: Li, linoleic acid (C18:2); O, oleic acid (C18:1); P, palmitic acid (C16:0); S, stearic acid (C18:0).

long‐chain saturated fatty acids generate a large portion of long chain trisaturated TAGs. These can only be forced to a limited extend and by aggressive processing to  co‐crystallize with mid‐melting TAGs composed of mixed saturated fatty acids. Similarly, the mixtures of straight palm oil and enzymatically interesterified mixtures can be put in context with the understanding of the phase behavior of cocoa butter TAGs and other subsystems. To illustrate that the interesterification might yield multiple solid phases, a rather atypical interesterification will be discussed for reasons of its simplicity. Namely the study of the mixing behavior of interesterified fats of fully hydrogenated canola oil (FHCO) and high‐oleic sunflower oil (HOSO) at a ratio of FHCO/HOSO = 1/1 (Ahmadi et al. 2008) is discussed. Table 3.5 shows that there are, seeing the nature of the starting material expectedly, only minute differences between the chemically and the enzymatically interesterified fat. Based on the TAG compositions shown in Table 3.5, two miscible solid phases will appear on crystallization. These are the mixed crystals of the high‐melting saturated TAGs, PPS and SSS, and the mixed crystals of the saturated‐unsaturated mixed‐acid TAGs such as SOS, POS, and POP. Obviously, the low‐melting TAGs of OOS and OOO are liquid at ambient temperatures. For the application of such a system great care has to be taken, that the crystallization process ensures adequate crystallization of the different solid phases, which will coexist in the final product.

3.5 ­Crystallization Properties The crystallization behavior of lipids is discussed excessively elsewhere in this book. However, some of the basic principles will be briefly discussed here with respect to their relevance for the content of this chapter. As pointed out previously, the execution of the crystallization process for lipid systems, which appear to have a complex crystallization behavior, is of utmost importance for the intermediate and final solid states. Already for systems that do not show polymorphic behavior as described by Los et  al. (2002), the supersaturation applied and hence also the cooling rate can

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influence the composition of the crystalline material generated. In short, higher supersaturation causes, according to the authors, a more chaotic embedding of material into the crystal lattice and hence generates energetically suboptimal mixed crystal compositions. The models applied further indicate that, taken the general state of miscibility is not changed during the process variation, the higher supersaturation causes a reduced level of solid material to be produced. With this in mind and the understanding of Ostwald’s rule of stages (Ostwald 1879), it is not surprising that which crystal structures appear is highly sensitive to process parameters and storage conditions. Because Ostwald’s rule can be well understood by considering the process of competitive nucleation between different polymorphic forms it appears to be important to understand nucleation and growth in depth to deliberately manage and apply polymorphic crystallization of lipids. Even though manufacturing processes often involve high cooling rates and significant shear rates, it should be noted here that these crystallization conditions primarily apply for the creation of the first metastable solid phase. Subsequent transitions to more stable polymorphs proceed under much less process induced driving forces because the liquid phase is depleted because of the presence of the metastable solid phase. In these, often spontaneous, transitions from a less stable to a more stable polymorph, the heat released and effects of local composition might play a role for the course of the transitions. Finally, it has to be acknowledged that the presence of interfaces and minor components can have a significant influence on both the crystallization process and the occurrence of different polymorphic forms.

3.6 ­Conclusions In this chapter, an attempt was made to comprehensively cover the solid‐liquid phase behavior of lipid aliphatic chain‐based molecules and its underlying principles up to the relevance in industrial applications. The theoretical framework outlined allows a better interpretation of experimental observations in rather pure academic systems but will benefit the interpretation in practice as well. The formulation of complete phase diagrams is however often hampered by the limited availability of experimental evidence. Even though this yields interpretations at times a bit speculative the framework discussed generates credibility through consistency in the application. In this light, it is important to discriminate between spontaneous transitions, occurring on transitions of a metastable solid to a more stable solid structure, and equilibrium or pseudo‐equilibrium transitions, such as melting of a metastable polymorph. Taken that the spontaneous transitions considered can actually only occur on temperature increase, the following discrimination also holds for heating processes. The former, spontaneous transition, is supposed to be exothermic and could be represented by a single line of temperatures while the latter two are endothermic and in binary systems related to the presence of a two‐phase region. Melt‐mediated solid‐solid transitions represent a mixed case with both phenomena superimposed. Regarding the different types of molecule classes discussed a clear systematic approach for the evolution of phase behavior was found. Interactions of the aliphatic chains are dominant contributors to mixing states from n‐alkane to TAG‐systems. Key

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

parameters in the assessment of the interaction are the chain‐length differences, with major disturbances already appearing for differences of two carbon atoms. The presence and configuration of unsaturated bonds in neighboring lipid chains is also highly important. Combining unsaturated and saturated chains in one leaflet is as unfavorable as disparities in the Δ ‐ chain lengths of neighboring unsaturated fatty‐acid chains. Additionally, the conformation of the glycerol backbone has to be taken into account to discriminate favorable from less favorable crystal structures. Phenomena occurring such as the formation of molecular compounds, hylotrops, miscibility, and immiscibility can be related to the geometrical considerations of the molecular packing allowing to forecast crystallization behavior. Furthermore, it is found repeatedly that in the intermediate composition range of mixed systems polymorphic forms occur, which do not appear under the same process conditions for the constituting pure components. In principle, the eutectic demixing primarily appears in the β polymorph, while α, γ, and β ’ structures seem to tolerate more deviations from the optimal packing. In general, care should be taken because the phase transitions observed are strongly depending on the processes applied to crystallize lipid mixtures. This influence can cause anomalies in the mixing behavior or result in long‐term kinetically frozen states generated according to Ostwald’s rule of stages or possible changes of the relative stability of polymorphic forms of mixed crystals. Because the phase behavior found for binary subsystems is also propagating into more complex and industrially relevant multicomponent systems, the understanding of the detailed crystallization behavior of lipids is also highly relevant for the practitioner. Dealing with product defects due to recrystallization phenomena, compounds or simple eutectoid demixing, the design of functional fat phases and the design of processes for fractionation and product manufacturing could greatly benefit from appreciation of the basic concepts outlined. Extrapolating into the future one could imagine that the problem of chocolate bloom is resolved because we will be able to manufacture chocolate with good organoleptic properties even though the cocoa butter is in the stable polymorph VI. Instead of trying to prevent recrystallization processes to avoid product defects, better control of spontaneous, pseudo‐equilibrium, and equilibrium transitions combined with the adequate processes could allow to use recrystallization deliberately. A fat phase that is deliberately driven into a metastable state of demixing could only on product application fully crystallize as miscible solid with much higher solid content to yield improved product properties. Less futuristic, better understanding of mixing/demixing phenomena can be applied to develop fractionation processes aiming at specific compositions of fractions and improved separation efficiencies. In general, to better exhaust the structuring potential of fats in food products new combinations of composition and processes need to be identified. This necessitates that the effort to understand the molecular interactions in different polymorphic forms is intensified through studies on model systems, with more detail than available currently. To design polymorphic manufacturing processes, the differences between spontaneous, pseudo‐equilibrium, and equilibrium transitions needs to be understood in much more detail with emphasis on the kinetics of appearance and transitions of phases. Lastly, a firm grip on the concept how mixing and processing parameters, primarily cooling rates but also shear rates, drive the occurrence of different solid phases is a prerequisite to develop the fat crystallization of the 21st century.

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­References Abes, M., & Narine, S. S. (2007) Crystallization and phase behavior of fatty acid esters of 1,3‐propanediol. I: Pure systems. Chemistry and Physics of Lipids. 149, 14–27. Abes, M., Bouzidi, L., & Narine, S. S. (2007) Crystallization and phase behavior of 1,3‐ propanediol esters. II. 1,3‐Propanediol distearate/1,3‐propanediol dipalmitate (SS/PP) and 1,3‐propanediol distearate/1,3‐propanediol dimyristate (SS/MM) binary systems. Chemistry and Physics of Lipids. 150, 89–108. Abes, M., Bouzidi, L., & Narine, S. S. (2008) Crystallization and phase behavior of fatty acid esters of 1,3 propanediol III: 1,3 propanediol dicaprylate/1,3 propanediol distearate (CC/SS) and 1,3 propanediol dicaprylate/1,3 propanediol dipalmitate (CC/PP) binary systems. Chemistry and Physics of Lipids. 151, 110–24. Ahmadi, L., Wright, A. J., & Marangoni, A. G. (2008) Chemical and enzymatic interesterification of tristearin/triolein‐rich blends: Chemical composition, solid fat content and thermal properties. European Journal of Lipid Science and Technology. 110, 1014–24. Bakhuis‐Roozeboom, H. W. (1901) Die heterogenen Gleichgewichte vom Standpunkte der Phasenlehre. Erstes Heft: Die Phasenlehre–Systeme aus einer Komponente. Bakhuis‐Roozeboom, H. W., Deuss, J. J. B., & Aten, A. H. W. (1911) Die heterogenen Gleichgewichte vom Standpunkte der Phasenlehre. Drittes Heft: Die tertiären Gleichgewichte. Bayés‐Garcia, L., Calvet, T., Cuevas‐Diarte, M. A., Ueno, S., & Sato, K. (2015) Phase behavior of binary mixture systems of saturated‐unsaturated mixed‐acid triacylglycerols: Effects of glycerol structures and chain‐chain interactions. Journal of Physical Chemistry B. 119, 4417–27. Boodhoo, M. V., Bouzidi, L., & Narine, S. S. (2009a) The binary phase behavior of 1, 3‐dipalmitoyl‐2‐stearoyl‐sn‐glycerol and 1, 2‐dipalmitoyl‐3‐stearoyl‐sn‐glycerol. Chemistry and Physics of Lipids. 160, 11–32. Boodhoo, M. V., Bouzidi, L., & Narine, S. S. (2009b) The binary phase behavior of 1,3‐ dicaproyl‐2‐stearoyl‐sn‐glycerol and 1,2‐dicaproyl‐3‐stearoyl‐sn‐glycerol. Chemistry and Physics of Lipids. 157, 21–39. Boodhoo, M. V., Kutek, T., Filip, V., & Narine, S. S. (2008) The binary phase behavior of 1,3‐dimyristoyl‐2‐stearoyl‐sn‐glycerol and 1,2‐dimyristoyl‐3‐stearoyl‐sn‐glycerol. Chemistry and Physics of Lipids. 154, 7–18. Bouzidi, L., Boodhoo, M. V., Kutek, T., Filip, V., & Narine, S. S. (2010) The binary phase behavior of 1,3‐dilauroyl‐2‐stearoyl‐sn‐glycerol and 1,2‐dilauroyl‐3‐stearoyl‐sn‐glycerol. Chemistry and Physics of Lipids. 163, 607–29. Bouzidi, L., & Narine, S. S. (2012) Phase behavior of saturated triacylglycerides: Influence of symmetry and chain length mismatch. In: Garti, N., & Widlak, N. (eds.), Cocoa butter and related compounds. Urbana, IL: AOCS Press, pp. 73–101. Chaiseri, S., & Dimick, P.S. (1989) Lipid and hardness characteristics of cocoa butters from different geographic regions. Journal of American Oil Chemists’ Society. 66, 1771–76. Costa, M. C., Rolemberg, M. P., Boros, L. A. D., et al. (2007) Solid‐liquid equilibrium of binary fatty acid mixtures. Journal of Chemical & Engineering Data. 52, 30–36. Costa, M. C., Rolemberg, M. P., Meirelles, A. J. A., Coutinho, J. A. P., & Krähenbühl, M. A. (2009a) The solid–liquid phase diagrams of binary mixtures of even saturated fatty acids differing by six carbon atoms. Thermochimica Acta. 496, 30–37.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Costa, M. C., Sardo, M., Rolemberg, M. P., et al. (2009b) The solid‐liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids. Chemistry and Physics of Lipids. 160, 85–97. Costa, M. C., Sardo, M., Rolemberg, M. P., et al. (2009c) The solid‐liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids: differing by four carbon atoms. Chemistry and Physics of Lipids. 157, 40–50. Coutinho, J., & Stenby, E. H. (1995) Phase equilibria in petroleum fluids: Multiphase regions and wax formation. Kgs. Lyngby: Technical University of Denmark, Department of Chemical Engineering. Craven, R. J., & Lencki, R. W. (2013) Polymorphism of acylglycerols: A stereochemical perspective. Chemical Reviews. 113, 7402–20. Craven, R. J., & Lencki, R. W. (2011) Binary phase behavior of diacid 1,3‐diacylglycerols. Journal of the American Oil Chemists’ Society. 88, 1125–34. De Bruijne, D. W., Hendrickx, H. A. C. M., Anderliesten, L., & De Looff, J. (1993) Mouthfeel of foods. In: Dickinson, E., & Walstra, P. (Eds.), Food colloids cociety, polymers: stability and mechanical properties. Cambridge, UK: Royal Society of Chemistry, Cambridge, pp. 204–13. De Hoog, E. H. A., Prinz, J. F., Huntjens, L., Dresselhuis, D. M., & Van Aken, G. A. (2006) Lubrication of oral surfaces by food emulsions: The importance of surface characteristics. Journal of Food Science. 71 (7), 337–41. Dorset, D. L. (2005) Binary and multicomponent solids of n‐paraffins. In: Dorset, D. L. (ed.) Crystallography of the polymethylen chain: An inquiry into the structure of waxes. New York: Oxford University Press, pp. 73–108. Engström L. (1992) Triglyceride systems forming molecular compounds. European Journal of Lipid Science and Technology. 94, 173–81. Floeter, E., Hollanders, B., de Loos, T. W., & Arons, D. S. (1998) The effect of the addition of water, propane, or docosane on the vapour‐liquid and solid‐fluid equilibria in asymmetric binary n‐alkane mixtures. Fluid Phase Equilibrium. 143, 185–203. Goodman, D. A., Cahn, J. W., & Lawrence, L. H. (1981) The centennial of the Gibbs‐ Konovalov rule for congruent points: Its underying theroetical basis and its application to phase daigramm evaluation. Bulletin of Alloy Phase Diagrams. 29–34. Grunenberg, A., Henck, J.‐O., & Siesler, H. W. (1996) Theoretical derivation and practical application of energy/temperature diagrams as an instrument in preformulation studies of polymorphic drug substances. International Journal of Pharmaceutics. 129, 147–58. Hillert, M. (1982) Prediction of slopes of phase boundaries. Bulletin of Alloy Phase Diagrams. 3, 4–8. Himawan, C., MacNaughtan, W., Farhat, I. A., & Stapley, A. G. F. (2007) Polymorphic occurrence and crystallization rates of tristearin/tripalmitin mixtures under non‐ isothermal conditions. European Journal of Lipid Science and Technology. 109, 49–60. Himawan, C., Starov, V. M., & Stapley, A. G. F. (2006) Thermodynamic and kinetic aspects of fat crystallization. Advances in Colloid and Interface Science. 122, 3–33. Ikeda, E., Ueno, S., Miyamoto, R., & Sato, K. (2010) Phase behavior of a binary mixture of 1,3‐dipalmitoyl‐2‐oleoyl‐sn‐glycerol and 1,3‐dioleoyl‐2‐palmitoyl‐sn‐glycerol in n‐dodecane solution. Journal of Physical Chemistry B. 114, 10961–69. Ikeda‐Naito, E., Hondoh, H., Ueno, S., & Sato, K. (2014) Mixing phase behavior of 1,3‐ dipalmitoyl‐2‐oleoyl‐sn‐glycerol (POP) and 1,2‐dipalmitoyl‐3‐oleoyl‐rac‐glycerol (PPO) in n‐dodecane solution. Journal American Oil Chemists’ Society. 91, 1837–48.

101

102

Crystallization of Lipids

Inoue, T., Hisatsugu, Y., Ishikawa, R., & Suzuki, M. (2004a) Solid‐liquid phase behavior of binary fatty acid mixtures: 2. Mixtures of oleic acid with lauric acid, myristic acid, and palmitic acid. Chemistry and Physics of Lipids. 127, 161–73. Inoue, T., Hisatsugu, Y., Suzuki, M., Wang, Z., & Zheng, L. (2004b) Solid‐liquid phase behavior of binary fatty acid mixtures: 3. Mixtures of oleic acid with capric acid (decanoic acid) and caprylic acid (octanoic acid). Chemistry and Physics of Lipids. 132, 225–34. Inoue, T., Hisatsugu, Y., Yamamoto, R., & Suzuki, M. (2004c) Solid‐liquid phase behavior of binary fatty acid mixtures: 1. Oleic acid/stearic acid and oleic acid/behenic acid mixtures. Chemistry and Physics of Lipids. 127, 143–52. Inoue, T., Motoda, I., Hiramatsu, N., Suzuki, M., & Sato, K. (1992) Phase behavior of binary mixtures of cis‐monounsaturated fatty acids with different ω‐chain length. Chemistry and Physics of Lipids. 63, 243–50. Kellens, M., & Calliauw, G. (2013) Oil modification processes. In: Hamm, W., Hamilton, R. J., Calliauw, G. (eds.) Edible oil processing. Chichester, UK: John Wiley & Sons Ltd., pp. 153–96. Kellens, M., Gibon, V., Hendrix, M., & De Greyt, W. (2007) Palm oil fractionation. European of Journal Lipid Science Technology. 109, 336–49. Kellens, M., Meeussen, W., Gehrke, R., & Reynaers, H. (1991) Synchrotron radiation investigations of the polymorphic transitions of saturated monoacid triglycerides. Part 2: Polymorphism study of a 50:50 mixture of tripalmitin and tristearin during crystallization and melting. Chemistry and Physics of Lipids. 58, 131–44. Kitaigorodsky, A. I. (1973) Molecular crystals and molecules. New York: Academic Press. Kobayashi, M., Kaneko, F., Sato, K., & Suzuki, M. (1986) Vibrational spectroscopic study on polymorphism and order‐disorder phase transition in oleic acid. Journal of Physical Chemistry. 90, 6371–78. Koyano, T., Hachiya, I., & Sato, K. (1992) Phase behavior of mixed systems of SOS and OS0. Journal of the American Chemical Society. 96, 10514–20. Ksiazczak, A., Moorthi, K., & Nagata, I. (1994) Solid‐solid transition and solubility of even n‐alkanes. Fluid Phase Equilibrium. 95, 15–29. Lillford, P. (2000) The materials science of eating and food breakdown. Bulletin of Materials Science. 25, 38–43. Los, J. H., Enckevort, W. J. P. Van, Vlieg, E., & Gandolfo, F. G. (2002) Metastable states in multicomponent liquid. Solid systems II : Kinetic phase separation. Journal of Physical Chemistry B. 106, 7331–39. MacNaughtan, W., Farhat, I. A., & Himawan, C. (2006) A differential scanning calorimetry study of the crystallization kinetics of tristearin‐tripalmitin mixtures. Journal of the American Oil Chemists’ Society. 83, 1–9. Marikkar, J. M. N., Saraf, D., & Dzulkifly, M. H. (2013) Effect of fractional crystallyzation on composition and thermal behavior of coconut oil. International Journal of Food Properties. 16, 1284–92. Minato, A., Ueno, S., & Smith, K. (1997a) Thermodynamic and kinetic study on phase behavior of binary mixtures of POP and PPO forming molecular compound systems. Journal of Physical Chemistry B. 101, 3498–505. Minato, A., Ueno, S., Yano, J., et al. (1997b) Thermal and structural properties of sn‐1, 3‐dipalmitoyl‐ binary mixtures examined with synchrotron radiation X‐Ray diffraction. Journal of the American Oil Chemists’ Society. 74, 1213–20.

Molecular Interactions and Mixing Phase Behavior of Lipid Crystals

Minato, A., Ueno, S., Yano, J., et al. (1996) Synchrotron radiation X‐ray diffraction study on phase behavior of PPP‐POP binary mixtures. Journal of the American Oil Chemists’ Society. 73, 1567–72. Mondieig, D., Rajabalee, F., Metivaud, V., Oonk, H. A. J., & Cuevas‐Diarte, M. A. (2004) n‐alkane binary molecular alloys. Chemistry of Materials. 16, 786–98. Ostwald, W. (1879). No Title. Zeitschrift für Physikaliche Chemie. 22, 289. Pickering, S. U. (1907) Emulsions. Journal of the Chemical Society. 91, 2001–21. Poling, B. E., Prausnitz, J. M., & O’Connell, J. P. (2004) The properties of gases and liquids. New York: McGraw‐Hill. Prinz, J. F., & Lucas, P. W. (1997) An optimization model for mastication and swallowing in mammals. Proceedings of the Royal Society of London, Series. B, Biological Science. 264, 1715–21. Rousset, P., Rappaz, M., & Minner, E. (1998) Polymorphism and solidification kinetics of the binary system POS‐SOS. Journal of the American Oil Chemists’ Society. 75, 857–64. Sandler, S. I. (2006) Chemical, biochemical, and engineering thermodynamics, 4th ed. Hoboken, NJ: Wiley. Sasaki, M., Ueno, S., & Sato, K. (2012) Polymorphism and mixing behavior of major triacylglycerols of cocoa butter. In: Garti, N., & Widlak, N. (eds.) Cocoa butter and related compounds. Urbana, IL, AOCS Press, pp. 151–72. Sato, K. (2001) Crystallization behaviour of fats and lipids—a review. Chemical Engineering Science. 56, 2255–2265. Sato, K., & Ueno, S. (2001) Molecular interactions and phase behavior of polymorphic fats. In: Garti, N., Sato, K. (eds.), Crystallization processes in fats and lipid systems. New York, Marcel Dekker, pp. 177–209. Sato, K., Yano, J., Kawada, I., et al. (1997) Polymorphic behavior of gondoic acid and phase behavior of its binary mixtures with asclepic acid and oleic acid. Journal of the American Oil Chemists’ Society. 74, 1153–59. Schaum, K. (1898) Ueber hylotrop‐isomere Körperformen. Justus Liebigs Annalen de Chemie. 205–28. Schreinemakers, F. A. H. (1911) Die ternären Gleichgewichte. In: Die Heterogenen Gleichgewichte Vom Standtpunkte Der Phasenlehre. Braunschweig. Small, D. M. (1986) Substituted aliphatic hydrocarbons, alcohols and acids. In: Small, D. M. (ed.) Physical chemistry of lipids. New York: Plenum, pp. 233–84. Smirnov, W. J., & Kurnakov, N. S. (1910) Bestimmte Verbindungen mit veränderter Zusammensetzung der festen Phase I. Leitfähigkeit und Härte des Systems: Magnesium‐ Silber., Bericht des Polytechnischen Instituts St. Petersburg. Smith, K. W., Bhaggan, K., & Talbot, G. (2013) Phase behavior of symmetrical monounsaturated triacylglycerols. European Journal of Lipid Science and Technology. 115, 838–46. Smith, K. W., Bhaggan, K., Talbot, G., & van Malssen, K. F. (2011) Crystallization of Fats: Influence of Minor Components and Additives. Journal of the American Oil Chemists’ Society. 88, 1085–101. Takeuchi, M., Ueno, S., Floeter, E., & Sato, K. (2002) Binary phase behavior of 1,3‐ distearoyl‐2‐oleoyl‐sn‐glycerol (SOS) and 1,3‐distearoyl‐2‐linoleoyl‐sn‐glycerol (SLS). Journal of the American Oil Chemists’ Society. 79, 627–32.

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Takeuchi, M., Ueno, S., & Sato, K. (2003) Synchrotron radiation SAXS/WAXS study of polymorph‐dependent phase behavior of binary mixtures of saturated monoacid triacylglycerols. Crystal Growth & Design. 3, 369–74. Timms, R. E. (2003) Confectionery fats handbook, properties, production and application. Bridgwater, UK: Oily Press. Uehara, H., Suganuma, T., Negishi, S., et al. (2008) Physical properties of two isomers of conjugated linoleic acid. Journal of the American Oil Chemists’ Society. 85, 29–36. van Mechelen, J. B., Peschar, R., & Schenk, H. (2006) Structures of mono‐unsaturated triacylglycerols. I. The β1 polimorph. Acta Crystallographica Section B. 62, 1121–30. Vereecken, J., Meeussen, W., Foubert, I., et al. (2009) Comparing the crystallization and polymorphic behaviour of saturated and unsaturated monoglycerides. Food Research International. 42, 1415–25. Walstra, P., Kloek, W., & van Vliet, T. (2001) Fat crystal networks. In: Garti, N., & Sato, K. (eds.) Crystallization processes in fats and lipid systems. New York, Marcel Dekker, pp. 289–328. Yoshimoto, N., Nakamura, T., & Sato, K. (1991) Phase properties of binary mixtures of petroselinic acid/oleic acid and asclepic acid/oleic acid. Journal of Physical Chemistry B. 3384–90. Zhang, L., Ueno, S., Miura, S., & Sato, K. (2007) Binary phase behavior of 1,3‐ dipalmitoyl‐2‐oleoyl‐sn‐glycerol and 1,2‐dioleoyl‐3‐palmitoyl‐rac‐glycerol. Journal of the American Oil Chemists’ Society. 84, 219–27. Zhang, L., Ueno, S., Sato, K., Adlof, R. O., & List, G. R. (2009) Thermal and structural properties of binary mixtures of 1,3‐distearoyl‐2‐oleoyl‐glycerol (SOS) and 1,2‐ dioleoyl‐3‐stearoyl‐sn‐glycerol (sn‐OOS). Journal of Thermal Analysis and Calorimetry. 98, 105–11. Zou, X., Huang, J., Jin, Q., et al. (2013) Lipid composition analysis of milk fats from different mammalian species: Potential for use as human milk fat substitutes. Journal of Agricultural and Food Chemistry. 61, 7070–80.

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4 Fundamental Aspects of Crystallization of Lipids Hironori Hondoh, Satoru Ueno, and Kiyotaka Sato

4.1 ­Introduction In crystallization, a step‐wise evolution including cluster formation (clustering), nucleation, and growth occurs under various external factors (temperature, pressure, solute concentration, etc.), as illustrated in Chapter 1. A prerequisite for the initiation of crystallization is to achieve a driving force that originates from the deviation between thermodynamic equilibrium and nonequilibrium conditions in the crystallizing systems. Given the driving force, the crystallization proceeds continuously from nucleation to crystal growth under isothermal or nonisothermal conditions until aging of the end products stabilizes the crystal network formation. Several approaches have been attempted to describe the overall crystallization processes carried out under isothermal and nonisothermal conditions, including the processes of homogeneous/heterogeneous, secondary nucleation, and crystal growth. Semi‐empirical models describing the overall crystallization processes have been proposed using various approaches to appropriately accommodate crystallization‐specific lipid systems (Dibildox‐Alvarado & Toro‐Vazquez 1998; Foubert et al. 2002, 2003, 2006; Los et al. 2002a, 2002b; Rousset 2002; Marangoni et al. 2006; Vereecken et al. 2009; Le Reverend et al. 2009; Hjorth et al. 2015a, 2015b). In this chapter, we discuss crystallization from a fundamental point of view by discussing the driving forces for crystallization, nucleation, and crystal growth of lipids separately. We begin with structure models of the liquid states of lipids, then discuss the driving force for crystallization, nucleation, and crystal growth. The formation of crystal networks and the effects of various external factors such as temperature variation, additives, shear, sonication, and emulsification are described in other chapters.

4.2 ­Physical and Structural Properties of Lipid Liquids From a macroscopic point of view, liquids of matter are thought to be isotropic and the molecules in the liquid state to behave with random Brownian motion. In the cases of lipids, however, many researchers have worked to figure out the molecular‐level anisotropic Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, First Edition. Edited by Kiyotaka Sato. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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properties of lipids in the liquid state, conceiving the idea that lipids are structured in a liquid state. One may assume that strongly anisotropic molecular interactions involving van der Waals interactions among the hydrophobic moieties and polar interactions among carboxyl (fatty acids) or glycerol groups (acylglycerols) may be operating and that the crystallization behavior of lipids can be affected by modifying the structured liquid. Research on the liquid structure of lipids, however, should be conducted cautiously for the following reasons. 1) Experiments to successfully determine the liquid structure of lipids are difficult to conduct, similarly to other liquid materials. The true features of a liquid structure may not be uncovered with only one method; various methods must be combined. 2) Experiments clarifying the liquid structure must employ a large amount of highly pure samples because there is a possibility that impurities or other minor components having concentrations even at the level of ppm may affect the liquid structures. 3) A theoretical study involving molecular dynamics or molecular mechanics cannot be performed without simplifying the molecular shapes of lipids. In cases where the lipids include glycerol groups and long‐chain fatty‐acid moieties, the molecular weights are so large and the molecular interactions are so complex that it becomes difficult to avoid the possibility of overlooking complicate molecular interactions operating among real lipid structures. Even under these limitations, experiments and theoretical studies have been performed to explore the structured liquid of lipids from various viewpoints that will be highlighted in this section. 4.2.1  Preheating Effects Many technologists working on lipid crystallization have a common sense that the lipid samples should be heated to temperatures far above the melting temperature (Tm) of the crystallizing lipid material (pre‐heating). Doing so increases supercooling (ΔT = Tm – Tc, Tc: crystallizing temperature), and the crystallization can be tailored by various external factors because crystallization is initiated after a certain induction time when the temperature is lowered below Tm. When crystallization occurs quickly with small ΔT, controlling the crystallization becomes difficult. The influence of pre‐heating before crystallization is called the memory effect because the pre‐heating may eliminate the effects of memory after the melting of crystallized samples. For example, Hernqvist and Larsson (1982) claimed that a fat should be heated at least 30° C above its Tm to avoid any influence of “memories” on crystallization. Three possibilities can be thought to explain the preheating effects. 1) Molecular‐level anisotropic structures are present in the liquid of lipids by its original nature, and such structures are strengthened in the liquid by melting the crystalline materials. 2) Soon after melting, the lamellar‐type structures of the lipid crystals still preserved in the liquid state dissociate at elevated temperatures. 3) There is no anisotropic structure in pure liquid in its original nature. Instead, certain composites made up of lipid molecules and other high‐melting minor components are formed and decompose at elevated temperatures far above the Tm of lipids.

Fundamental Aspects of Crystallization of Lipids

Evaluation and justification of each possibility explaining the preheating effect may be open to future research, and we here refer to previous studies about the preheating effects on the crystallization of lipids. Van Malssen et al. (1996) studied the crystallization of the β form of cocoa butter (CB) by using different CB samples produced in different countries, which differ slightly in the fatty‐acid compositions of the triacylglycerols (TAGs) of CB and other minor components. They heated the CB samples above 30° C and then cooled them after keeping them at preheating temperature. They then observed the crystallization of polymorphic forms of CB with X‐ray diffraction (XRD) in‐situ during further cooling at 25° C. The rate of crystallization of CB is too low to crystallize directly without using a tempering or crystal‐seeding technique. Therefore, they decided that, if the β form of CB was observed by this method, it would be because of the memory effect. Without this effect, β’ of CB was crystallized at 25° C. Table 4.1 indicates the β‐memory point temperature (β‐MPT), which is defined as the maximum temperature to cause the direct crystallization of the β form of CB at 25° C within 45 minutes after preheating at β‐MPT and subsequent cooling. Crystallization of CB occurred in the β’ polymorph at 25° C, when the CB samples were heated above β‐ MPT and then cooled. Tm of the β polymorph (more precisely Form V) of CB is around 32–33° C, depending on the area of production. In fact, CB samples having all origins except for Brazil exhibited memory effects. These results were explained by the presence of a higher concentration of stearic acid–containing TAGs. A memory effect of this type may be categorized as possibility 3 above. The effects of preheating on ΔT were examined for fatty acids (Yoshimoto & Sato 1994; Iwahashi et al. 1991). Fatty acids exist mostly as dimers in their liquid state and in  nonpolar solvents, as determined by combined vapor‐pressure osmosis and near‐ infrared spectroscopic measurements (Iwahashi et al. 1995). The temperature dependence of the molecular conformation and liquid structure of oleic acid in a neat liquid was studied by measuring Tc, density, viscosity, self‐diffusion coefficient, and other spectroscopic properties (NMR, infrared spectrum, fluorescence polarization, electron spin resonance; Iwahashi et al. 1991). It was determined that oleic acid has three liquid structures depending on temperature: (1) the first structure consists of clusters of a quasi‐smectic liquid crystal in a range of temperatures from Tm (16° C) to 30° C. The clusters are randomly oriented and are smaller than the wavelength of visible light because the samples of oleic acid are always a transparent clear liquid. (2) The second structure presumably consists of clusters having a less ordered structure Table 4.1  β‐memory point temperature (β‐MPT) of cocoa butter of different country origins. Country of Origin

β‐MPT (±0.25° C)

Country of Origin

β‐MPT (±0.25° C)

Peru Bahia, Brasil Equatorial Guinea Sierra Leone Liberia Ivory coast

34.5 32.5 36.0 35.5 35.0 36.0

Ghana Lome, Togo Lagos, Nigeria Cameroon Congo Malaysia

35.5 35.5 35.5 34.5 35.5 38.0

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Crystallization of Lipids

(a)

(b) 3.6 Radius, a /10–10m

10 Tc (° C)

108

Oleic acid

8 6 4

Tetradecane 20

40

3.4 3.2 3.0

60 Tp (° C)

80

100

20

40 60 Temperature (° C)

80

Fig. 4.1  (a) Relationship between crystallization temperature (Tc) and preheating temperature (Tp) of oleic acid and tetradecane. ● cooling from liquid after melting of crystal; ◻, cooling from liquid formed after condensation of gas, (b) Stokes radius.

between 30° C and 55° C. (3) The third structure appears to be an isotropic liquid above 55° C. The presence of the three liquid structures was indicated by the measurements of Tc of oleic acid performed with differential scanning calorimetry (DSC) using highly pure samples (>99.9%). A sample in a DSC pan (~1 mg) was heated to the preheating temperature (Tp) and kept at this temperature for 15 minutes, then cooled at 2° C/min to observe the exothermic peaks of crystallization. The relationship between Tc and Tp is depicted in Fig. 4.1(a). Tc depends on Tp; it remains constant (about 10.4° C) up to Tp = 30° C, then decreases sharply, and again remains constant (4.7° C) above Tp = ~50° C. This relationship suggests the presence of three different liquid structures at temperatures below 30° C, between 3° C and 50° C, and above 50° C. Such a relation holds for the liquid samples of oleic acid formed by both melting from crystals and condensation of a gas phase. As a reference, Tc of tetradecane was not influenced by Tp, as seen in Fig. 4.1(a). The distinct difference between oleic acid and tetradecane presumably results from the different mobilities of those molecules in their liquids; specifically, tetradecane should have a higher mobility than oleic acid. In fact, the diffusion coefficient, D, of tetradecane obtained by NMR measurement is about 20 times larger than that of oleic acid. The values of the Stokes radius increases with increasing temperature up to 30° C, then becomes almost constant in the range of 30–55° C, and increases again above 55° C (Fig. 4.1b). Breakpoints exist at 30° and 55° C and obviously correspond to the Tc versus Tp relationship. Yoshimoto and Sato (1994) investigated the effects of Tp on the nucleation and growth of oleic acid polymorphs. As preheating temperatures of the melt increased, the crystallization temperatures of α‐form crystal decreased from 8.5° C to 3.6° C. Pre‐heating thus remarkably affected the nucleation of the α‐form but not the growth rate. XRD experiments were carried out to directly observe the long‐spacing peak of the lamellar structures in the liquid phase of oleic acid (Iwahashi et al. 1991). A clear diffraction peak of 1.73 nm, although quite weak, was detectable at 22° C. As this value corresponds to one monomer of oleic acid, they proposed a quasi‐smectic liquid crystalline model of oleic acid that may present below 30° C as seen in Fig. 4.2. The dynamic properties of the molecular movements of oleic acid and other fatty acids are discussed in a review by Iwahashi and Kasahara (2011).

C=C

C=C

C

O

CH

O H

O H O C

Fundamental Aspects of Crystallization of Lipids

C=C O H O

C C

O H O

CH

C=C

C

O H O

O H O

C

1.73 nm

Fig. 4.2  Quasi‐smectic liquid crystalline model of neat liquid of oleic acid.

4.2.2  Liquid Phases of Triacylglycerols The structures and properties of TAGs in the liquid state have been studied over many years. As a neutral lipid, a TAG molecule does not have intermolecular hydrogen bonding. However, one may assume that attractive dipole‐dipole interactions between the –C = O groups of the glycerol groups and lateral van der Waals interactions between the hydrocarbon chains of three fatty‐acid moieties may be strong enough to form lamellar‐type aggregates of TAGs in the liquid state. Based on XRD patterns from neat liquid of trimyristoyl‐glycerol, which revealed long‐spacing peaks of 3.2 nm with first‐ and third‐order reflections, the idea that pseudo‐lamellar domains persist in the liquid state was proposed as depicted in Fig. 4.3(a). Larsson (1972) claimed that molten TAG molecules having the tuning‐fork (or chair; see Chapter  2) conformation gather together and form a lamellar domain structure, and that the domains remain at 20° C to 40° C above Tm, decreasing in size with increasing temperature. Hernqvist (1984) supported the Larsson model using Raman spectroscopy measurements. The existence of liquid‐crystal‐like domains having a smectic structure in the liquid of TAG was supported by 13C NMR studies (Callaghan 1977; Callaghan & Jolley 1977). They examined longitudinal and transverse 13C spin relaxation times to investigate the molecular motion of tristearoyl‐glycerol over a wide range of temperatures in the melt from Tm (70° C) to ~200° C. The overall molecular rotational diffusion rate and the diffusion rate about successive bonds in the stearoyl chains were measured. An anisotropic rotor model was applied to analyze fast (8 109/sec) and slow (0.018 109/sec) molecular diffusion rates. The alignment of the former diffusion rate is close to the long‐chain axis of the tuning‐fork model. In addition, the two different relaxation times

109

110

Crystallization of Lipids

(a)

(b)

(c) h

h*

Y

Fig. 4.3  Structure models of liquid of triacylglycerol. (a) Smectic liquid crystal model, (b) nematic liquid crystal model, and (c) discotic model (Y conformer). (Smectiic model of h and h* is also shown).

for the two chemically equivalent primary glycerol carbons may support the existence of the tuning‐fork configuration in the melt. The structural properties of the liquid of TAGs were examined with vibrational spectroscopic methods, which are quite informative for assessing the structural properties of TAGs in the crystalline as well as liquid state. Da Silva et  al. (2008, 2009) studied liquid phase transitions using Raman spectroscopy. They observed the ratio of the absorption intensity of the symmetric methylene stretching mode to that of the anti‐ symmetric one (I[νs)CH2)]/I[νas)CH2)]) to identify the intermolecular order of the hydrocarbon chains of TAGs, and proposed that a solid‐like component exists 2° to 3° C above the Tm of the β form of TAG. Different opinions have been put forward regarding structure models of the liquid of TAGs. Using neutron diffraction techniques, Cebula et al. (1992) proposed a nematic liquid‐crystal model (Fig.  4.3b), presenting a contrast to the smectic‐type Larsson model. In this model, three hydrocarbon chains of TAG molecules are oriented to form domains having a nematic liquid‐crystal structure with a tuning‐fork (or chair) conformation. This model was later commented on by Larsson (1992). A model in which TAG molecules have splayed and disordered chains in the liquid state has been proposed (Corkery et al. 2007). Specifically, the TAG molecules take on a Y‐shaped conformation (Y‐conformers), in which the three soft hydrocarbon chains have an angle of 120 degrees between them (Fig. 4.3c); the Y‐conformer disks loosely attach to each other and stack into flexible, short cylindrical rods. It was also suggested that h‐conformers having a tuning‐fork (or chair) conformation may exist at low temperatures, and that Y‐conformers exist at high temperature. Pink et al. (2010) theoretically investigated the liquid of trilauroyl‐glycerol (LLL), examining the discotic model

Fundamental Aspects of Crystallization of Lipids

proposed by Corkery et  al. (2007) and the nematic model proposed by Cebula et  al. (1992). In their calculations, LLL molecules in a crystalline state are in an h‐conformation with extended, possibly all‐trans, chains at low temperatures. In the liquid state, the LLL molecules are in either a Y conformation or an h* conformation above Tm (319K; Fig. 4.3c). The h* conformation is an excited h‐conformation. They determined that the conversion of h to Y corresponds to the transformation from the crystal phase to the liquid phase of the discotic Y‐conformer model, whereas the conversion of h to h* corresponds to the transformation to the liquid phase of the nematic h‐conformer model. They used the experiment data for the enthalpy of melting and temperature dependence of the 1132 cm−1 Raman band as reference data for the assessing the calculation. The calculation was in favor of the discotic Y‐conformer model because this model led to reasonable agreement with the value of enthalpy of melting and the Raman band. A computer simulation was applied to TAG molecules in liquid, crystalline, and water‐adsorbed states (Yan et al. 1994; Engelsen 1994; Sum et al. 2003; Hall et al. 2008; Hsu & Violi 2009; Dibildox‐Alvarado et al. 2010a). It was indicated that the polar groups among the TAG molecules show a pronounced tendency toward aggregation in the liquid state (Sum et al. 2003; Hsu & Violi 2009). Computer simulations of PPP using atomistic molecular dynamics revealed the packing of TAG molecules and their molecular conformations in the liquid to‐crystalline phase transition (Hall et al. 2008). The simulation used 128 PPP systems and displayed the transition processes through a temperature quench from the isotropic liquid phase at 350K to 310K, which is below Tm of α (319K; Hagemann, 1988). Figure 4.4 presents snapshots of PPP molecules simulated (a)

(b)

(c)

(d)

Fig. 4.4  Snapshots of atomic molecular simulation patterns of PPP at (a) 350 K, 300 ns, (b) 300 ns, (c) 310 K, 420 ns, and (d) 310 K, 700 ns. With courtesy of Prof. I. Vattulainen (Hall et al. 2008). (See color plate section for the color representation of this figure.)

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Crystallization of Lipids

at 350K (300 ns) and at 310K from 300 ns to 700 ns, visualizing the ordering process of the PPP molecules in the lamellar structure after quenching at 310K (Hall et al. 2008). The 1ns running average density at 350K was 853.8 ± 0.3 kg/m3 (100–300ns), but it was 952.0 ± 0.6 kg/m3 (500–700ns) at 310K. Correspondingly, the time dependence of the order parameter, which is defined as the orientation order of the PPP molecules with respect to the average directions of the chains in the layered structure, increased from 0.4 at 350 ns to 0.9 at 500 ns. The rapid ordering process is divided into two parts: the system is in a metastable state and remains in the liquid phase right after the temperature quench. Once the ordering process is initiated, the system goes through the phase transition in about 200 ns. The prenucleation behavior of pure saturated and unsaturated TAGs and vegetable oils was examined by means of fluorescence polarization spectroscopy and molecular mechanics simulations (Dibildox‐Alvarado et  al. 2010a). The simulation and experiment results indicated that short‐range van der Waals interactions are responsible for an increase in the microviscosity of the fat/oil blends before crystallization, and that the presence of TAGs containing at least one palmitic‐acid moiety induces an increase in the microviscosity associated with pre‐nucleation.

4.3 ­Driving Forces for Crystallization The driving force of crystallization is the difference in the chemical potential μ, the Gibbs free energy per molecule, of the molecules in the crystal and in the gas or solution or liquid melt at the nonequilibrium conditions at which the crystallization takes place. As most of the crystallization of lipids occurs from the solution or liquid phase, we limit the present discussion to the driving forces for crystallization from the solution and liquid phases. Figure  4.5 illustrates the driving force (Δμ) for crystallization from the liquid and solution phases. The equilibrium conditions of the two phases correspond to the temperatures at Tm and Ts. When the temperature is reduced to Tc, the liquid becomes supercooled and the solution phase becomes supersaturated. In the solution phase, the

(a)

(b) liquid

∆µ

Solubility

Chemical Potential

112

crystal

Tc

Tm Temperature

Fig. 4.5  Driving force for crystallization.

Xc ∆µ XS

Tc

Ts

Temperature

Fundamental Aspects of Crystallization of Lipids

concentration of the solute (X in molar fraction) is Xc, and the saturated concentration is Xs at T = Tc. The driving forces for crystallization can then be expressed as follows. Hm T/Tm (from liquid phase) (4-1) k BTc ln(X c /X s ) k BTc ln (from solution phase),



Where ΔHm is the enthalpy of melting, ΔT is the supercooling, kB is the Boltzmann constant, χ is the supersaturation ratio (=Xc/Xs), and 1nχ is the degree of supersaturation. The driving force for the crystallization of lipids plays decisive roles in the crystallization because the rate of nucleation increases with increasing Δμ and the average sizes of crystals are reduced, and the values of Δμ differ among the polymorphic forms. Acevedo and Marangoni (2010) measured with transmission electron microscopy the length and width of nanometer‐scale β crystals of fully hydrogenated canola oil (FHCO) grown from high‐oleic sunflower oil (HOSO) at different supersaturations of lnχ. This crystallization system is actually a solution growth of tristearin from triolein as a solvent. The values of average crystal thickness and width were 148 nm and 63 nm at lnχ = 5.9 and 369 nm and 157 nm at lnχ = 4.8. A schematic illustration of the chemical potential and solubility of three typical polymorphic forms, α, β’, β, of TAGs is presented in Fig. 4.6. The three forms reveal monotropic polymorphism (see Chapter 2), having different Tm and solubility values over a wide range of temperature. Tm is highest for the most stable β form, intermediate for β’, and lowest for α. Correspondingly, the solubility is lowest for β, intermediate for β’, and highest for α. As an example, the driving forces for crystallization of the three polymorphic forms of tripalmitoyl‐glycerol (PPP) from the liquid phase are calculated as summarized in Table 4.2. The value of Δμ is largest for β, intermediate for β, and smallest for α. However, it is worthy to note that the ratio of Δμ between the polymorphic forms is minimized when Tc is lowered. For example, Δμ(β) / Δμ(α) is 10.3 at Tc = 325K whereas it becomes 4.8 at Tc = 320K and 3.1 at Tc = 310K. The same tendency is observed for β’ and β. (a)

(b)

Chemical Potential

α β′

𝛥T(α)

β

𝛥T(β′) 𝛥T(β)

Tc

Tm(α) Tm(β′) Temperature

Tm(β)

Solubility (Molar Fraction)

liquid

α Xc

β′

β

Xα Xβ′ Xβ Tc

Ts(α) Ts(β′) Ts(β)

Temperature

Fig. 4.6  (a) Melting temperature (Tm) and (b) solubility of three polymorphic forms of triacylglycerol.

113

114

Crystallization of Lipids

Table 4.2  Driving forces for crystallization of three polymorphs of tripalmitoyl‐glycerol (PPP) from melt. Tc = 310 (K) Form

Tm (K)

ΔHm (kJ/mol)

α

317.7

103.0

ΔT (K)

7.7

Δμ (kJ/mol)

2.5

Tc = 315 (K) ΔT (K)

2.7

Δμ (kJ/mol)

0.9

Tc = 320 (K) ΔT (K)

Δμ (kJ/mol)

—

—

β’

329.6

131.5

19.6

7.8

14.6

5.8

9.6

3.8

β

339.4

163.3

29.4

14.1

24.4

11.7

19.4

9.3

This  tendency becomes critically important when we consider the relative rate of nucleation of crystals among the three polymorphic forms with varying driving forces. Namely, the advantage of large driving force for the most stable β form compared with those of metastable α and β’ forms is minimized when the driving force is increased by decreasing Tc. The same conclusion may be drawn for crystallization from the solution phase.

4.4 ­Nucleation 4.4.1  Homogeneous versus Heterogeneous The actual crystal nucleation processes can occur in different manners. If the crystallization system does not contain any foreign particles and consists of crystallizing matter alone, homogeneous nucleation can occur. If foreign materials are present in the liquid and they intimately interact with the crystallizing matter, it is possible for the surfaces of the foreign material to cause nucleation (heterogeneous nucleation) at lower driving forces compared to homogeneous nucleation. Homogeneous and heterogeneous nucleation can be categorized as primary nucleation. In contrast, when crystals of the same material exist and act as attrition agents or seed crystals, secondary nucleation can occur. In the actual crystallization procedures of lipids, it is hardly possible for homogeneous nucleation to occur because of the presence of minor ingredients that may catalyze the nucleation. In addition, secondary crystallization often occurs in factory‐scale crystallization processes of lipids. Nucleation of crystals from the viewpoint of the classical nucleation theory (CNT) is illustrated in Fig. 4.7. According to classical nucleation theory (CNT; Kashchiev 2000;

Molecules

Crystal nucleus

Growing crystal

Fig. 4.7  Evolution from molecules to crystal nuclei and irreversibly growing crystals.

Fundamental Aspects of Crystallization of Lipids

Kashchiev & van Rosmalen 2003; Vekilov 2005; Povey 2014), the process represents stochastic clustering of molecules into nuclei which are nanoscopically small crystalline aggregates regarded as having minimum‐energy shape and size‐independent molecular volume and specific surface energy. Although the smallest nuclei (known also as embryos) tend to decay, after randomly surpassing the so‐called critical nucleus size, they become able of irreversibly growing to macroscopically large crystals. The rate of nucleation is defined as the number of supercritical nuclei appearing per unit volume and time. For its formation, the critical nucleus requires maximum work, which determines the activation energy of nucleation, the most important parameter in the nucleation rate. Although the activation energy of nucleation depends on a number of parameters, it is largely controlled by the crystal/liquid or crystal/solution interfacial energy and the driving force for nucleation (see Eq. 4‐1). Nonclassical nucleation theory (NCNT; Kashchiev 2000; Vekilov 2005; Whitelam 2010; Cabriolu et al. 2012; Prestipino et al. 2012; De Yoreo et al. 2015) does not simply assume the processes illustrated in Fig.  4.7. NCNT takes into account more complicated prenucleation stages and thermally fluctuating diffuse solid‐liquid interfaces of clusters and crystal nuclei, as well as incorporating monomeric as well as polymeric growth units during the transformation from clusters to crystal nuclei. In this chapter, we discuss nucleation based on CNT. A full discussion of the atomistic/molecular level kinetics and thermodynamics included in the crystal nucleation is beyond the scope of this chapter; readers are directed to the relevant book chapters and reviews (Kashchiev 2000; Aquilano & Squaldino 2001; Kashchiev & van Rosmalen 2003; Povey 2014). We consider here that a spherical three‐dimensional (3D) nucleus of crystal (A) having a radius of r is adsorbed on the surface of substrate (B), and that A and B are put in a supercooled liquid or supersaturated solution (Fig. 4.8). One may be doubtful about a spherical model of the nucleus because lipid crystals, including the nuclei, are all of polyhedral shape. However, the spherical‐shaped nucleus can be treated in simpler ways, and the understanding obtained with this model can be applied to polyhedral‐ shaped nuclei (Aquilano & Squaldino 2001).

(a)

θ=180°

(b)

θ=90°

∆G*

Liquid γA

θ=75°

Nucleus (A) θ Substrate (B)

γAB

γB

Liquid

Crystal

Fig. 4.8  (a) Relationship between specific interfacial energy of a nucleus (A) and a substrate (B) and (b) activation Gibbs free energy for nucleation with different values of contact angle (θ).

115

116

Crystallization of Lipids

In Fig. 4.8(a), the bottom surface of A is shared with the surface of B by adsorption. Specific interfacial energy between liquid versus A, liquid versus B, and A versus B are defined as γA, γB, and γAB. The equilibrium conditions among the specific interfacial energies are related in accordance with Young’s formula,

B

AB

A

cos (4-2)

When the nucleus does not adsorb on the substrate occurs, homogeneous nucleation takes place with the following activation Gibbs free energy for nucleation, ΔG*hom (Kashchiev 2000). G *hom



4f

3 A

2

2

/ 3

(4-3)

In Eq. (4‐3), f is a geometrical factor depending on the shape of the crystal nucleus (4π for spherical, 24 for cubic, etc.), Ω is the volume per molecule in the crystal nucleus, and Δμ is the driving force defined by Eq. (4‐1). For heterogeneous nucleation, the nucleation activation energy ΔG*het is given by (Kashchiev 2000) G *het



G *hom 1/2

3/4 cos

1/4 cos3

(4-4)

The variation in ΔG*het with varying values of contact angle θ is illustrated in Fig. 4.8(b), indicating that ΔG*het decreases with decreasing θ. This is because the work to create the interfaces of the crystal nucleus decreases with adsorption of the nucleus on the substrate, as seen in Fig. 4.8(b). With θ = 180°, however, the substrate has no affects, and ΔG*het = ΔG*hom. Having ΔG*, we can obtain the nucleation rate J as a function of crystallization temperature Tc using the following equation (Kashchiev 2000): J J0 exp

G * /k BTc

zN 0 exp



G * /k BTc ,

(4-5)

in which z is the so‐called Zeldovich factor, N0 is the number density of molecules in the liquid or the solution, ν is the frequency of attaching a molecule to the critical nucleus, and J0 = zN0ν is the nucleation rate preexponential factor. Combining Eqs. (4‐1), (4‐3), and (4‐5), and with γA = γ, we obtain the rate of heterogeneous nucleation: Jhet

N 0 exp N 0 exp

4/3 4/3

f

3

kBT f

Tm2 H T

3

kBT

3

Tm2 ln

2

2

1/2 3 cos

1/4 cos3

from liquid (4-6)

1/2 3/4 cos

1/4 cos3

from solution

The rate of homogeneous nucleation is obtained by setting θ = 180° in Eq. (4‐6), as mentioned previously. The critical nucleus radius r*, which is the minimum radius for the cluster to become a stable nucleus, is expressed as (Kashchiev 2000)

r* 2

/

.

(4-7)

Fundamental Aspects of Crystallization of Lipids

Nucleation Rate (Nuclei mm–3sec–1)

120

80

40

0 46

48 50 Temperature (° C)

52

Fig. 4.9  Rate of nucleation of tripalmitoyl‐glycerol (PPP) crystals. ◻, Kellens et al. (1992); ●, Stapley et al. (2009). Adapted from Stapley et al. (2009).

The value of r* is determined by Δμ for a given set of Ω and γ. It is almost impossible to directly and precisely observe the true features of the critical nuclei occurring in liquid states with the experiment techniques so far available. This is because the critical nuclei must grow quite rapidly soon after the size of r* is attained, and the experimentally visible crystals must have far exceeded r*. Experiment‐based measurements of the rate of crystal nucleation with varying values of Δμ = ΔHmΔT / Tm were accomplished by performing isothermal crystallization of PPP from the melt per Kellens et al. (1992) and Stapley et al. (2009), as portrayed in Fig. 4.9. In the two experiments, the liquid was rapidly cooled to different Tc, and the nucleation rate was obtained by counting the number of nuclei appearing per unit of time and unit of observed volume. The polymorphic form of PPP observed over the range of Tc in Fig. 4.9 was β’ (Stapley et al. 2009). The results obtained by the two groups are in good agreement, clearly revealing nonlinear dependence of the nucleation rate on ΔT = Tm – Tc. The nucleation rates of palm oil also increased approximately exponentially and much greater numbers of crystals having a smaller final average crystal size were formed with decreasing Tc (Harrison et al. 2016). Another approach to obtaining the important variables of ΔG* and γ appearing in the nucleation rate J is to measure the induction time for nucleation, τi. τi can be monitored with DSC during isothermal conditions at Tc, to which the liquid is rapidly quenched from above Tm (Ng 1990; Kloek et al. 2000a, 2000b; Toro‐Vazquez et al. 2000; Dibildox‐ Alvarado et al. 2010b). For this purpose, it is necessary to obtain the thermodynamic variables of ΔHm and Tm and to measure the τi values at different Tc. As τi is reciprocally proportional to a power of J (Kashchiev & van Rosmalen 2003), ΔG* and γ can be calculated from the slope(s) of the linear regression of log (τi) vs. 1 / TcΔT2. Actually, the experimentally detected value of τi may include the true induction time for nucleation as well as the time delays needed to monitor nucleation with the experiment techniques (e.g., the time needed to detect the release of latent heat of crystal nucleation with DSC or to let the nuclei grow to optically visible size with microscopic

117

118

Crystallization of Lipids

Table 4.3  Induction time (τi) and activation Gibbs energy for nucleation (ΔG*) of PPP and SSS in triolein. PPP ΔT

τi (min)

SSS ΔG*

ΔT

τi (min)

ΔG*

23.7

2.04

1.07

24.2

3.56

2.01

22.7

2.50

1.16

23.2

4.97

2.19

21.7

2.66

1.27

22.2

4.88

2.39

20.7

2.61

1.40

21.2

5.56

2.62

19.7

2.98

1.54

20.2

8.47

2.88

18.7

4.79

1.71

19.2

7.84

3.19

Dibildox‐Alvarado et al. 2010b.

methods). Kloek et al. (2000) examined the induction times of isothermal crystallization of fully hydrogenated palm oil in sunflower oil mixtures and obtained a γ of 3.4 to 3.9 mJ/m2 for the heterogeneous nucleus formation of β’ of hydrogenated palm oil. Dibildox‐Alvarado et al. (2010b) measured τi of the β form of PPP and SSS in the solvents of OOO, high‐oleic safflower oil and soybean oil at the solute‐to‐solvent ratio of 25:75. The values of τi and ΔG* of PPP and SSS in OOO are listed in Table 4.3. It is evident that τi and ΔG* increased as ΔT decreased and that τi was longer for SSS‐OOO blends than for PPP‐OOO blends. 4.4.2  Polymorph‐Dependent Nucleation Kinetics CNT predicts that the nucleation rates of polymorphic forms are determined by the values of ΔG*, assuming that the preexponential kinetic factor in Eq. (4‐5) is common to the polymorphic forms. All of the variables included in ΔG* (Eq. 4‐3), however, are polymorph‐dependent because the shape of the critical nucleus, the surface (interface) energy γA, the molecular volume of the critical nucleus, and Δμ differ among the polymorphic forms. In addition, γAB may also be polymorph dependent, if foreign matter acting in the heterogeneous nucleation processes prefers specific polymorphic forms. Among these, however, we may assume that the most decisive variables are γ and Δμ (Aquilano & Squaldino 2001). The values of Δμ are largest for β, in‐between for β’ and smallest for α when Tc is fixed below Tm(α), as seen in Figs. 4.5(a) and 4.6(a) and in Table 4.2. This may indicate that the rate of nucleation is highest for the most stable β form. However, we may assume that the values of γ are smallest for α, in‐between for β’, and largest for β. ΔG* is proportional to γ3 / Δμ2 (Eq. 4‐3), and the values of ΔG* for the three polymorphs may increase in the order of α, β’, and β because of the prevailing factor of γ3 over Δμ2, as illustrated in Fig. 4.10(a). The rate of nucleation of the three polymorphs examined at different Tc is as presented in Fig. 4.10(b). The polymorph‐dependent nucleation rates illustrated in Fig.  4.10(b) are in good agreement with the experiment results. Specifically, the increase in the cooling rate during nonisothermal crystallization may cause preferred nucleation of metastable

Fundamental Aspects of Crystallization of Lipids

(a)

(b) βʹ

β

α

∆G# Nucleation rate

α Supercooled Liquid

α βʹ β

βʹ

β

∆G Tm(α)

Tm(βʹ)

Tm(β)

Temperature

Fig. 4.10  Polymorph‐dependent nucleation rates of α, β’, and β. (a) Crystal free energy (ΔG) and activation free energy for nucleation (ΔG*) and (b) nucleation rate at different temperatures.

(a)

(b)

Fig. 4.11  Polarized optical micrographs of crystallization of spherulite of tripalmitorl‐glycerol (PPP). (a) α form at Tc = 315K and (b) β’ form at 325 K.

polymorphs, as confirmed for PPP and tristearoyl‐glycerol (SSS) by Himawan et  al. (2007) and for other TAGs by Bayés‐García et al. (see Chapter 6). For isothermal crystallization, Fig. 4.11 presents polarized optical microscopy (POM) images of spherulite crystals of PPP, which were taken after rapidly quenching the molten liquid to Tc (unpublished data). Many tiny spherulite patterns were observed for α at Tc = 315K, and several large spherulite patterns were observed for β’ at Tc = 325K. In both cases, no nucleation of β was observed. Figure 4.12 illustrates the inverse of the induction time (τi−1) for three polymorphic forms of PPP obtained during isothermal crystallization after quenching the PPP liquid at different Tc (Sato & Kuroda 1987). The values of τi were obtained by observing the occurrence of the crystal in‐situ with POM at a fixed Tc. The polymorphic form was determined by XRD soon after crystallization was confirmed. α was crystallized at very  large τi−1 values (small (τi values) at Tc just below Tm(α) with no appreciable

119

Crystallization of Lipids α form

10–1

βʹ form β form τ–1 (sec–1)

120

20–1

50–1 100–1 40

50 Tm(α)

60 Tm(βʹ )

Tm(β)

Temperature (° C)

Fig. 4.12  Inverse of induction time (τ) for crystallization of tripalmitorl‐glycerol (PPP) measured at different crystallization temperatures.

supercooling. τi−1 decreased for β’ and β compared to α over a range of temperatures between Tm(α) and Tm(β’), β’ occurred in a lower temperature range, whereas β prevailed at higher temperatures, with β crystallized exclusively above 53° C. This behavior agrees well with the illustration in Fig. 4.10(b), in which the nucleation rate of β’ becomes higher than that of β with decreasing Tc between Tm(α) and Tm(β’). Kellens et al. (1990) observed that the Tc ranges of the preferred crystallization for the three polymorphs of PPP are below 43° C for α, 43° C 

–0.09

SAXS

–0.13 –0.17

2L3 (3.9 nm) βʹ

–0.21

24° C 4.1 nm 2L1

(b)

3L (6.2 nm) α

–0.25

–15

5

–5

2L1 (4.1 nm) 2L2 (4.8 nm) βʹ

15

25

α

35

Temperature (° C) 23° C 4.8 nm 2L2

13° C 6.2 nm 3L001

5.5° C 3.9 nm 2L3

(c)

βʹ

WAXS traces of β

3.2 nm 3L002

–15

Liquid

–10

–15 –10

–5

–5

0

0 5 10

15

15

20

20 25

1.8

1.6

1.4

1.2

1.0

0.30

30

0.25

0.20

0.15

0.10

25

q (Å–1)

T (° C)

10

T (° C)

5

0.05

288

30

q (Å–1)

Fig. 10.4  Crystallisation of anhydrous milk fat characterized on cooling at 0.1 °C/min. (a) Differential scanning calorimetry (DSC) thermogram, and synchrotron radiation X‐ray diffraction patterns recorded (b) at small angles (SAXS) and (C) at wide angles (WAXS). Adapted from Lopez et al. (2001a).

different β’‐2 L crystals successively melt without any changes in the structural parameters. The HMP TAG crystals correspond to a β’‐2 L (4.15 nm) organization that disappears around 40 °C. The absence of polymorphic transformation on heating of AMF is the signature of stable TAG crystals close to equilibrium. This study showed that a TAG molecular segregation occurs in milk fat on slow cooling from the melt. AMF crystallizes and melts in several independent steps corresponding to the phase separation of several groups of TAGs. Each TAG fraction acts as an independent solid solution. The ability of dry fractionation to separate such TAG crystal species into so‐called olein and stearin fractions after slow cooling rates confirms that they correspond to different TAG compositions (Kaylegian & Lindsay 1995). On cooling of AMF from the melt at the rates of 1–3 °C/min (Fig. 10.5a), TAG molecules sequentially crystallize in α form under three different lamellar structures (Lopez et al. 2005a). From about 17 °C, the successive formation of the α‐2 L (4.6 nm) and α‐2 L (3.8 nm) crystals has been characterized, and from 14 °C TAG molecules crystallize in a α‐3 L (7.2 nm) organization. A time‐dependent sub‐α ↔ α reversible transition was

Crystallization Properties of Milk Fats

(a)

(b)

α

traces of sub-α

3L

α β 3L 3.6 nm

1.2

1.4

1.6 1.8 2.0 q (Å–1) 3L 3L 3L 2.4 nm 1.4 nm 1.8 nm

2L 3.8 nm

–10 0 10 20 30 40 50

T (° C)

liquid

3L 7.2 nm

3L 6.7 nm

3L

2L 4.6 nm

3L

1.2

1.4

1.6 1.8 q (Å–1) 3L

–10

–10

0

0

10

10

20

20 30

30 40

40

50

60

0.40

0.35

0.25

0.30

0.20

0.10

0.15

50 0.05

2.0 3L

T (° C)

2L 3.8 nm 2L 4.6 nm

–10 0 10 20 30 40 50 60

T (° C)

liquid

T (° C)

3L 7.2 nm

0.05

0.15

0.25

0.35

0.45

2L (4–4.15 nm)

q (Å–1)

q (Å–1)

(d)

–0.05

2L 4.6 nm α 2L 3.8 nm α

–0.10

–0.15

–10 –5

3L 7.2 nm α

0

Heat flow (AU) Endo > (E-1)

Heat flow (AU) Endo ->

(c)

5

10

15

20

Temperature (° C)

0.80

0.60

0.40

0.20

25

30

–10

3L(7.2 nm) α

0

2L (4.15 nm) βʹ 2L βʹ 2L (4 nm) (3.8 nm) βʹ

liquid

2L (4.6 nm)

10

20

30

40

50

Temperature (° C)

Fig. 10.5  Crystallization properties of anhydrous milk fat. Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small and wide (insert) angles (a) on cooling at 3 °C/min from 60° to –10 °C, and (b) subsequent heating at 2 °C/min. Differential scanning calorimetry (DSC) thermograms recorded (c) on cooling and (d) on heating. DSC and SR‐XRD were measured simultaneously. Adapted from Lopez et al. (2005a).

observed at –10 °C. DSC recordings show two successive exotherms that have been attributed to the successive crystallization of TAG in α‐2 L crystals and α‐3 L crystals (Fig. 10.5c). Subsequent heating at 2 °C/min has shown numerous structural rearrangements of the α‐2 L and α‐3 L crystals and the formation of β’‐3 L (6.7 nm) and β’‐2 L (4–4.15 nm) crystals that takes advantage of the melting of the other crystalline structures (Fig. 10.5b). The recording of an exotherm in the DSC thermogram confirms α to β’ polymorphic transition occurring on heating of AMF (Fig. 10.5d). For temperatures above 20 °C, the remaining β’‐2 L (4–4.15 nm) crystals progressively melt until their ­disappearance at about 39 °C (Fig. 10.5d). Such rearrangements of TAG molecules into a β’ lamellar organization are facilitated by the presence of the liquid TAG phase. The complex melting behavior confirms that the TAG crystals formed on cooling at 1–3 °C/ min are not thermodynamically stable forms. The crystallization properties of milk TAGs were studied after quenching from the melt (cooling at ~1000 °C/min), to characterize the most unstable crystalline structures and their reorganization as a function of time (Lopez et al. 2001b, 2002a). The samples were cooled rapidly from 60° to 4 °C or –8 °C to ensure crystallization of TAG molecules.

289

290

Crystallization of Lipids

In the set of experiments performed after quenching of AMF to –8 °C, it was possible to identify the formation of α‐2 L (4.7 nm) and α‐3 L (7 nm) crystals thanks to the brightness of synchrotron X‐ray beam which allows fast recordings (Fig. 10.6a and b). The α‐2 L (4.7 nm) crystals were very unstable because they disappeared during 20 minutes isothermal recording and progressively converted into 3 L (Fig.  10.6c; Lopez et  al. 2001b). On subsequent heating of the AMF sample, the α‐3 L (7 nm) crystals melted. From about 11 °C, TAG molecules in the solid phase formed β’‐2 L (3.7 nm) crystals (Fig. 10.7). The exotherm recorded at around 11 °C in the DSC thermogram confirms the α to β’ polymorphic transition that occurs on heating of AMF (Fig.  10.7b). The thickness of this β’‐2 L lamellar structure increased up to 4.1 nm until its final melting, showing structural reorganizations as a function of the increase in temperature. In a second set of experiments, AFM was quenched at 4 °C, and the thermal and structural changes were characterized as a function of time in isothermal conditions, as shown Fig. 10.8(a) (Lopez et al. 2002a). During the 30 minutes following quenching, structural changes occurred, the α‐3 L (7 nm) structure melted and the 3 L (6.6 nm) and 2 L (3.9 nm) structures corresponding to β’ and β polymorphs were formed. Isothermal DSC recorded simultaneously to XRD experiments showed ­exothermic signals with a release of the heat of crystallization as a function of time, corresponding to polymorphic transition from α to β’and β of milk TAGs (Fig. 10.8c). The nucleation time (the time at which a peak starts forming), time of maximum crystallization rate (the time of peak maximum), and heat of crystallization (proportional to peak area) can all be determined from the DSC thermogram. After 4 days storage of AMF at 4 °C, the crystals were organized in 2 L (4 nm) and 3 L (5.4 nm) lamellar structures with the coexistence of α, β’1, β’2 and β polymorphic forms, as shown Fig. 10.8(d) (Lopez et al. 2002a). As a summary concerning the crystallization behavior of AMF, DSC, and XRD investigations showed that milk TAGs segregate as a function of temperature and display a complex polymorphism. Depending on the cooling rate, six different types of crystals were identified, several of them in coexistence, and their time‐ and temperature‐dependent evolutions were quantitatively monitored. They correspond to lamellar structures with 2 L (4–4.8 nm) and 3 L (5.4–7.2 nm) organizations of TAG (Fig. 10.9a). At least five crystalline subcell species were observed at wide angles: α and sub‐α, two β’ and one β (Fig. 10.9b). All these crystalline structures coexist with a liquid phase even at low temperature (i.e., 4 °C). The comparison of the small number of  crystal type formed to the large number of TAGs present in milk fat (Fig.  10.1b) provides evidence that mixed crystals are formed in AMF (i.e., co‐crystallisation of different TAG molecules). On cooling, the first longitudinal organizations of TAG molecules correspond to 2 L structures with long spacings of about 3.8–4.8 nm. These crystalline varieties may correspond to crystallization of HMP fractions of TAGs. These 2 L crystals are generally formed by TAGs with saturated and similar chain length FAs, such as MPP and PPP (Fig. 10.9c). Then, crystallization of 3 L structures (about 6.2–7.2 nm) occur. Triple chain length stackings may correspond to crystallization of unsaturated TAG molecules (e.g., PPO) or to that of TAGs with FA chains of different lengths, like BPP (Fig. 10.9c). On heating, the crystals formed on cooling (2 L, 3 L) melt and recrystallizations take place with the formation of a β’‐2 L (4–4.15 nm) structure. The increase in the thickness of the β’‐2 L crystals on heating is attributed to progressive and selective melting of the TAGs with the shortest FA chains so that the

(a)

(b) 3e+04

2000

2L 4.7 nm

3L001

2e+04

0.42 nm

2.37 nm

7 nm

0 0.00

α

0 0.06

0.13 0.19 q (Å–1)

(c)

0.26

1.1

1.2

1.4

1.5

1.6

1.7

q (Å–1)

(d)

3.6 nm 3L001 7 nm

1.3

0.32

2L 4.7 nm

2L 4.7 nm

3L001 7 nm

3.6 nm 2.37 nm

2.37 nm t = 21 min.

20 min 10 min

t = 5 min.

time

t = 11 min.

5 min

t = 1 min. 0.00

0.06

0.13

0.19 q (Å–1)

0.26

0.32

time

1000

0.42 nm I (UA)

I (UA)

3L002 3.6 nm

1 min 0.00

0.06

0.13

0.19 q (Å–1)

0.26

0.32

Fig. 10.6  Crystalline structures formed at –8 °C by milk triacylglycerols after quenching from 60 °C and evolutions as a function of time in isothermal conditions. Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded (a) at small angles and (b) at wide angles for anhydrous milk fat (thick lines) and cream (thin lines). Selected SR‐XRD patterns recorded at small angles as a function of time at –8 °C just after quenching from 60 °C (c) of anhydrous milk fat and (d) cream samples. Adapted from Lopez et al. 2000, 2001b.

(a)

α

βʹ

–5

0

re ( eratu Temp

5

10 15 20

liquid

25

30

° C)

35 40

1.7

1.6

1.4

1.5 q

1.3

1.2

1.1

(Å–1)

Temperature (° C)

2L 40 35 30 25 20 15 10 5 0 –5 0.10

0.05

3L001

(b)

0.15 0.20 q (Å–1) 3L002

0.25

0.30

0.35

3L003

Heat flow (AU) Endo>

2.0

1.5

1.0

2L 4.7 nm α 3L 3L 7 nm 7 nm 2L + α 2L 3.7 nm 3.6 nm α βʹ 0

10

2L 4 nm

2L 4.1 nm

βʹ

20 30 Temperature (° C)

Liquid 40

60

Fig. 10.7  Melting behavior of anhydrous milk fat during heating after quenching from 60° to –8 °C. (a) Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small and wide (insert) angles on heating at 2 °C/min from –8° to 60 °C. (b) Differential scanning calorimetry (DSC) thermogram recorded simultaneously to XRD experiments on heating. Adapted from Lopez et al. (2001b).

Crystallization Properties of Milk Fats

(a)

(b) βʹ

β α

3L002 3L001 7 nm

3L002

50

40

40 30

Time (min)

3L001 6.6 nm

Time (min)

60

2L 3.9 nm

20 10

1.2

1.6 q (Å–1)

30 20 10

2.0

3L003

30

1.6

1.2

2.0

q (Å–1) 30

25

15 10

Time (min)

20

25 20 15 10

5 0.1

0.2

3L001 7 nm

0.3 q (Å–1)

3L002

0.4

0.5

5

0 0.1

3L005

3L003

0.2

0.3

0.4

(d)

18.0

βʹ1 α

4000

βʹ2

I (Counts)

3000

5

10

2000

10.8

β

βʹ1 β2

7.2

3.6

3L 5.4 nm

0.0

1.1

1.3

1.5

1.7

1.9

2.1

q (Å–1)

1000

βʹ 15

2L 4 nm

I (Counts)

Heat flow (AU) Endo >

14.4

0

0

0.5

q (Å–1)

(c)

α

Time (min)

2L 3.9 nm

βʹ

α

2.9 nm 20

Temps (min)

25

30

0.02

0.10

0.17

0.25

0.32

0.40

q (Å–1)

Fig. 10.8  Isothermal evolutions of synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small and wide angles at 4 °C after rapid quenching from 60 °C of (a) anhydrous milk fat (AMF) and (b) cream samples. (c) Differential scanning calorimetry (DSC) recordings of AMF and cream samples obtained simultaneously with X‐ray diffraction (XRD) experiments. The signal jumps observed on the left side of the DSC recordings correspond to the equilibration of the microcalorimeter after sample introduction. (d) Small and wide (insert) angle SR‐XRD patterns recorded at 4 °C after storage of cream (thin line) and anhydrous milk fat (thick line) samples at this temperature for 135 and 105 h, respectively, following a rapid quenching from 60 °C. Adapted from Lopez et al. (2002a).

solid phase composition enriches with longer FA chains and higher melting points TAGs. Recrystallization also occurs during isothermal storage. The stable crystalline structures take advantages of the melting of the metastable crystals which melt first or of some kind of Ostwald ripening occurring thanks to the liquid phase that coexists with the solid phases. The mixed TAG crystals formed in milk fat organize in the less

293

294

Crystallization of Lipids

(a)

(b) 2L 4 nm 3L 5.4 nm

βʹ1 isotherm 4° C

α

4° C

2L 4.15 nm 2L 3.9 nm

βʹ

traces of β

2L 4.8 nm

3L002 3.2 nm

3L001 6.2 nm 3L001 7.2 nm

α

β

2.9 nm

2L 4.6–4.7 nm

3L002

cooling rate

0.1° C/min

–0.1° C/min

1° C/min

α α

3L003 2.4 nm

3° C/min

sub α

–1° C/min –3° C/min

2.4 nm

3L001 7 nm 0.08

0.02

βʹ2

quenching

Quenching

0.14

0.20

0.26

0.32

1.2

1.4

1.6

1.8

q (Å–1)

q (Å–1)

(c) Longitudinal organization

Lateral chain packing

Double-chain length (2L)

α form

b a

c a

4.6 – 4.8 nm

4.2 – 4.0 nm βʹ form

Triple-chain length (3L) Saturated FA 5.4 – 7.2 nm

Unsaturated FA Saturated FA

Saturated FA

β form

Short-chain FA Saturated FA

Fig. 10.9  Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at –8 °C (a) at small angles and (b) at wide angles after either cooling of anhydrous milk fat at different rates as indicated on the figure or isothermal conditioning at 4 °C during 5 days. (c) Lamellar structures formed by anhydrous milk fat molecules in the solid state (fatty acids [FAs]are drawn as straight lines) and acyl chain lateral packings corresponding to α, β’, and β polymorphic forms. Adapted from Lopez et al. (2005a).

polymorphic forms β’ and α because molecular packing is not dense. Low amount of β crystals have been identified in milk fat, even after long time storage at low temperature. It has been reported on milk fats from ­individual cows with large differences in their TAG profiles that the presence of β polymorphs is dependent on TAG composition and that cooling rate and tempering are not critical factors in the formation of β

Crystallization Properties of Milk Fats

polymorphs (Tzompa‐Sosa et al. 2016). The high concentration of unsaturated TAGs with a carbon number 52–54 and the presence of a substantial amount of liquid TAGs may be equally important for the formation of β polymorphs. 10.3.3  Effect of Shear on AMF Crystals Of particular interest are the AMF crystallization studies carried out under shear (Grall & Hartel 1992; Van Aken & Visser 2000; Vanhoutte et al. 2002; Mazzanti et al. 2009; see Chapter 7). Shear affects the crystallization process as a whole, by enhancing molecular diffusion and favoring the rearrangement of TAG molecules in the melt, which helps to overcome the kinetic barriers for nucleation and growth. The studies on shear effects demonstrated the formation of smaller TAG crystals and narrower distribution sizes at higher shear rates and attributed this to higher nucleation rates and breakdown of milk fat crystals. Under slow cooling rates and mixing speeds, little effect was found from the agitation speed on the kinetics of milk fat crystallization (Vanhoutte et  al. 2002). A detailed synchrotron XRD study on the kinetics of crystallization of AMF performed in a Couette cell showed a shear‐induced acceleration of the α to β’ form transition and the presence of crystalline orientation (Mazzanti et  al. 2009). Shear (i.e., agitation of milk fat) affects the crystallization kinetics and the microstructure with consequences on the mechanical properties of the fat crystal network obtained. Agitation enhances nucleation, which leads to the formation of numerous small crystals with a softening of the material. 10.3.4  Effect of Minor Lipid Compounds TAGs represent generally 97–98% of milk fat. The balance is composed mainly by minor lipids such as free fatty acids (FFAs), monoacylglycerols (MAGs), diacylglycerols (DAGs), and phospholipids that can influence the mechanisms of TAG crystallization in AMF (i.e., the nucleation stage, the crystal growth, or the polymorphic behavior). Most of the experiments investigating the effect of minor components on milk fat crystallization are performed under isothermal conditions and the crystallization behavior is monitored by DSC and pulsed nuclear magnetic resonance (pNMR). The crystallization process is described by the Avrami and the Gometz models which are fitted by nonlinear regression. It has been shown that removal of minor lipid components from milk fat has no effect on melting point, equilibrium solid fat content, polymorphic forms, microstructural crystal network, and mechanical properties (Wright et al. 2000; Wright & Marangoni 2003). However, the minor components affect the crystallization kinetics of milk fat and delay the onset of crystallization at low degrees of supercooling (Wright et al. 2000). Milk fat DAGs have been reported to have an inhibitory effect on the crystallization of milk TAGs without modifying the microstructure of crystals. It was suggested that structural complementary between DAGs and crystallizing TAGs allowed the TAGs to co‐crystallize within early seed crystals and subsequent further delay TAG crystallization (Wright et al. 2000). Other studies showed that the effect of DAGs and MAGs on the crystallization behavior of milk fat depends on temperature and concentration. Moreover, the type of esterified FAs and the polar head of the amphiphilic molecules determine to what extent the partial glycerides MAGs and DAGs influence the

295

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Crystallization of Lipids

nucleation and crystal growth of TAGs (Foubert et  al. 2004). For example, stearic acid (C18:0)–based MAGs and DAGs enhance heterogeneous nucleation at low temperatures, while at higher temperatures an interaction with the crystal growth predominates. Oleic acid (C18:1c9)–based MAGs and DAGs have an effect on the nucleation process, although no interaction with the crystal growth was observed (Foubert et al. 2004). The effects of MAGs on milk TAG crystallization in recombined cream revealed differences as a function of the FA esterified (Fredrick et  al. 2013). MAGs with C18:0 formed on ­cooling a two‐dimensional crystal at the surface of the emulsion droplet, which induced interfacial heterogeneous nucleation and an acceleration of TAG crystal growth and α to β’ polymorphic transition. On the contrary, MAGs with C18:1c9 did not affect the crystallization behavior, whereas MAGs with C12:0 showed intermediate behavior. None of the MAGs influenced the solid fat content after storage for 5 days at 5 °C. The observed differences in nucleation mechanisms and crystallization kinetics may influence the microstructural arrangement of the milk TAGs inside the emulsion droplets and consequently the partial coalescence rate and hence the whipping properties of recombined creams. Phospholipids were shown to delay the onset time of AMF crystallization under isothermal conditions at 25 °C by their fast adsorption on the growth sites of crystals inducing steric hindrance (Vanhoutte et al. 2002). The effect of FFAs on milk fat crystallization has been demonstrated under isothermal conditions at 25 °C. The addition of short‐chain FFAs increased the induction time of milk fat ­crystallization, whereas the addition on saturated long‐chain FFAs accelerated the crystallization kinetics with consequences on the microstructure of milk fat crystals (Bayard et al. 2017). As a conclusion, the molecular interactions between the minor lipids and milk TAGs able to affect milk fat crystallization depend on the temperature, on their concentration and on the FA composition (mainly the chain length similarity) of the phospholipids, FFAs, MAGs, and DAGs.

10.4 ­Crystallization of TAGs in Bovine Milk Fat Globules and Emulsion Droplets Milk and many dairy products are O/W emulsions (e.g., cream, cheeses). Studying the crystallization of milk TAGs in milk fat globules and processed emulsion droplets is of prime importance because it affects many properties in the following: ●● ●● ●● ●●

resistance of fat globules to disruption and then to coalescence, susceptibility of fat globules to churning for the manufacture of butter, partial coalescence and stability of whipped cream, and texture, rheological properties and mouth feel of high‐fat content food products (e.g., cream, butter, cheeses products).

It is therefore important to understand better the physical properties of TAGs in milk fat globules (e.g., their thermal and crystallographic properties) for industrial applications and to improve the quality of food products. Moreover, it is interesting to compare crystallization of milk TAGs dispersed in emulsion, such as milk or cream (which is the high concentration of fat globules from milk to reach at least 30% fat) in which fat globules are surrounded by a membrane rich in phospholipids, with crystallization of bulk milk fat (AMF). It is also important to know the crystallization properties of emulsion droplets as a function of their size.

Crystallization Properties of Milk Fats

The crystallization properties of milk TAGs in emulsion were studied at different scale levels by using polarized light microscopy and TEM, DSC, and XRD. Microscopy techniques showed that the morphology and the location of TAG crystals within milk fat globules are affected by the cooling rates and tempering. After rapid cooling of milk, numerous small crystals of needle type are formed with no preferred orientation. At a slow cooling rate, fat globules display needle‐shaped crystals that can deform the biological membrane surrounding milk fat globules (Fig. 10.10). After long storage at low temperature (4–7 °C) or after tempering, TAG crystals are of needle type with a radially organized crystallization. Bhandari’s group performed excellent experiments using cryo‐TEM, showing the stacking of individual lamellar layers with 3.8–4.2 nm thickness formed by milk TAG molecules at the periphery of droplets in nano‐emulsions upon cooling at 4 °C (Fig. 10.11; Truong et al. 2015). The examination of the crystallization behavior and TAG polymorphism in emulsion by using XRD is much more challenging than for AMF and especially difficult because (1) both small and wide angle XRD should be considered at the same time and ­compared to determine the evolution of each of the species as a function of time, (2) the X‐ray intensity diffracted by each of the crystalline structures is proportional to the fraction of particular crystal in the structure, (3) the whole XRD signal is largely absorbed by the surrounding water and its solutes (e.g., casein micelles, minerals, lactose), and (4) the small‐angle XRD peak broadening results from the crystallization constraints in dispersed systems and to the lower size of the crystals. The use of DSC coupled to SR‐ XRD is a suitable way allowing identification of the crystalline structures formed by TAG molecules as a function of temperature and time in dispersed systems such as

(a)

(b)

(c)

Fig. 10.10  Polarized light microscopy images of TAG crystals within milk fat globules formed after cooling at different rates from 60° to −5 °C, (a) cooling at 5 °C/min, (b) quenching, (c) cooling at 0.5 °C/ min, and (d) images taken during cooling of milk at 1 °C/ min, the intermediate temperatures are indicated in the figure. TAG crystals can deform the milk fat globule membrane as indicated by arrows. Adapted from Lopez (2011). (See color plate section for the color representation of this figure.)

297

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Crystallization of Lipids

(a)

(b)

50 nm

20 nm

Fig. 10.11  Cryo‐transmission electron microscopic (cryo‐TEM) micrographs of nano‐emulsions containing crystalline particles of high melting point fraction of milk fat called stearin, upon crystallization at 4 °C. (a) Stacking of individual TAG lamellar layers formed in the stearin nano‐ emulsions after cooling at 1 °C/min. (b) Thickness of single lamellae including light and dark layers in the stearin nano‐emulsions. Adapted from Truong et al. (2015).

milk fat globules (Lopez et  al. 2000, 2001c, 2002a, 2002b) and emulsion droplets of ­various sizes (Lopez et al. 2002b; Michalski et al. 2004; Bugeat et al. 2011). The investigations of milk TAG crystallization within emulsion droplets is not performed below about –10 °C to avoid the formation of ice crystals that could alter the physical stability of the emulsion. 10.4.1  Effect of Cooling Rate and Tempering The crystallization behavior of milk fats in emulsion is influenced by changing the cooling rate, more remarkably than that in a bulk phase (see Chapter 6). It is also affected by tempering (i.e., successive cooling and heating). Therefore, we discuss here the effects of cooling rate on the crystallization properties of milk fat dispersed in milk fat globules or lipid droplets, which are crystallized at the rates of cooling in the range of 0.1° to 1000 °C/min from 60 °C and subjected to subsequent heating at the rate of 2 °C/min. Slow cooling of cream (0.1–0.15 °C/min) leads to the DSC recording of a single broad exotherm corresponding to crystallization of TAGs in fat globules (Fig. 10.12b). However, precise XRD study allowed isolating four polymorphic forms that are successively formed during the cooling process (Fig. 10.12a; Lopez et al. 2001c). On cooling from the melt, the 2 L (4.7 nm) crystals were first formed from 22 °C, then the 2 L (4 nm) crystals were observed from 20 °C. From 16 °C, the formation of 3 L (7.1 nm) and 3 L (6.5 nm) organizations were reported. On slow cooling of cream (0.1–0.15 °C/min), nucleation occurs in the α form, then both the α and β’ polymorphic forms coexist until the end of cooling. On subsequent heating of the cream, the successive melting of the 3 L (7.1 nm), 3 L (6.5 nm), and 2 L (4.7 nm) organizations occurred (Fig.  10.12c). The β’‐2 L (4 nm) predominantly occurred during the cooling and heating processes, and its lamellar thickness increased to 4.16 nm on heating. The DSC curve recorded on heating of the cream showed three endotherms (Fig. 10.12d). The LMP endotherm corresponds

Crystallization Properties of Milk Fats

(a)

(c)

βʹ

α 3L1(002)

βʹ 3L1(002)

3L1(001)

20° C 2L2

71.3Å

40Å Liquid

2L1 3L2

Liquid

6

46.5 Å

65 Å

16 26

3L2(002)

1.1

36

1.3

1.1 3L1(003)

1.5 q (Å–1)

1.3

1.7

2L1 3L2

1.7

10

16

20

0.4

T (° C)

27

0.3

0

6 T (° C)

22° C

0.2

1.5 q (Å–1)

2L2

3L1(005) –5

0.1

–2 3 8 13 18 23 28 33 38

–4

T (° C)

3L1(001)

α

T (° C)

12° C

30

37

40

47

50

2L

60

0.5

0.25

0.15

q (Å–1)

0.05

q (Å–1)

(b)

(d)

1.5

MMP 1.0

HMP

Endo > (UA)

Endo > (UA)

LMP 0.5

0.0

3L2 2L (65 Å) 2L2 2L1 (40 Å) (41.6 Å) (46.5 Å)

3L1 (71.3 Å)

0

10

20

Temperature (° C)

30

40

–0.5 –10

α + βʹ 0

α + βʹ 10

20

βʹ

30

40

Liquid

50

60

Temperature (° C)

Fig. 10.12  Crystallization of triacylglycerols within milk fat globules characterized on cooling at 0.1 °C/ min. Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded (a) on cooling and (c) on subsequent heating at 2 °C/min. Differential scanning calorimetry (DSC) thermograms recorded (b) on cooling and (d) on heating. DSC and SR‐XRD were measured simultaneously. HMP, high melting point; LMP, low melting point; MMP, medium melting point. Adapted from Lopez et al. (2001c).

to the melting of the 3 L (7.1 nm) crystals, the MMP endotherm was related to the melting of both the 3 L (6.5 nm), and the 2 L (4.7 nm) crystals, the HMP endotherm was attributed to the melting of the β’‐2 L crystals (Fig. 10.12d). The comparison of the crystallization properties of milk TAGs dispersed within fat globules and in anhydrous state revealed the following differences: ●●

The initial crystallization temperature of TAGs within milk fat globules was depressed compared to bulk TAGs (Tonset = 20.4° vs. 25.7 °C), showing that cream requires a much higher supercooling than does AMF. The differences in supercooling can

299

300

Crystallization of Lipids

●●

largely be explained by the theory of nucleation for TAG crystallization. In cream, milk TAGs are divided into numerous fat globules that are isolated from the others by the aqueous phase and by the biological membrane stabilizing the fat globule surface (Lopez 2011). This means that, if TAG crystallization occurs in one fat globule, it will not easily spread to the TAGs in the surrounding fat globules, at least when no shear is applied to the system. In AMF, once TAG crystallization begins, it rapidly spreads throughout the whole system because of the processes of secondary nucleation and crystal growth. Such an effect of emulsification on TAG crystallization rates in oil droplets in the O/W emulsion are discussed in Chapter 15. Different TAG crystals were formed (nucleation in α form in cream vs. in β’ form in AMF; Lopez et  al. 2001c), revealing the high role played by the dispersion of milk TAGs on their crystallization properties.

On cooling of cream from the melt at the rates of 1–3 °C/min (Fig. 10.13a and b), milk TAGs sequentially crystallize within milk fat globules in three different lamellar structures (Lopez et al. 2002b). From about 19 °C, α‐2 L (4.7 nm) and α‐2 L (4.2 nm) crystals were formed, and below 15 °C, the crystallization of TAGs in the α‐3 L (7.1 nm) organization was recorded (Fig. 10.13a). On subsequent heating from –10 to 60 °C at 2 °C/min (Fig. 10.13c and d), the α‐3 L (7.1 nm) crystals melt at about 16 °C, and some TAGs are involved in the formation of α‐3 L (6.5 nm) organization before its melting at about 20 °C. The α‐2 L (4.7 nm) and α‐2 L (4.2 nm) structures also melt around 20 °C. Between about 17° and 20 °C, crystallization of a new lamellar structure β’‐2 L (3.9 nm) occurred with structural reorganization leading to an increase of its thickness up to 4.17 nm. This high‐melting point organization of TAGs in their solid state, likely formed by the reorganization of the TAGs initially incorporated in the crystals, progressively melted from about 21 °C and disappeared above 38 °C. On cooling at 1–3 °C/min, similar crystalline structures are formed by TAGs within milk fat globules of cream (Fig.  10.13) and in AMF (Fig. 10.5). However, the thickness of the α‐3 L organization is slightly thicker in AMF (7.25 vs. 7.21 nm) and the small‐angle XRD peak widths were larger in cream, showing defects of longitudinal stacking in α‐3 L crystals within milk fat globules. DSC curves recorded on cooling showed differences between cream and AMF (Figs. 10.13b and 10.5c). Crystallization of TAGs in AMF is induced at higher temperature with a sharp exotherm at about 18 °C, related to crystal growth of α‐2 L crystals. Tempering of milk fat globules and emulsion droplets (i.e., successive cooling and heating) at controlled temperatures allows tailoring TAG crystals. In the previous paragraph, we discussed the formation of α‐3 L (7.1 and 6.5 nm) and α‐2 L (4.7 and 4.2 nm) crystals within milk fat globules after cooling at 1–3 °C/min from the melt. The subsequent heating of the cream to about 17–20 °C leads to the melting of these TAG crystals and to crystallization of a new lamellar structure β’‐2 L (3.9 nm). The stabilization of temperature at about 20 °C and then cooling of the cream to 4 °C favors the growth of β’‐2 L (3.9 nm) crystals. Tempering of cream leads to reorganization of TAGs within emulsion droplets and can have implications on the physical stability and functional properties of milk fat globules (e.g., in the manufacture of butter, whipped cream). In the dairy industry, milk and cream are often submitted to high‐temperature thermal treatments (e.g., pasteurization) and rapidly cooled in a tank for storage before processing. Understanding the crystallization behavior of milk TAGs within milk fat globules during this thermal history is therefore of industrial relevance. The most

Crystallization Properties of Milk Fats

(a)

(c)

sub-α

sub-α

α

α 3L002 3.6 nm –10 0 10 20 30 40 50

2L 4.7 nm

1.1

1.3

3L003 2.4 nm

T (° C)

liquid

1.5 1.7 1.9 2.1 q (Å-1)

liquid

1.1

3L005 1.4 nm –7

T (° C)

33

2L 4.7 nm

6.5 nm

3L1(003)

20 30 40

42

50

52 0.4

2.1

0

T (° C)

23

0.3

1.9

2L 4.2 nm

10

13

0.2

1.5 1.7 q (Å-1)

3L005 3

0.1

1.3

–10 0 10 20 30 40 3L 2(002) 50 T (° C)

2L 4.2 nm 3L001 7.1 nm

3L1(001) 3L1(002) 7.1 nm

β′

60

0.5

0.5

0.4

0.3

0.1

0.2

2L

q (Å-1)

q (Å-1)

(b)

(d)

2L 4.7 nm α 2L 4.2 nm

–10

3L 7.1 Å α

–20 –10

Heat flow (AU) Endo >

Heat flow (AU) Endo >

0

0

10

20

30

Temperature (° C)

40

0.12

0.11

0.10 –10

3L(7.1 nm) α

0

2L(4.2 nm) β′ 2L(4.7 nm) 2L(4.2 nm) 3L(6.5 nm) 2L(39.5 Å) β′ α+β′

10 20 30 40 Temperature (° C)

Liquid

50

Fig. 10.13  Crystallization of triacylglycerols within milk fat globules characterized on cooling at 1 °C/ min. Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small and wide (insert) angles (a) on cooling at 1 °C/min from 60° to –10 °C, and (c) on subsequent heating at 2 °C/min. Differential scanning calorimetry (DSC) thermograms recorded (b) on cooling and (d) on heating. DSC and SR‐XRD were recorded simultaneously. Adapted from Lopez et al. (2005a).

unstable crystalline structures of the TAGs formed within milk fat globules were studied after quenching from the melt down to –8 °C or 4 °C (~1000 °C/min), as shown Fig. 10.6(a). Their reorganization was characterized as a function of time (Fig. 10.6d) and on subsequent heating (Lopez et al. 2000, 2002a). The most unstable TAG crystals formed in milk fat globules correspond to α‐2 L (4.7 nm) and α‐3 L (7 nm) lamellar structures, as in AMF (Fig. 10.6a). Due to the rapid cooling, the crystallization starts in the metastable α‐polymorph. The α‐2 L organization is unstable and disappears during a 20‐minute conditioning in isothermal conditions (Fig. 10.6d). In a first series of experiments, the cream was heated at 2 °C/min. On heating, the α‐2 L (4.7 nm) and α‐3 L (7 nm) lamellar structures progressively melted, and from 13 °C a new lamellar organization was formed, β’‐2 L (3.8 nm), and was the single solid TAG

301

302

Crystallization of Lipids

organization until its final melting above 38 °C. The crystallization occurring in ­emulsion is similar to in anhydrous state, showing that the metastable TAG molecular packings obtained after quenching from the melt are not drastically affected by the dispersion state. However, the width of small‐angle XRD peaks indicated that TAG crystallization is more disordered in emulsion, which has been attributed to the constraints because of the interface curvature in the emulsion droplets. In a second series of experiments, the cream and AMF were quenched from 60° to 4 °C and stored in isothermal conditions for comparison, as shown Fig. 10.8(b) (Lopez et  al. 2002a). After quenching at 4 °C, similar liquid to solid TAG phase transition occurred after quenching to –8 °C, but the α‐2 L (4.7 nm) structure remained less than 1 minute since the higher liquid TAG phase amount favored the α‐2 L (4.7 nm) to α‐3 L (7 nm) transition. During isothermal storage at 4 °C, crystallization and polymorphic evolutions occurred. The α‐3 L (7 nm) structure transformed as a function of time into 2 L (3.9 nm) and 3 L (6.6 nm) lamellar structures through α → α + β’ + β secondary exothermic transitions. This secondary process is faster in AMF (within 30 minutes) than in cream (>1 hour). The delayed polymorphic evolution observed in milk fat globules shows that the dispersion state of TAGs plays a role in the transition process and could be explained by a lack of stable nuclei in each fat globule at 4 °C as compared to AMF. Similar conclusions were drawn by comparing the isothermal crystallization behavior at 5 °C of milk fat in bulk and emulsified state in natural and recombined creams (Fredrick et al. 2011). It is important to note that this exothermic transition leads to an increase in temperature during the storage of cream at the industrial level. After 4 days storage at 4 °C, similar TAG crystals were characterized within milk fat globules and in AMF, that is, the coexistence of 2 L (4 nm) and 3 L (5.4 nm) lamellar structures corresponding to α, β’1, β’2, and β polymorphs (Fig. 10.8d). This is in line with Söderberg et  al. (1989) who reported that the main structure formed by the TAGs in milk fat globules after long‐time storage at low temperature was a bilayer with a thickness of 3.9 nm. Also, these TAG crystals are similar to those characterized in butter (Buldo et  al. 2013; Ronholt et  al. 2014). At 4 °C, the high melting point ­fractions (HMPF) and the medium melting point fractions (MMPF) of milk fat will  crystallize and constitute the solid TAG phase, whereas the low melting point fractions (LMPF) is present is the liquid TAG phase. Therefore, the solid TAG phase primarily consists of TAGs containing three long chain saturated FAs and TAGs ­containing two long‐chain saturated FAs and a long chain unsaturated FA or a short‐ chain saturated FA (Fig. 10.2) The existence of an isothermal polymorphic evolution both in milk fat globules and in AMF was demonstrated (Lopez et al. 2002a). Density measurements were sensitive to the α to β’ and β’ to β polymorphic transitions occurring within milk fat globules following the quenching of cream at 4 °C (Lopez et al. 2002a). The increase in density, that corresponds to an increase in the compactness of milk TAGs, is explained by a reduction of the subcell volume from about 25.5 A3 (α form) to 23.5 A3 (β’ form) and a possible increase in the amount of TAG crystallized. After 6 days of storage at 4 °C, the cream and AMF were heated to 60 °C at 2 °C/min. The 3 L crystals corresponding to α, β’2, and β polymorphs melted first, and from about 23 °C, the solid TAG phase corresponded to a β’1‐2 L (4 nm) organization until its final melting. The DSC curves recorded simultaneous on heating showed two main endotherms corresponding to the successive melting of 3 L and 2 L crystals.

Crystallization Properties of Milk Fats

As a summary concerning the crystallization behavior of milk TAGs in cream (i.e., concentrated milk fat globules), DSC curves recorded on cooling show two overlapped exothermal events (i.e., a small event as a result of the crystallization of 2 L forms and a broad event related to crystallization of 3 L structures identified thanks to the coupling with XRD). Whatever the cooling rate of cream, the DSC curve recorded on subsequent heating exhibits three main endotherms more or less overlapped corresponding to the LMPF, MMPF, and HMPF of TAGs (Lopez et al. 2002b). An exothermic event corresponding to α → β’ polymorphic evolution can be recorded between the first two endotherms after fast cooling of milk fat globules. As for AMF, the LMPF corresponds to melting of 3 L lamellar structures, the MMPF to the melting of 2 L lamellar structures formed on cooling. The HMPF corresponds to the progressive melting of the β’ 2 L lamellar structures formed during heating until final melting of TAGs dispersed within fat globules of cream. These studies dedicated to the crystallization properties of milk TAGs in emulsion showed that milk TAGs are partially crystallized and that the solid TAG phase displays a complex polymorphism that is temperature and time dependent. The polymorphic transitions that occur in the solid TAG phase are favored by the liquid TAG phase. Figure 10.14 shows the crystalline structures formed within milk fat globules at –8 °C after cooling at different rates and the crystals formed upon storage at 4 °C. As for AMF, the comparison of the small number of crystal type formed to the large number of TAGs present in milk fat provides evidence that mixed crystals are formed within milk fat globules (i.e., co‐crystallization of different TAG molecules). On cooling, the first longitudinal organizations of TAG molecules dispersed within fat globules correspond to 2 L structures with long spacings of about 4–4.2 nm and 4.6–4.8 nm. These crystalline varieties may correspond to crystallization of HMPF of milk TAGs. These 2 L crystals are generally formed by TAGs with saturated and similar chain length FAs, such as MPP and PPP (Fig.  10.9c). Then, crystallization of 3 L structures (about 7–7.2 nm) occurs. The formation of a 3 L (6.5 nm) structure was only observed on slow cooling of milk fat globules (0.15 °C/min). Triple chain length (3 L) stackings may correspond to crystallization of unsaturated TAG molecules or to that of TAGs with FA chains of different lengths, like BPP and OPP (Fig. 10.9c). Whatever the cooling rate, initial crystallization occurs in a hexagonal packing (α form). Then, as a function of the decrease in temperature, the formation of β’ form and the coexistence of α and β’ polymorphic forms was observed. Traces of β form crystals were only recorded on slow cooling of milk fat globules (0.15 °C/min) and after at least 3 days storage at 4 °C. According to Lopez et al. (2002a), stabilization of the TAG crystals is only attained after at least 4 days storage at 4 °C. On heating, the crystals formed on cooling (α 2 L, α 3 L) melt and recrystallizations take place within milk fat globules with the formation of a β’‐2 L (4 nm) structure, accompanied or not by α‐3 L (5.4 nm). Recrystallization also occurs during isothermal storage. The stable crystalline structures take advantages of the melting of the metastable crystals, which melt first or of some kind of Ostwald ripening occurring within the milk fat globules thanks to the liquid TAG phase that coexists with the solid TAG phases. The β’‐2 L mixed crystals selectively melt starting with the TAGs with shorter chains as shown by a progressive increase on their thickness. As for AMF, low amount of β crystals have been identified in milk fat globules, even after long time storage at low temperature. The mixed TAG crystals formed in milk fat organize in the less polymorphic forms β’ and α because molecular packing is not dense.

303

Crystallization of Lipids

(a) 2L 4 nm 3L 5.4 nm 3L(002) 3L 7–7.2 nm I (UA)

4° C

3L(002)

2L 4–4.2 nm 2L 4.6–4.8 nm

3L(003)

3L(005)

3° C/min 1° C/min

3L2 6.5 nm

3L2(002) 0.15° C/min quenching

0.05

0.25

0.15

0.35

0.45

0.55

q (Å–1)

(b) β2′ β ′ 1

α

β

I (UA)

304

4° C

sub-α

3° C/min

sub-α α β′

1° C/min α 0.15° C/min quenching 1.2

1.3

1.4

1.5

1.6

1.7

q (Å–1)

Fig. 10.14  Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at –8 °C: (a) at small angles and (b) at wide angles after either cooling of milk fat globules concentrated in cream at different rates as indicated on the figure or isothermal conditioning at 4 °C during 5 days. Adapted from Lopez et al. (2002b).

10.4.2  Effect of the Size of Milk Fat Globules and Lipid Droplets Studies on milk fat globules and protein‐coated lipid droplets showed that the temperature of the initiation of TAG crystallization is lowered with decreasing size (Fig. 10.15) because of increased supercooling (Lopez et al. 2002b; Michalski et al. 2004; Truong et al. 2014). This can be explained by the theory of nucleation for crystallization. When

Crystallization Properties of Milk Fats

(a) 22 21

Tonset (° C)

20 19 18 17 16 15 0

1

2

3

4

5

6

7

d (μm)

(b) 0.01

–0.11 (E-1) –0.18

15

Tonset (° C)

Heat flow (AU) Endo >

–0.05

1.25 μm 0.67 μm

–0.24

0.45 μm

13 12 11 10

0.38 μm –0.30

14

0 0.2 0.4 0.6 0.8 1 1.2 1.4 d (μm)

0

10

20

30

Temperature (° C)

Fig. 10.15  Effect of the size of emulsion droplets on the initial temperature of milk fat crystallization (Tonset). (a) Tonset as a function of the size of natural milk fat globules selected by microfiltration, adapted from Michalski et al. (2004). (b) Differential scanning calorimetry (DSC) thermogram recorded on cooling of protein‐coated milk fat droplets of different sizes and changes in Tonset as a function of size (insert). Adapted from Lopez et al. (2002b).

the liquid TAGs are divided in droplets, as in an emulsion, not all the TAG droplets may contain the catalytic impurities required to start heterogeneous nucleation. The number of catalytic impurities per unit volume may be far too low to produce nuclei in every emulsion droplet, and considerable supercooling may occur. Then, it is accepted that, on cooling, TAG crystallization in the smaller globules is delayed compared to the larger

305

306

Crystallization of Lipids

ones. Assuming that there is no difference of composition from a milk fat globule to another, the major influence in TAG crystallization properties is that of fat droplet size. It was reported that milk TAGs dispersed in small milk fat droplets have a lower final melting temperature compared to large droplets, in relation with the crystal structure (Bugeat et al. 2011). Furthermore, small droplets (0.18 µm) have a lower melting enthalpy than larger droplets (1.7 µm), which could be related to a lower solid TAG content in small droplets (Bugeat et al. 2011). Truong et al. (2014) also reported a strong tendency toward decreasing proportion of milk fat crystallinity with smaller droplet size stabilized by dairy proteins. To elucidate the effects of emulsion droplet size on the organization of TAG molecules, crystallization behavior of protein‐coated lipid droplets homogenized at different pressures were examined (Lopez et al. 2002b; Bugeat et al. 2011; Truong et al. 2014). On cooling at 1 °C/min from 60° to –7 °C, similar lamellar structures were formed by milk TAG molecules in their solid state whatever the size of the emulsion droplets from 1.3 to 0.4 µm, that is, α‐3 L (7.2 nm) crystals (Lopez et al. 2002b). However, a decrease in diffraction intensities along with broaden of the SAXS peak width in smaller droplet size were observed, showing that crystallization in small milk TAG droplets is more disordered than in large droplets and in AMF or that the size of TAG crystals confined in lipid droplets is smaller (Lopez et al. 2002b). After 48‐hour storage of dairy emulsions at 4 °C, Bugeat et al. (2011) identified the coexistence of up to four different types of TAG crystals within emulsion droplets whatever their size in the range 1.7 to 0.2 µm, (i.e., 2 L and 3 L corresponding to β’1, β’2, β1, and β2 polymorphs). It was observed that the confinement of milk TAG molecules in small emulsion droplets enhanced the segregation of some types of TAGs and the formation of β polymorph, at the expense of β’ polymorphs (Bugeat et al. 2011). The crystallization properties of TAG within native milk fat globules selected as a function of their size—small (1 µm) and large (7 µm) milk fat globules—revealed differences (Michalski et al. 2004). XRD permitted the identification of different crystallization behavior in natural milk fat globules with different sizes, which could be implicated in the manufacture of dairy products involving tempering periods in the technological process (e.g., butter, ice‐cream, whipped products).

10.5 ­Crystallization Properties of Milk Fat in Dairy Products The thermal properties and crystallization behavior of milk fat in complex food products (e.g., butter, whipped cream, ice cream, cheese) have been investigated because TAG crystals can impact the physical stability, texture, sensorial properties, and acceptability by the consumer. During manufacture of butter, milk fat globules concentrated in cream are first ­subjected to a specific time–temperature program to obtain partially crystallized fat globules and subsequently the cream is exposed to a severe mechanical agitation in which fat globules destabilize through a mechanism known as partial coalescence for which TAG crystals are indispensable. Butter consists of a continuous fat phase in which water droplets, residual milk fat globules and a network of fat crystals are ­dispersed (Lopez et al. 2015). The mechanical properties of butter (i.e., its consistency, spreadability, firmness), appearance, and mouth feel depend not only on the ratio of

Crystallization Properties of Milk Fats

solid to liquid TAG but also, to a large degree, on the size, shapes and spatial distribution of the TAG crystal network, that depend on the milk TAG composition and crystallization behavior of TAG molecules. Both compositional and processing conditions influence crystallization of TAG in butter. The FA composition of butter, which can be affected by seasonal variations and cow diet (Fig. 10.1a), affects its crystallization and melting properties with consequences on the texture (Lopez et  al. 2007). Butter produced during the period of the year when cows are fed a maize‐silage based diet (i.e., during the winter) tends to have a higher level of palmitic acid C16:0 and less oleic acid C18:1c9 than butter produced when the cows are fed a grass‐based diet (i.e., during spring; Fig. 10.1a). This results in a firmer consistency of butter in winter. Processing conditions (temperature, cooling rate, scale of operation, agitation, storage conditions) can impact crystallization and ultimately the rheology of butter (Ronholt et al. 2014). For example, rapid cooling of cream leads to the formation of many small TAG crystals, a higher solid fat content and a firmer texture of butter. It is well‐known that thermal kinetics (i.e., temperings, cold‐warm‐cold processes also called physical ripening) are applied to cream to control the solid‐to‐liquid fat ratio and to govern the size and orientation of TAG crystals in milk fat globules before churning. Such treatment of cream before butter manufacturing largely determines the final textural characteristics of the butter. The rheological properties of milk fat and butter, in connection with the TAG crystal networks, are detailed elsewhere (Wright & Marangoni 2006). Among dairy products, cheeses have received a special attention to better understand the role played by the physical properties of milk fat, especially the formation of TAG crystals. However, studies of the crystallization properties of milk fat in as such complex food products as cheeses remain scarce. It has been demonstrated that the liquid to solid milk TAG phase transition recorded by DSC on cooling of hard‐type cheeses is sensitive to the microstructure of fat within the protein matrix, especially the destabilization of fat globules and the formation of nonemulsified fat during the manufacture of cheese (Lopez et al. 2006a). When cheese fat is dispersed in fat globules, the DSC profile is close to the recordings of cream (e.g., a single broad exotherm). When nonemulsified fat is formed within the cheese matrix, the DSC profile evolves toward the behavior of AMF (e.g., two successive exotherms). Dairy products are stored at low temperature (e.g., 4°–7 °C in the fridge), which raises questions about the crystallization of milk fat. Using synchrotron radiation XRD, Lopez et al. (2008) revealed the coexistence of several types of TAG crystals within hard‐type cheese stored in the fridge and identified 2 L (4.1 nm) and 3 L (5.5 nm) longitudinal organizations of TAG molecules corresponding to the coexistence of α, β’1, β’2, and β polymorphic forms. These results obtained in cheese are in line with the TAG crystals formed on long storage of AMF and cream at 4 °C. On heating of cheese previously stored at 4 °C, the DSC profile showed the three endotherms corresponding to the LMPF, MMPF, and HMPF of milk fat (Fig.  10.2). Similar milk TAG crystals and melting behavior have been characterized within the small size droplets of processed cheese (below 1 µm; Gliguem et  al. 2011). The final melting point of milk fat within cheese, about 41 °C, is higher than the temperature of digestion in the gastrointestinal tract of humans. The TAGs that remain in their solid state above 37 °C, estimated to be about 3% of milk fat (Lopez et al. 2006a) and composed by long‐chain saturated FAs such as palmitic acid, could impact the digestibility of milk fat consumed in cheese.

307

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Crystallization of Lipids

The texture of processed cheese is an important parameter affecting its acceptability by the consumer. Both microstructure and rheological properties of spreadable ­processed cheeses are strongly dependent on the properties of fat, mainly the amount and type of TAG crystals that can be formed at the temperature of storage and cheese consumption. A study combining DSC, XRD, and rheology as a function of temperature demonstrated the influence of milk TAG crystallization, melting, and polymorphism upon the viscoelastic properties of processed cheese (Gliguem et al. 2009). On cooling at 2 °C/min from 60 °C (Fig. 10.16), the crystals formed by milk TAGs within processed cheese were observed from about 15 °C and corresponded to α‐3 L (7.2 nm) structures. These results were consistent with previous observations stating that crystallization in milk fat globules (4 µm in diameter) gives rise to a α‐2 L (4.2–4.7 nm) structure followed by α‐3 L (7.1 nm) structure, while in fat droplets ranging from 1.25 to 0.38 µm only the α‐3 L (7.2 nm) structure was observed (Lopez et  al. 2002b). The formation of α‐3 L (7.2 nm) crystals and the absence of α‐2 L crystals in processed cheese may be related to the small size of fat droplets (0.7 µm). The crystallization of milk TAGs within the processed cheese matrix, characterized simultaneously by XRD and DSC (Fig. 10.16a and b), was related to an increase in the viscoelastic moduli G’ and G” (Fig. 10.16c). These results showed that milk TAG crystals contribute in the firmness of processed cheese at low temperature (e.g. below 15 °C; Gliguem et al. 2009). On subsequent heating of the processed cheese, the α‐3 L (7.2 nm) crystals melted and from about 12 °C, the formation of β’‐2 L (4.1 nm) crystals was characterized until its final melting above 38 °C. The successive melting of the 3 L and 2 L TAG crystals embedded in the fat droplets was related to successive decrease in the viscoelastic moduli recorded on heating of processed cheese. These studies combining different biophysical techniques allowed the identification of the milk TAG crystals formed within complex dairy products (i.e., butter, cheeses) and revealed polymorphic evolutions on heating. Moreover, the impact of milk fat crystals on the rheological properties fat‐rich products was demonstrated.

10.6 ­TAG Compositions Affecting Crystallization Properties of Milk Fat The FA and TAG composition of milk can be tailored for technological, nutritional, and health reasons (e.g., increase in unsaturated FA and decrease in palmitic acid). In this respect, numerous techniques have been applied including physical, chemical, and dietary manipulation by means of feeding dairy animals. Technological treatments are often applied to have desired functionalities (e.g., improved cold spreadability of butter) and expend the use of milk fat in the food industry. Milk TAGs also exhibit compositional ­differences between the mammal origin of the milk (e.g., bovine, goat, sheep, human). The variations in TAG composition affect the crystallization and melting properties of milk fats. 10.6.1  Technological Process: Dry Fractionation Among the technological processes, hydrogenation, interesterification, or blending of milk fat with fats from other origin (e.g., vegetable oil) can be used to tailor the FA and TAG composition but will not be discussed in this chapter.

100 80 60

0 –10

HF (cumulative integration; %)

3L002 3L003 23.8 Å

–8.6

0

5

10 15 20 Temperature (° C)

25

30

–0.14 –0.15 –5

0

5

10 15 20 Temperature (° C)

25

30

–0.16

14.9 5

20.7 25.6

3000

10

2000

5

1000

0 –10

–5

0

5

10

15

20

25

0 30

G″ (Pa)

G′ (Pa) (Thousands)

15

7.1 12.0

12.0

6

4

4000

G′ (Pa) G″ (Pa)

2.2

7.1

(c) Viscoelastic properties 20

–2.7

2.2

7 –0.13

–5.4

–8.6 –5.4 –2.7

8 –5

α

T (° C)

9

: 3L001 : 3L002 : 3L003

(b) Thermal properties 100 80 60 40 20 0 –10

WAXS

3 0.10

0.20 q (Å–1)

0.30

Temperature (° C)

20

SAXS

36 Å

HF (AU)

40

3L001 72 Å

3L001 3L002 3L003

Intensity (cps)

% maximal intensity

(a) Structural properties

14.9 20.7 25.6

30.6

30.6

35.5

35.5

0.8 1.0 1.2 1.4 1.6 1.8 q (Å–1)

Temperature (° C)

Fig. 10.16  Characterization of the crystallization of milk fat in processed cheese on cooling at 2 °C/min showing the consequences on the rheological properties. (a) Evolution as a function of temperature of the maximal intensities of the synchrotron radiation X‐ray diffraction (SR‐XRD) peaks recorded at small angles and corresponding to crystallization in α‐3 L (7.2 nm) structures as shown in the right part. (d) Differential scanning calorimetry (DSC) curve recorded simultaneously on cooling and its cumulative integration as a function of temperature. (c) Changes in the viscoelastic moduli G’ and G” of processed cheese on cooling. SAXS, small angle X‐ray scattering; WAXS, wide‐angle X‐ray scattering. Adapted from Gliguem et al. (2009)

310

Crystallization of Lipids

Dry fractionation (i.e. the crystallization of milk fat from the melt and the subsequent filtration of the slurry) is a common industrial process to obtain milk fat fractions with different TAG compositions and physical properties (Kaylegian & Lindsay 1995). Dry fractionation is based on the different thermal (crystallization and melting) properties of TAGs resulting from their different FA compositions. At a fixed temperature during the fractionation process, the solid fraction is called stearin, and the liquid fraction is called olein. Fractionation of milk fat and recombination of the fractions in various proportions allow to control and to improve the thermal and physical properties (e.g., its consistency and the development of cold spreadable butter; Kaylegian & Lindsay 1995). Milk fat fractions are employed for pastry‐making, as chocolate bloom inhibitor, butter flavor‐rich concentrates and for improving the rheology of reduced fat cheese curds. Many studies have focused on the milk fat fractionation process, on the chemical and thermal characteristics of the separated fractions, and on the phase behavior of milk fat and its fractions (Timms 1980; Marangoni & Lencki 1998; van Aken et al. 1999). So far only a few studies have been reported about the structural characteristics of milk fat fractions investigated using time‐resolved SR‐XRD as a function of temperature. The chemical composition, crystallization properties, and melting behavior of milk fat and its primary fractions, stearin and olein fractions, obtained by dry fractionation at 21 °C were characterized (Lopez et  al. 2006b; Lopez & Ollivon 2009). Compared to whole milk fat, the stearin fraction was enriched in TAGs with (1) three saturated long‐chain FAs (SSS, PSS, PPS, PPP, MSS, MPS, MPP, SSL), (2) one or two saturated medium‐chain FAs and a saturated long‐chain FAs (MMS, MMP, LaPS, LaPP, LaMP, CPS, CPP, CMS), and (3) one monounsaturated long‐chain FA and two saturated long‐chain FAs (PoSS, PPoS, LaOS, PSO). The olein fraction was enriched in TAGs with (1) two monounsaturated long‐chain FA (SOO, PoSO, POO, MOO, PPoO), (2) one monounsaturated and two medium‐chain FAs (CMO, CPO, LaMO, MMO, LaPO), and (3) a short chain FA and two saturated long‐chain FAs (BMP, BPS, BPP, BMS, CaMP, CaPP, CaMS) or one monounsaturated FA (BPO, BMO) or two monounsaturated long‐chain FAs (BOO). On cooling from the melt at 1 °C/min, milk fat showed the formation of two α‐2 L (4.7 and 4.2 nm) and one α‐3 L (7.2 nm) lamellar structures, as previously reported and ­discussed (Lopez et al. 2001a; Fig. 10.5a). In similar experimental conditions, the stearin fraction started to crystallize at 26 °C with the formation of two main lamellar structures, α‐2 L (4.8 nm; molecules arranged perpendicular to the methyl end group plane) and β’‐2 L (4.2 nm; tilt of the chains; Fig. 10.17a). Then, from about 13 °C, a low amount of 3 L (6.8 nm) crystals that may correspond either to α or β’ polymorphs were formed. In the olein fraction cooled in similar experimental conditions, α‐3 L (7.2 nm) lamellar structures started to crystallize from 13 °C (Fig.  10.17b). The thickness of this α‐3 L structure corresponds to the packing of short and medium chain FAs with a mean number of atoms of carbon of about 13.5 in two layers, and to the packing of unsaturated and saturated long‐chain FAs with a mean number of atoms of carbon of 18 in the third layer (Fig.  10.17c; Lopez et  al. 2006b). The different type of TAG crystals formed in stearin and olein fractions, as compared to whole milk fat, result from their different TAG composition. On subsequent heating at 2 °C/min, the final temperature of melting recorded for stearin fraction, milk fat and olein fraction were 44°, 37.5°, and 22 °C, respectively (Lopez & Ollivon 2009). The structure of TAG crystals networks observed at the microscale level using polarized light microscopy, after cooling from the melt at 1 °C/min, corresponded to

Crystallization Properties of Milk Fats

(a) Stearin fraction

α

(b) Olein fraction β′

3L002

2L1 4.8 nm 2L2 4.2 nm T (° C)

–7 3 12

3L001 7.3 nm

3L001 7.2 nm

Liquid

21 31 40

1.4

1.6

1.8

2.0

4

T (° C)

T (° C)

23 33 43 52 0.3 q (Å-1)

0.4

0.5

–7 –2 3 8 13 18 23 28 33 38 0.0

0.1

1.4

1.6

1.8

2.0

2.2

q (Å-1)

3L003 3L004

2L

2.2

q (Å-1)

0.2

Liquid

1.2 1.2

12

0.1

–8 –3 2 6 11 15 20 25 30

50

3L002

–7

0.0

T (° C)

α

0.2

0.3 q (Å-1)

0.4

3L005

0.5

Longitudinal organization

Lateral chain packing

(c) Organization of TAG in the crystals b a

Hexagonal subcell Orthorhombic perpendicular subcell (β’ form) (α form)

A

13.5 carbons

B

18 carbons

7.2 nm

13.5 carbons

c

c

4.8 nm

4.2 nm

a

Double-chain length (2L)

a

Double-chain length (2L)

Triple-chain length (3L)

Fig. 10.17  TAG crystals formed in stearin fraction and olein fraction on cooling at 1 °C/min from 60° to –7 °C. Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small and wide (insert) angles as a function of temperature (a) for stearin fraction and (b) for olein fraction. (c) Proposed TAG packing in the main crystals formed on cooling. The α − 3 L (7.2 nm) crystals in olein fraction correspond to the packing of short and medium‐chain length fatty acids in layers A with unsaturated and saturated long‐chain fatty acids in layers B. Adapted from Lopez et al. (2006b).

spherulitic organizations in milk fat and stearin fraction, and needle‐shape crystals were formed in the olein fraction (Lopez & Ollivon 2009). The microstructure and ­crystallization kinetics of binary and ternary mixtures of milk fat fractions during ­isothermal crystallization at 5°, 15°, and 20 °C were characterized using polarized light microscopy and the Avrami model (Ramel & Marangoni 2016). Results showed that for

311

312

Crystallization of Lipids

both binary and ternary mixtures, high concentrations of the high‐melting fraction result in the formation of rod or needle‐like crystals (i.e., one‐dimensional growth and low ­values of Avrami index, n, while at relatively higher concentrations of the MMPFs and LMPFs, multidimensional crystal growth is favored (i.e., higher n values). On the effect of temperature, for binary mixtures, it was found that at high undercooling conditions (5 °C) one‐dimensional growth is favored while for ternary mixtures, increasing the crystallization temperature (i.e., decreasing supersaturation) from 15° to 20 °C results in large differences in crystal structure. Ramel and Marangoni (2016) were therefore able to propose a concentration–temperature map for different fat crystal structures in milk fat. 10.6.2  Dietary Manipulations Seasonal variations in the diet of cows naturally occurring in some countries (e.g., maize‐based diet in winter vs. fresh grass based diet in spring) affect the FA and TAG composition of milk (Fig. 10.1) and have consequences on the crystallization properties of milk fat and final texture of fat‐rich products. Thus, the control of fat‐rich product quality (e.g., butter) in different seasons is a real challenge for the industrials. The composition of milk fat can also be modified by specific feeding strategies and alter consequently the physical and functional properties of high‐fat content dairy products. For example, feeding cows with highly unsaturated oils or whole oilseeds can reduce the level of saturated FAs while simultaneously increasing the unsaturated fatty acid (UFA) content. Several studies reported the improvement in the spreadability and softer texture of winter butter and milk fat in general through changes in the feed of the cow by adding unsaturated oils or fresh grass (Wright & Marangoni 2006; Couvreur et al. 2006). Yogurt, ice cream, and cheeses made from milk enriched in UFAs have been reported to show a softer texture than the products made from control milk. The relationship between the FA composition of milk fat and the texture of dairy products has been demonstrated. However, few authors studied the effect of cow diet on the crystallization properties of milk fat enriched in UFAs (Smet et al. 2010; Bugeat et al. 2011, 2015). An increased amount of UFAs in milk TAGs was reported to decrease the solid fat content at 5 °C from 60% (control TAGs; 28% UFAs) down to 46% (UFA‐enriched TAGs; 39% UFAs; Smet et al. 2010). Upon isothermal crystallization monitored by pNMR, higher content of the UFAs resulted in a slower nucleation, a longer induction time to crystallization and a lower solid fat content at the end of crystallization, although crystallization occurred according to similar α to β’ polymorphic transition (Smet et al. 2010). Bugeat et  al. (2015) compared the crystallization properties of control milk TAGs (29% UFAs) and UFA‐enriched TAGs (51% UFAs obtained with a linseed oil rich diet) using the coupling of DSC with SR‐XRD (Fig. 10.18). On cooling from the melt at 3 °C/ min, both milk TAG mixtures started to crystallize from about 16 °C in α‐2 L (4.5– 4.9 nm) structures then formation of α‐3 L structures occurred with a higher thickness (7.55 nm vs. 7.15 nm) and a delay (Tonset = 8.5 °C vs. 12.1 °C) for UFA‐enriched TAGs that result from a higher amount of C18:1c9. Groups of TAG molecules with high crystallization temperature (HCT; α‐2 L crystals) and low crystallization temperature (LCT; α‐3 L crystals) segregated on cooling. On subsequent heating, melting of TAG crystals and formation of a new 3 L (6.5–8.0 nm) and β’‐2 L (4.0–4.4 nm) crystals associated with polymorphic reorganizations have been characterized. Increase in thickness of the lamellar structures was characterized for UFA‐enriched TAGs as compared to control

Crystallization Properties of Milk Fats

(a) sub-α α *

3L001 (7.15 nm)

Temperature (° C)

2L (4.6– 3.9 nm)

3L003 (2.4 nm)

Temperature(°C)

3L002 (3.6 nm)

liquid

0 10 20 30 40

1.3 3L005 3L004 (1.4 nm)

1.4

1.6

1.5

1.7

1.8

q(Å–1)

0 10 20 30 40

0.1

0.5

0.4

0.3

0.2

q (Å–1)

(b) α 3L002 (3.8 nm)

2L2 2L1 (4.9– (4.5–4.1 nm) 3L003 4.8 nm)

(2.5 nm)

Temperature(°C)

3L001 (7.55 nm)

*

liquid 0 10 20 30 40

3L005

1.3

1.4

1.5

1.6

1.7

1.8

q (Å–1)

Temperature (° C)

3L004 (1.5 nm) 0 10 20 30 40

0.1

0.2

0.3 q (Å–1)

0.4

0.5

Fig. 10.18  Comparison of the crystallization behavior of unsaturated fatty acid (UFA)‐enriched TAGs and control TAGs examined on cooling at 3 °C/min from 60° to –5 °C using the coupling of synchrotron radiation X‐ray diffraction (SR‐XRD) and differential scanning calorimetry (DSC). SR‐XRD patterns recorded at small and wide (insert) angles during cooling of (a) control TAGs and (b) UFA‐enriched TAGs.

313

Crystallization of Lipids

(c) 0.018

3L002 (3.6 nm)

0.016

Intensity (AU)

3L002 (3.8 nm)

0.012

0.006

0.001

3L001 (7.15 nm)

0.01 0.008

sub-α α

0.0015

0.014

0.0005

2L (3.9 nm)

3L001 (7.5 nm)

0 0.05

0.1

1.3

0.2

0.15

(d)

0.25 0.3 q (Å–1)

1.6

1.5 1.4 q (Å–1)

3L003 3L003 (2.4 nm) (2.5 nm)

2L1 (4.8 nm)

0.002

Liquid TAG

0 1.2

2L2 (4.1 nm)

0.004

1.7

3L005 3L005 (1.4 nm) (1.5 nm)

0.35

0.4

0.45

UFA-enriched TAG 0.00 Heat flow (AU) Endo

314

control TAG

–0.02

α–2L1

–0.04

α–2L2

–0.06

α–3L

–0.08 –0.10

–5

0

5

10

15

20

25

Temperature (° C)

Fig. 10.18 (Cont’d)  (c) XRD patterns recorded at –5 °C for control (thin line) and UFA‐enriched (thick line) TAGs after cooling. (d) DSC curves recorded simultaneously to X‐ray diffraction (XRD) experiments. Adapted from Bugeat et al. (2015).

TAGs, demonstrating differences in the FA composition of the crystals. Interestingly, the melting profile of the UFA‐enriched TAGs was mainly altered in the range 11 to 21 °C, corresponding to the MMPF, and not in the HMPF because the final melting temperatures of both the control and the UFA‐enriched TAGs were similar. It has also been demonstrated with the same milk fats that the enrichment of UFAs in TAGs decreases the solid fat content and affects the type of crystalline structures that are formed within emulsion droplets upon storage at 4 °C (Bugeat et al. 2011). Control TAGs were crystallized in 2 L (3.95 nm) and 3 L (5.66 nm) lamellar structures with four polymorphic forms (β1, β, β’1, β’2), whereas UFA‐enriched TAGs were crystallized in 2 L (4.18 nm) lamellar structures displaying three polymorphic forms (β1, β’1, β’2). The absence of 3 L crystals in the UFA‐enriched TAG emulsions was because of decrease in the melting point of these TAG crystals rich in UFAs (Saturated‐Unsaturated‐ Unsaturated TAGs vs. Saturated‐Saturated‐Unsaturated TAGs in control milk fat) that remain in the liquid TAG phase upon storage of the emulsion droplets at 4 °C.

Crystallization Properties of Milk Fats

As a conclusion, the enrichment of milk TAGs in UFAs affects both their crystallization and melting behaviors. 10.6.3  Milk Fat from Various Mammal Species Most of the studies about milk fat crystallization have been performed with bovine milk because it represents about 84% of the global worldwide milk production (information from the International Dairy Federation). However, the FA and TAG composition of milk depend on mammal species and changes in milk fat composition can affect the crystallization and melting properties between milk fats from various origins (i.e., goat, sheep, water buffalo, donkey, horse, camel; Smiddy et  al. 2012). The crystallization properties of milk TAGs have been characterized by the coupling of DSC and SR‐XRD in AMF and fat globules from goat milk (Ben Amara‐Dali et al. 2007, 2008), dromedary milk (Karray et al. 2005; Lopez et al. 2005b), and human milk (Lopez et al. 2013). Goat milk fat globules have a mean diameter of about 3–3.5 µm. They are rich in saturated FAs, about 70% of total FAs, and the five FAs C10:0, C14:0, C16:0, C18:0, and C18:1c9 account for more than 75% of total FAs (Ben Amara‐Dali et al. 2008). The most important TAGs present in goat’s milk fat are medium chain FAs (C8:0, C10:0, C12:0) and C18:1c9 as unsaturated FA. The molecular organization of the solid TAG phase formed within goat milk fat globules was investigated on cooling at the rates of 0.1 °C/min (slow cooling) and 1000 °C/min (quenching) and on subsequent heating at 1 °C/min. The lamellar structures 3 L (6.9–7.0 nm) and 2 L (3.7–4.5 nm) were characterized and the five polymorphic forms α, sub‐α, β’1, β’2, and β were identified. The two main types of crystals correspond to a segregation of goat TAG molecules in the solid state as a result of different compositions, as observed for bovine TAGs. Polymorphic transitions were observed within goat’s milk fat globules as a function of time after quenching from the melt and as a function of temperature on heating. Increasing the knowledge about physical properties of goat’s milk fat is essential to improve the quality of existing dairy products and to increase the technical application of goat’s milk fat crystallization to contribute in the development of new food products. Human milk contains about 3% to 5% fat dispersed in fat globules having a mean diameter of about 5 µm. Human milk TAGs, that contain 48% to 57% saturated FAs with about 28% of C16:0, contribute some 40 %to 55% of the total energy intake for the breast‐fed infants. The efficient digestion of TAGs is therefore of primary importance for the optimal growth of newborns. However, storage of breast milk in the fridge at 4 °C leads to the partial crystallization of TAGs within milk fat globules (Lopez et al. 2013). Microscopic observations of breast milk stored at 4 °C revealed the nonspherical distorted shape of fat globules due to the presence of TAG crystals (Fig. 10.19a). SR‐XRD experiments allowed the identification of the TAG crystals that are formed within breast milk fat globules upon storage at 4 °C (i.e., β‐2 L [4.17 nm] lamellar ­structures; Fig.  10.19b). The β crystals correspond to the most thermodynamically stable polymorphic form of TAGs with a compact organization of the FA chains. The crystals formed in human milk fat globules upon storage at 4 °C are different from those characterized in bovine milk, which confirms that the chemical composition of milk TAGs govern their crystallization properties. The final melting point of the β‐2 L (4.17 nm) human TAG crystals was 41.1 ± 1.6 °C, which is above the in‐body temperature of milk digestion by newborns. The presence of solid TAGs in the core of milk fat globules after storage at 4 °C raises the question of the action of the digestive lipolytic enzymes on a solid substrate, on their

315

Crystallization of Lipids

(a)

Breast milk fat globules deformed by fat crystals TAG crystals

Liquid TAG

5 μm Organization of TAG crystals revealed by X-ray diffraction 400

SAXS

WAXS

45 40

350 300

0.46 nm β polymorphic form

35

2L001

Intensity (AU)

(b)

Intensity (AU)

316

4.2 nm

250

30 25 20 15

200

10

150

0

5 1

100

2

1.5 q(Å–1)

2L002 2L003

50 0

0.2

0.4

0.6

0.8

q(Å–1)

Fig. 10.19  TAG crystals formed within human milk fat globules upon storage at 4 °C. (a) Polarized light microscopy images showing the deformation of milk fat globules by TAG crystals. (b) Identification of the organization of TAG molecules performed using synchrotron radiation X‐ray diffraction (SR‐XRD) at both small angle X‐ray scattering (SAXS) and wide (insert) angle X‐ray scattering (WAXS). Adapted from Lopez et al. (2013).

solubilization, and then on the absorption and metabolism of milk lipids. The influence of the physical state of TAGs and particularly the proportion of solid TAGs and type of crystals on lipid digestion and absorption remains poorly documented. Lopez et  al. (2013) hypothesized that crystallization of milk TAGs could decrease the amount of usable fat for the recipient infant in the case breast milk is consumed at a temperature below the final melting temperature of TAG crystals. Warming breast milk at about 45–50 °C (i.e. above the final melting point of human β‐2 L TAG crystals) is then important to ensure breast milk TAG digestibility.

10.7 ­Liquid TAG Phase For temperatures above the melting point of milk TAGs, the liquid TAG phase exhibits an organization. Within milk fat globules, the synchrotron radiation X‐ray patterns of milk TAGs in their liquid state correspond to scattering peaks at both small and wide

Crystallization Properties of Milk Fats

(a)

(b) 600

stearin fraction olein fraction anhydrous milk fat

Counts (AU)

480 360

d: 2.33 ± 0.02 nm

240 120 0 0.00

c

0.10

0.20

0.30

0.40

0.50

a

q (Å–1)

Fig. 10.20  Structural information on the liquid phase of milk TAG molecules. (a) Synchrotron radiation X‐ray diffraction (SR‐XRD) patterns recorded at small angles with milk fat, olein fraction and stearin fractions at 60 °C. (b) Proposed structure for the molecular packing of TAG molecules in their liquid state, as seen in the ca projection. Short‐, medium‐, and long‐chain fatty acids; saturated fatty acides; and unsaturated fatty acids are stacked in monolayers between glycerol groups. Adapted from Lopez and Ollivon (2009).

angles, respectively centered at 2.24 nm and 0.45 nm (Lopez et al. 2001a). In AMF and the primary fractions, stearin and olein, the synchrotron radiation X‐ray scattering from the liquid‐crystalline organization of milk TAGs in their liquid state recorded at 60 °C was centered at 2.33 nm (SAXS) and about 0.45 nm (WAXS; Lopez & Ollivon 2009). Differences were characterized as a function of the FA composition of TAGs, with a higher thickness d for UFA‐enriched TAGs compared to control TAGs (d = 2.26 ± 0.01 vs. 2.21 ± 0.01 nm; Bugeat et  al. 2015). The thickness value d of 2.21– 2.33 nm supports the existence of liquid‐crystalline like lamellae and corresponds to the stacking of TAGs in a single layer of the acyl chains along the long‐chain axis integrating FAs with different chain length (from 4 to 18 atoms of carbon) and unsaturation (Fig.  10.20). The scattering peak recorded at about 0.45–0.46 nm corresponds to a ­disordered mesophase with short range order of the FA chains. These SR‐RD data support evidence that complex TAG blends such as milk TAGs display anisotropy with a lamellar ordering in the liquid state.

10.8 ­Conclusions Crystallization of TAGs is a complex phenomenon, especially for milk fat because of its extremely wide FA composition. As reviewed in this chapter, extensive research has provided considerable insight into the crystallization properties of milk fat in anhydrous state, in emulsion (natural milk fat globules, processed lipid droplets, recombined cream), and in complex dairy products (butter, cheeses). This chapter highlighted from scientific points of views that the crystallization properties of milk fat are affected by (1) its FA and TAG composition, (2) cooling rates and tempering, (3) shear, (4) presence

317

318

Crystallization of Lipids

of minor lipid compounds (FFAs, MAGs, DAGs, phospholipids), (5) its dispersion state (i.e., anhydrous bulk versus emulsified in numerous droplets). Polymorphic evolutions have been characterized as a function of temperature on heating and in isothermal ­conditions (e.g., after rapid cooling from the melt). Understanding the functional properties of milk TAG crystals networks requires investigations at several scale levels (microscopic level, nanoscale, molecular scale) performed as a function of temperature and time in isothermal conditions and then the combination of complementary ­techniques (rheology, polarized light microscopy, electron microscopy, NMR, DSC, XRD including ultra‐small angle X‐ray scattering [USAXS], SAXS, and WAXS). Undoubtedly, pursuit of fundamental knowledge in the area of TAG crystallization can yield fascinating new insights that will further increase the values of milk fat in food and other applications.

­References Bayard, M., Leal‐Calderon, F., & Cansell, M. (2017) Free fatty acids and their esters modulate isothermal crystallization of anhydrous milk fat. Food Chemistry. 218, 22–29. Ben Amara‐Dali, W., Lesieur, P., Artzner, F., et al. (2007) Anhydrous goat’s milk fat: Thermal and structural behaviors studied by coupled DSC and X‐ray diffraction. 2. Influence of cooling rate. Journal of Agricultural and Food Chemistry. 55, 4741–51. Ben Amara‐Dali, W., Lopez, C., Lesieur, P., & Ollivon, M. (2008) Crystallization properties and polymorphism of triacylglycerols in goat’s milk fat globules. Journal of Agricultural and Food Chemistry. 56, 4511–22. Bugeat, S., Briard‐Bion, V., Pérez, J., et al. (2011) Enrichment in unsaturated fatty acids and emulsion droplet size affect the crystallization behaviour of milk triacylglycerols upon storage at 4 °C. Food Research International. 44, 1314–30. Bugeat, S., Perez, J., Briard‐Bion, V., et al. (2015) Unsaturated fatty acid enriched vs. control milk triacylglycerols: Solid and liquid TAG phases examined by synchrotron radiation X‐ray diffraction coupled with DSC. Food Research International. 67, 91–101. Buldo, P., Kirkensgaard, J. J. K., & Wiking, L. (2013) Crystallization mechanisms in cream during ripening and initial butter churning. Journal of Dairy Science. 96, 6782–91. Campos, R., Narine, S. S., & Marangoni A. G. (2002) Effect of cooling rate on the structure and mechanical properties of milk fat and lard. Food Research International. 35, 971–82. Couvreur, S., Hurtaud, C., Lopez, C., Delaby, L., & Peyraud, J. L. (2006) The linear relationship between the proportion of fresh grass in the cow diet, milk fatty acid composition, and butter properties. Journal of Dairy Science. 89, 1956–69. Foubert, I., Vanhoutte, B., & Dewettinck, K. (2004) Temperature and concentration dependent effect of partial glycerides on milk fat crystallization. European Journal of Lipid Science and Technology. 106, 531–39. Fredrick, E., Van de Walle, D., Walstra, P., et al. (2011) Isothermal crystallization behavior of milk fat in bulk and emulsified state. International Dairy Journal. 21, 685–95. Fredrick, E., Moens, K., Heyman, B., et al. (2013) Monoacylglycerols in dairy recombined cream: I. The effect on milk fat crystallization. Food Research International. 51, 892–98. Gliguem, H., Ghorbel, D., Lopez, C., et al. (2009) Crystallization and polymorphism of triacylglycerols contribute to the rheological properties of processed cheese. Journal of Agricultural and Food Chemistry. 57, 3195–203.

Crystallization Properties of Milk Fats

Gliguem, H., Lopez, C., Michon, C., Lesieur, P., & Ollivon, M. (2011) The viscoelastic properties of processed cheeses depend on their thermal history and fat polymorphism. Journal of Agricultural and Food Chemistry. 59, 3125–34. Grall, D. S., & Hartel, R. W. (1992) Kinetics of butterfat crystallization. Journal of American Oil Chemists’ Society. 69, 741–47. Karray, N., Lopez, C., Lesieur, P., & Ollivon, M. (2005) Dromedary milk fat: Thermal and Structural Properties 2. Influence of cooling rate. Lait. 433–51. Kaylegian, K. E., & Lindsay, R. C. (1995) Handbook of Milk Fat Fractionation Technology and Application. Champaign, IL, AOCS Press. Lopez, C. (2011) Milk fat globules enveloped by their biological membrane: Unique colloidal assemblies with a specific composition and structure. Current Opinion in Colloid and Interface Science. 16, 391–404. Lopez, C., & Ollivon M. (2009) Triglycerides obtained by dry fractionation of milk fat. 2. Thermal properties and polymorphic evolutions on heating. Chemistry and Physics of Lipids. 159, 1–12. Lopez, C., Lesieur, P., Keller, G., & Ollivon, M. (2000) Thermal and structural behavior of milk fat: 1. Unstable species of cream. Journal of Colloid and Interface Science. 229, 62–71. Lopez, C., Lavigne, F., Lesieur, P., Keller, G., & Ollivon, M. (2001a) Thermal and structural behavior of anhydrous milk fat: 2. Crystalline forms obtained by slow cooling. Journal of Dairy Science. 84, 2402–12. Lopez, C., Lavigne, F., Lesieur, P., Keller, G., & Ollivon, M. (2001b) Thermal and structural behavior of milk fat: 1. Unstable species of anhydrous milk fat. Journal of Dairy Science. 84, 756–66. Lopez, C., Lesieur, P., Bourgaux, C., Keller, G., & Ollivon M. (2001c) Thermal and structural behavior of milk fat: 2. crystalline forms obtained by slow cooling of cream. Journal of Colloid and Interface Science. 240, 150–61. Lopez, C., Bourgaux, C., Lesieur, P., & Ollivon, M. (2002a) crystalline structures formed in cream and anhydrous milk fat at 4 °C. Lait. 82, 317–35. Lopez, C., Bourgaux, C., Lesieur, P., et al. (2002b) Thermal and structural behavior of milk fat: 3. Influence of cream cooling rate and droplet size. Journal of Colloid and Interface Science. 254, 64–78. Lopez, C., Lesieur, P., Bourgaux C., & Ollivon, M. (2005a) Thermal and structural behavior of anhydrous milk fat. 3. Influence of cooling rate. Journal of Dairy Science. 88, 511–26. Lopez, C., Karray, N., Lesieur, P., & Ollivon, M. (2005b) Crystallisation and melting properties of dromedary milk fat globules studied by X‐ray diffraction and differential scanning calorimetry. Comparison with anhydrous dromedary milk fat. European Journal of Lipid Science and Technology. 107, 673–83. Lopez, C., Briard‐Bion, V., Camier, B., & Gassi, J.‐Y. (2006a) Milk fat thermal properties and solid fat content in Emmental cheese: a differential scanning calorimetry study. Journal of Dairy Science. 89, 2894–910. Lopez, C., Bourgaux, C., Lesieur, P., Riaublanc, A., & Ollivon, M. (2006b) Milk fat and primary fractions obtained by dry fractionation 1. Chemical composition and crystallisation properties. Chemistry and Physics of Lipids. 144, 17–33. Lopez, C., Bourgaux, C., Lesieur, P., & Ollivon, M. (2007) Coupling of time‐resolved synchrotron X‐ray diffraction and DSC to elucidate the crystallisation properties and polymorphism of triglycerides in milk fat globules. Lait. 87, 459–80.

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Lopez, C., Briard‐Bion, V., Beaucher, E., & Ollivon, M. (2008) Multiscale characterization of the organization of triglycerides and phospholipids in Emmental cheese: From the microscopic to the molecular level. Journal of Agricultural and Food Chemistry. 56, 2406–14. Lopez, C., Briard‐Bion, V., Bourgaux, C., & Perez, J. (2013) Solid triacylglycerols within human fat globules: Crystals with a melting point above in‐body temperature formed upon storage of breast milk at low temperature. Food Research International. 54, 1541–52. Lopez, C., Briard‐Bion, V., & Ménard, O. (2014) Polar lipids, sphingomyelin and long‐chain unsaturated fatty acids from the milk fat globule membrane are increased in milks produced by cows fed fresh pasture based diet during spring. Food Research International. 58, 59–68. Lopez, C., Cauty, C., & Guyomarc’h, F. (2015) Organization of lipids in milks, infant milk formulas and various dairy products: role of technological processes and potential impacts. Dairy Science Technology. 95, 863–93. Marangoni, A. G., & Lencki, R.W. (1998) Ternary phase behaviour of milk fat fractions. Journal of Agricultural and Food Chemistry. 46, 3879–84. Mazzanti, G., Marangoni, A. G., & Idziak, S. H. J. (2009) Synchrotron study on crystallization kinetics of milk fat under shear flow. Food Research International. 42, 682–94. Michalski, M.‐C., Ollivon, M., Briard, V., Leconte, N., & Lopez C. (2004) Native fat globules of different sizes selected from raw milk: Thermal and structural behaviour. Chemistry and Physics of Lipids. 132(2), 247–61. Ollivon, M., Keller, G., Bourgaux, C., et al. (2006) DSC and high resolution X‐ray diffraction coupling. Journal of Thermal Analysis and Calorimetry. 85, 219–24. Ramel, P. R., & Marangoni, A. G. (2016) Engineering the microstructure of milk fat by blending binary and ternary mixtures of its melting fractions. RSC Advances. 6, 41189–94. Ramel, P. R., Peyronel, F., & Marangoni, A. G. (2016) Characterization of the nanoscale structure of milk fat. Food Chemistry. 203, 224–30. Ronholt, S., Kirkensgaard, J. J. K., Mortensen, K., & Knudsen, J. C. (2014) Effect of cream cooling rate and water content on butter microstructure during four weeks of storage. Food Hydrocolloids. 34, 169–76. Smet, K., Coudijzer, K., Fredrick, E., et al. (2010) Crystallization behaviour of milk fat obtained from linseed‐fed cows. Journal of Dairy Science. 93, 495–505. Smiddy, M. A., Huppertz, T., & van Ruth, S. M. (2012) Triacylglycerol and melting profiles of milk fat from several species. International Dairy Journal. 24, 64–69. Söderberg, I., Hernqvist, L., & Buchheim, W. (1989) Milk fat crystallization in natural milk fat globules. Milchwissenschaft. 44, 403–6. Ten Grotenhuis E., van Aken, G. A., van Malssen, K. F., & Schenk H. (1999) Polymorphism of milk fat studied by differential scanning calorimetry and real‐time X–ray powder diffraction. Journal of American Oil Chemists’ Society. 76, 1031–39. Timms R. E. (1980) The phase behaviour and polymorphism of milk fat, milk fat fractions and fully hardened milk fat. Australian Journal of Dairy Technology. 35, 47–53. Truong, T., Bansal, N., Sharma, R., Palmer, M., & Bhandari, B. (2014) Effects of emulsion droplet sizes on the crystallisation of milk fat. Food Chemistry. 145, 725–35. Truong, T., Morgan, G. P., Bansal, N., Palmer, M., & Bhandari, B. (2015) Crystal structures and morphologies of fractionated milk fat in nanoemulsions, Food Chemistry. 171, 157–67.

Crystallization Properties of Milk Fats

Tzompa‐Sosa, D. A., Ramel, P. R., van Valenberg, H. J. F., & van Aken, G. A. (2016) Formation of b polymorphs in milk fats with large differences in triacylglycerol profiles. Journal of Agricultural and Food Chemistry. 64, 4152–57. Van Aken, G. A., & Visser, K. A. (2000) Firmness and crystallization of milk fat in relation to processing conditions. Journal of Dairy Science. 83, 1919–32. Van Aken, G. A., ten Grotenhuis, E., van Langevelde, A. J., & Schenck, H. (1999) Composition and crystallization of milk fat fractions. Journal of the American Oil Chemists’ Society. 76, 1323–31. Vanhoutte, B., Dewettinck, K., Foubert, I., Vanlerberghe, B., & Huyghebaert, A. (2002) The effect of phospholipids and water on the isothermal crystallisation of milk fat. European Journal of Lipid Science and Technology. 104, 490–95. Wright, A. J., & Marangoni, A. G. (2003) The effect of minor components on milk fat microstructure and mechanical properties. Journal of Food Science. 68, 182–86. Wright, A. J., & Marangoni, A.G. (2006) Crystallization and rheological properties of milk fat. In: Fox, P. F., & McSweeney, P. L. H. (eds.), Advanced dairy chemistry, Vol. 2 Lipids, 3rd ed. New York, Springer, pp. 245–332. Wright, A. J., Hartel, R. W., Narine, S. S., & Marangoni A. G. (2000) The effect of minor components on milk fat crystallization. Journal of the American Oil Chemists’ Society. 77, 463–75.

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11 Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications Maria L. Herrera and Silvana Martini

11.1 ­Introduction Sunflower oil is obtained from the seeds of Helianthus annuus (sunflower). Sunflower oil is the fourth‐major oilseed crop worldwide after palm, soybean, and rapeseed with approximately 16 million metric tons produced in July 2016 as reported by the U.S. Department of Agriculture. The world’s largest sunflower oil producers now are Ukraine, Russia, and Argentina with production levels of approximately 5,000, 4,000, and 1,000 thousand metric tons, respectively, in 2016 (http://apps.fas.usda.gov/ psdonline/circulars/oilseeds.pdf ). Sunflower oil is broadly used for retailing and as frying oil. In Western countries, it is a common ingredient of salads and cooking recipes (Salas et al. 2015). This oil predominantly consist of linoleic acid (67%), oleic acid (19%), with low content of linolenic (1%), palmitic (7%), and stearic acids (4%), although high oleic versions of the oil are also available with contents of oleic acid as high as 81% (O’Brien 2009). Fernández‐Moya et al. (2005) described new sunflower lines with medium, high, and very high stearic acid contents. These lines were generated on both, a normal and a high‐oleic background, by crossing and recombining the original mutant lines (nongenetically modified plants) between themselves or with standard and high‐oleic sunflower lines (Salas et al. 2005). It was reported that oils with high content of stearic acid at the sn‐1 and sn‐3 position are resistant to high temperatures because of their saturated characteristics (Valenzuela et  al. 2011). The TAG species present in the oil are of great relevance because they determine its physical, chemical, and nutritional properties. In particular, high stearic high oleic sunflower oil (HSHO‐SFO) could be an adequate substitute for partially hydrogenated fats in frying applications (Di Rienzo et al. 2008). For applications that require solids, such as shortening and cocoa butter alternatives, HSHO‐SFO can be fractionated to obtain fractions with desired melting point, solid fat content, and appropriate saturated/unsaturated/saturated (SUS) TAG content. This chapter will describe the research related to the crystallization behavior of HSHO‐SFO fractions for use as shortenings and cocoa butter alternatives as well as the blend of sunflower oil with other fats. Crystallization of Lipids: Fundamentals and Applications in Food, Cosmetics, and Pharmaceuticals, First Edition. Edited by Kiyotaka Sato. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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11.2 ­High Stearic High Oleic Sunflower Oil One of the alternatives developed to replace trans‐fat is the development of new oils through plant breeding using biotechnology and conventional techniques (see Chapter 1). These breeding strategies can be used to modify fatty‐acid composition of oils and to improve their functionality in foods. Stearic acid–rich fats were developed using these techniques because stearic acid has been proven to have low impact on consumer health, good plasticity, and stability (Crupkin & Zambelli 2008). Available scientific information confirms with reasonable evidence that consumption of less than 7% of the total energy of stearic acid does not modify lipid profile, thrombotic factors, and hemodynamic and cardiovascular risk molecular markers (Kris‐Etherton et  al. 2005; Crupkin & Zambelli 2008; Hunter et al. 2010). This aspect distinguishes stearic acid from other saturated fatty acids present in diets, such as palmitic, lauric, and myristic because stearic acid ranks among monounsaturated acid, such as oleic acid, whereas other fatty acids are considered hypercholesterolemic (Valenzuela et al. 2011). Common sunflower lines have demonstrated great plasticity and genetic variability, which has made it possible to produce mutant lines with altered fatty‐acid composition (Hazebroek 2000). Among these new mutants, lines producing HSHO‐SFO have been developed (Fernández‐Moya et al. 2005). Indeed, high stearic hybrids that produce oils ranging from 17% to 22% stearic acid on a high oleic background are under development, and some are currently being cultivated (Salas et  al. 2011). Although liquid at ambient temperature, HSHO‐SFO can be fractionated to produce stearins enriched in stearic acid. The chemical composition and crystallization behavior of HSHO‐SFO stearins will be described in the sections that follow. 11.2.1  Fractionation of HSHO‐SFO Fractionation is a physical process that involves the separation of a multicomponent mixture into a solid phase (stearin) and a liquid phase (olein). Fractionation is the result of two processes: a crystallization step and a separation or filtration step. The basis resides in the solubility of TAG species in the liquid phase at controlled temperature. There are two main types of fractionation processes: dry and solvent fractionation. Dry fractionation is cheaper than solvent fractionation and environmentally friendly. On the other hand, solvent fractionation is more efficient to separate olein and stearin fractions. Two types of stearins can be obtained from HSHO‐SFO by either dry (soft stearin [SS]) or solvent fractionation (hard stearin [HS]). SS is produced by crystallizing the oil under controlled temperature and agitation conditions (18 °C and 30 rpm). HS is then separated from the SS by dissolving the oil in hexane and storing the micelles without stirring at 5 °C (HS). The stearin is then collected by vacuum filtration of the precipitates. The melting point of HSHO‐SFO stearins was determined using AOCS Method Cc 1‐25 (Martini et al. 2013). Capillary melting points of SS and HS were 30.3° ± 0.6 °C and 35.1° ± 0.4 °C (means and standard deviations), respectively. The fatty acid (FA) and TAG compositions of SS and HS were determined by gas chromatography (GC) using a unit equipped with a flame ionization detector (FID) and on‐ column injector (Rincón Cardona et al. 2013). Table 11.1 shows the percentages of FA present in these samples. The main fatty acids for SS included stearic acid (28 %) and oleic acid (59 %). Palmitic, linoleic, arachidic, and behenic acids were minor components

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

Table 11.1  FA composition of hard and soft HSHO‐SFO stearins. FA

Soft Stearin (wt.%)

Hard Stearin (wt.%)

C16:0 (palmitic)

5.3 ± 0.3

4.8 ± 0.3

C18:0 (stearic)

28.1 ± 0.9

46.9 ± 0.9

C18:1 (oleic)

58.8 ± 0.8

39.9 ± 0.5

C18:2 (linoleic)

3.0 ± 0.2

0.9 ± 0.1

C20:0 (arachidic)

2.0 ± 0.1

3.1 ± 0.1

C22:0 (behenic)

2.8 ± 0.1

4.4 ± 0.2

S

38.2

59.2

MU

58.8

39.9

PU

3.0

0.9

Rincón‐Cardona et al. 2013. Copyright (2013) with permission from Elsevier. FA, fatty acid; HSHO‐SFO, high stearic high oleic sunflower oil; MU, monounsaturated; PU, polyunsaturated; S, saturated. Values of S, MU, and PU are the sum of averages.

and they were present in percentages of approximately 5%, 3%, 2%, and 3%, respectively. For HS, stearic acid was present in a percentage of approximately 47%, and oleic acid content was approximately 40%. Palmitic, linoleic, arachidic, and behenic acids had contents of approximately 5%, 1%, 3%, and4 %, respectively. The TAGs composition is presented in Table 11.2. The main TAGs for SS included POSt (7%), StOSt (24%), StOO (22%), StOA (4%), and StOB (3%). For HS, the main TAGs included POSt (9%), StOSt (55%), StOO (7%), StOA (8%), and StOB (9%), where P stands for palmitic acid, St for stearic acid, O for oleic acid, B for behenic acid, and A for arachidic acid. The monounsaturated FA contents for SSs and HSs were 58.8% and 39.9%, respectively, and saturated‐oleic‐saturated (SOS‐type) TAGs (POP, POSt, StOSt, StOA, and StOB) were 38.8% and 81.4%, for SSs and HSs, respectively. For SS, these values do not exactly agree with European Union standards to qualify as a cocoa butter equivalent (CBE) material. On the contrary, values for HS reach these requirements because they have less than 45% unsaturated fatty acids (UFAs) and more than 65% of SOS‐type TAGs. In short, dry fractionation produced SS fractions enriched in saturated FA that can be used for manufacturing structured fats or spreads. These stearins can also be used as an intermediate step in the production of HSs appropriate for confectionary fat formulation. SS and HS have a simple chemical composition that resembles cocoa butter composition to some extent. Compared with many fats, cocoa butter has a simple FA composition being largely composed of palmitic, stearic, oleic, and linoleic acids, being linoleic acid in a much lower content. This simple FA composition results in an equally simple TAG composition with three predominating TAGs: POP, POSt, and StOSt. Although TAG contents depend on cocoa butter origin, most of the studies report values from 18% to 19%, 38% to 40%, and 26% to 31%, for these three main TAGs, respectively (Talbot 2012). Contrary to SS and HS, cocoa butter contains low quantities of arachidic or behenic TAG in percentages less than 1%. Considering the difference between the

325

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Crystallization of Lipids

Table 11.2  TAG composition of hard and soft HSHO‐SFO stearins. TAG

Soft Stearin (wt.%)

Hard Stearin (wt.%)

POP

0.6 ± 0.1

0.4 ± 0.2

POSt

7.4 ± 0.3

9.3 ± 0.3

POO

5.4 ± 0.7

1.6 ± 0.3

POL

0.5 ± 0.2

0.2 ± 0.1

StOSt

23.7 ± 0.6

54.6 ± 0.9

StOO

22.3 ± 0.5

7.5 ± 0.5

OOO

22.8 ± 0.5

6.1 ± 0.3

StOL

2.1 ± 0.1

0.8 ± 0.2

OOL

3.3 ± 0.2

0.9 ± 0.2

OLL

0.7 ± 0.3

0.3 ± 0.1

StOA

3.6 ± 0.4

8.0 ± 0.2

OOA

1.6 ± 0.2

0.5 ± 0.1

StOB

3.5 ± 0.1

9.2 ± 0.4

OOB

2.3 ± 0.3

0.7 ± 0.2

SUS

38.8

81.4

SUU

34.3

11.2

UUU

26.9

7.3

Rincón‐Cardona et al. 2013. Copyright (2013) with permission from Elsevier. A, arachidic acid (C20:0); B, behenic acid (C22:0); HSHO‐SFO, high stearic high oleic sunflower oil; L, linoleic acid (C18:2); O, oleic acid (18:1); P, palmitic acid (C16:0); S, saturated; St, steric acid (C18:0); U, unsaturated.

chemical composition of SS or HS and cocoa butter a different crystallization behavior among these fats could be expected. The following sections describe crystallization and polymorphic behaviors of stearins of HSHO‐SFO crystallized under different processing conditions. 11.2.2  Crystallization Behavior The crystallization behavior of SS and HS fractions was evaluated by measuring the equilibrium solid fat content (SFC) using the pretreatment (tempering) described in the AOCS official methods Cd 16b‐93 I and II (AOCS 1997). For method I, the tempering procedure was as follows: melt the sample and keep for 15 minutes at 100 °C followed by 5 minutes at 60 °C; place sample at 0 °C for 90 ± 5 minutes, and finally, place the ­sample at each crystallization temperature for 60 minutes. Method II included an additional step: after placing the sample at 0 °C it was kept at 26 °C for 40 ± 0.5 hours, and again it was placed at 0 °C for 90 ± 5 minutes. SFC was determined at different temperatures as shown in Tables 11.3 and 11.4. Method I is usually chosen when the fat does not exhibit polymorphic behavior, whereas method II is selected for fats having various crystalline cells, some of which are

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

Table 11.3  SFC of soft stearin determined with the method Cd 16b‐93 part I and II of the American Oil Chemists’ Society (1997). SFCa

a

Temperature

Method I

Method II

5 °C

53,3

44,7

10 °C

48,1

38,8

15 °C

39,9

29,1

20 °C

28,1

24,6

25 °C

22,5

19,3

Values differing more than 1% are significantly different. SFC, solid fat conent.

Table 11.4  SFC of hard stearin determined with the method Cd 16b‐93 part I and II of the American Oil Chemists’ Society (1997). SFCa

a

Temperature

Method I

Method II

10 °C

83,0

77,0

15 °C

80,4

75,8

20 °C

75,3

74,0

25 °C

70,0

71,6

30 °C

62,1

62,8

Values differing more than 1% are significantly different. SFC, solid fat content.

unstable and are transformed easily into a more stable form depending on the conditions under which crystallized. It may be observed that values measured for SS by method II are significantly lower than those obtained by the method I (Table  11.3). During SFC determination using method II fat crystallizes in equilibrium conditions, and therefore the most stable polymorphic form is obtained. As the nuclear magnetic resonance (NMR) detector has a different response factor for each crystal form, working in a standardized way with the same experimental protocol allows obtaining comparable results between laboratories. The fact that SFC values obtained with method I are higher indicates that the crystalline form obtained is not the same as for method II. Method I did not allow crystallization of the stearins in the most stable form. The results indicate that these systems are highly polymorphic in nature and method II would be best suited for determining equilibrium SFC. Table 11.4 summarizes SFC measured by methods I and II for HS. Values obtained for both methods are significantly different at 10° and 15 °C. Most likely the most stable form of HS needed more time to crystallize

327

Crystallization of Lipids

at those temperatures. At higher temperatures, values for both methods were within the experimental error. This indicated that SS and HS showed different crystallization behaviors. Crystallization kinetics of SS and HS was evaluated by measuring actual SFC values as a function of time under isothermal conditions (Fig. 11.1). Bulk fat sample (4 mL) was placed in NMR tubes and heated at 80 °C for 30 minutes to destroy any crystal memory. Samples were then kept at 60 °C for 30 minutes and crystallized to selected crystallization temperatures. SFC was measured at time intervals for 120 minutes. Figure  11.1 reports the results obtained for SS (Fig. 11.a) and HS (Fig. 11.1b). Samples were crystallized at 10 °C/min and zero time was considered as the moment sample reached crystallization temperature. When SS crystallized at 5 °C, SFC versus time curves showed hyperbolic shape indicating that crystallization in this condition was very fast (Fig. 11.1a). SFC after 120 minutes at 5 °C was close to 40%. This value was the 91% of SFC value measured using method II. Similar results were obtained for the other temperatures except for 19 °C. At this temperature, SFC after 120 minutes was 2% of SFC by method II. It is likely that the polymorphic form obtained at this temperature needs (a) 45 40

SFC (%)

35 30 25

5° C

20

16° C

15

18.5° C

10

19° C

5 0

0

10 20

30 40

50 60 70 80 90 100 110 120 130 Time (min)

(b) 90 80 70 SFC (%)

328

60 50 40

10° C

30

23° C

20

24° C

10

25° C

0

0

10 20 30 40 50 60 70 80 90 100 110 120 130 Time (min)

Fig. 11.1  Solid fat content (SFC) as a function of time for (a) soft stearin (SS) and (b) hard stearin (HS) of high stearic high oleic sunflower oil (HSHO‐SFO), which are isothermally crystallized at different temperatures.

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

more time than 120 minutes to be noticeable. For HS (Fig. 11.1b), SFC after 120 minutes at 10° and 24 °C were 96 and 99%, respectively, of values of SFC by method II. At 25 °C, SFC value was 75% of SFC equilibrium value, and at 30 °C, value of SFC after 120 ­minutes was lower than 1% indicating that HS did not crystallize in those conditions. However, SFC at 30 °C as measured by method II was 62%. The different behavior found between samples crystallized under equilibrium and isothermal conditions may be indicative of a complex polymorphic behavior. 11.2.3  Polymorphic Behavior Data obtained by NMR suggested that HSs and SSs of HSHO‐SFO are polymorphic in nature. To describe the polymorphic behavior, stearins were isothermally crystallized to different temperatures and polymorphic forms were characterized by X‐ray scattering using synchrotron light. Results showed that SS and HS crystallized in five polymorphic forms (Rincón Cardona et al. 2013; Martini et al. 2013). For both stearins, at all crystallization temperatures the first polymorphic form that crystallized was the α‐form. Then, depending on selected crystallization conditions, two β’ forms appeared. The more unstable polymorph was called β’2 and the more stable form, β’1. Two β forms crystallized during storage: β2 at shorter times (less than 24 hours) and β1 at longer storage time (more than 48 hours) (Rincón Cardona et al. 2013; Martini et al. 2013). Stearins had a different polymorphic behavior than cocoa butter. They fractionated and more than one polymorphic form were present at the same time. In addition, some polymorphs such as the α form underwent a transition to a more stable form (i.e., β’2; however, other polymorphs such as β’2 form did not transform into β’1 form). Most likely β’2 and β’1 were formed by different solid solutions (Rincón Cardona et al. 2013). HSHO‐SFO contained arachidic and behenic TAG that were not present in cocoa butter. For this reason these stearins did not behave as a pure compound because it is unlikely that AOSt and BOSt TAG were fully compatible with StOSt and POSt in the β’ or β forms. Examples of polymorphic forms obtained for HSHO‐SFO stearins are presented here. For SS, the most unstable β’ form, the β’2 form did not crystallize above 14 °C. Figure 11.2 shows as an example representative images of the morphology of α (Fig. 11.2a) and β’2 (Fig. 11.2b) crystals. The α crystals are characterized by a needle shape and were homogeneously distributed in the microscope field. The β’2 crystals were called so because of the two orthorhombic cells found for SS; this β’ form crystallized at lower temperatures. Microscopic image showed needles agglomerated forming well‐defined spherulites. Figure 11.3 shows the small angle X‐ray scattering (SAXS) spectra (Fig. 11.3a) and the wide angle X‐ray scattering (WAXS) spectra (Fig. 11.3b) of SS crystallized at 15 °C for 40 minutes. At the moment the sample reached crystallization temperature (t = 0), no presence of crystalline material was detected indicating that crystallization process took place under isothermal conditions. After 3 minutes at 15 °C, SAXS and WAXS spectra showed the start of crystallization. During the first 15 minutes of crystallization, the SAXS patterns displayed a single peak with a q of 1.22 nm−1 (d = 5.15 nm; Fig. 11.3a) whereas WAXS patterns shown in Fig.  11.3(b) displayed a clear single peak at q = 14.97 nm−1 (d = 0.42 nm). As previously described, these X‐ray diffraction (XRD) patterns are consistent with a bilayer (2 L) lamellar packing arrangement and a hexagonal subcell or α polymorph (Lopez et al. 2001a, 2001b; Mazzanti et al. 2004; Cisneros

329

330

Crystallization of Lipids

(a)

(b)

Fig. 11.2  Polarized light microscopy images of crystals obtained for soft stearin (SS) of high stearic high oleic sunflower oil (HSHO‐SFO) isothermally crystallized at 10 °C. (a) 3 min. (b) 15 min.

et al. 2006; Rincón Cardona et al. 2013). After 15 minutes at 15 °C, other signals, corresponding to a second polymorphic form, appeared. SAXS patterns showed an intense signal with a q value of 1.39 nm−1 (d = 4.52 nm) together with a weak signal at q = 4.15 nm−1 (d = 1.51 nm). In WAXS patterns, two new signals at q = 14.13 nm−1 and 16.20 nm−1 (d = 0.44 nm and d = 0.39 nm) were observed. This polymorphic form corresponded to a β’‐phase. As this β’ form was obtained at longer times or higher temperatures than the β’ form shown in Fig. 11.2, it was called β’1. Figure 11.4 shows polarized light microscopy (PLM) images of crystals corresponding to X‐ray patterns report in Fig. 11.3. After 3 minutes at 15 °C, α crystals were noticeable. They grew and showed a needle shape (Fig. 11.3a). Crystals were isolated and randomly distributed in the microscope field. After 15 minutes at 15 °C (11.3b), β’1 crystals appeared. They formed big, well‐organized geometric clusters of homogeneous crystals. As in the case of SS, the α form of HS also showed needle shape crystals. β’2 polymorph was characterized by ill‐defined spherulites. These crystals showed two signals in the WAXS zone: one at 0.43 and the other at 0.38 nm. The morphology of β’2 crystals of HS did not exactly match the one of β’2 crystals of SS. HS also had a third type of crystal morphology characterized by well‐defined spherulites with distinctive Maltese crosses shapes. X‐ray patterns showed two signals in the WAXS region at 0.44 and 0.39 nm characteristics of the β’1 form (Martini et al. 2013). β crystals did not crystallized after 120 min of isothermal crystallization. They needed more time to crystallize. As an example, Fig. 11.5 shows the crystals obtained when HS was isothermally crystallized at 25 °C for (Fig. 11.5a) 6 hours and (Fig. 11.5b) 1 week. Both images are in the same scale. After 6 hours at crystallization temperature, crystals were characteristics of β2 form as indicated by the strong signal at 0.46 nm present in WAXS patterns. Their morphology was similar to the β’1 form. However, crystals had very distinctive concentric lines or patterns that were not observed in the β’1 crystals (Martini et al. 2013). After 1 week of storage at 25 °C, an ill‐defined agglomeration of crystals with no apparent organization crystallized. These crystals corresponded to a β1 polymorph characterized by a strong signal at 0.46 nm. Even though β2 and β1 polymorphs had the same signals in the WAXS spectra, their SAXS spectra were significantly different with signals at 6.06 and 3.02 nm for the β2 polymorphism and 6.49 and 3.71 nm for the β1 polymorphic form (Rincón Cardona et al. 2013).

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

(a) β′1

Intensity (arb. u.)

1,6

0,8

0,0 α Time (min) 3 q (nm –1)

(b) β′1

α

0,5 0,4 0,3

Intensity (arb. u.)

0,2 0,1 0,0

Time (min) 16

14 q

(nm –1)

Fig. 11.3  Three‐dimensional plots of (a) small angle X‐ray scattering(SAXS) and (b)wide angle X‐ray scattering (WAXS) measurements of soft stearin of high stearic high oleic sunflower oil (HSHO‐SFO) crystallized at 15 °C for 40 min.

Figure 11.6 shows PLM images of HS crystallized at 26 °C for 90 minutes. More than one polymorphic form was present at the same time. The α polymorph was still present, even at this high temperature, together with β’1 and β1 forms. This could be a disadvantage from the practical point of view. TAG composition, tempering regime, the presence of other lipids or additives, and mechanical treatment (e.g., shear, agitation) influence how a lipid solidifies from the melt. In the food industry, these parameters are all used to direct polymorphic behavior and morphological development in fats. Polymorphism of fats is a fundamental property that determines food products appearance. For example, in margarine production, maintaining β′‐crystallinity is imperative to preserve smooth texture and acceptable spreadability. In chocolate manufacturing, careful tempering is used to promote the crystallization of the metastable β‐V form of cocoa butter, responsible for much of chocolate’s organoleptic and shelf life properties

331

332

Crystallization of Lipids

(a)

(b)

Fig. 11.4  Polarized light microscopy (PLM) images of crystals obtained for soft stearin (SS) of high stearic high oleic sunflower oil (HSHO‐SFO) isothermally crystallized at 15 °C. (a) 3 min. (b) 15 min.

(a)

(b)

Fig. 11.5  Polarized light microscopy images of hard stearin (HS) of high stearic high oleic sunflower oil (HSHO‐SFO) isothermally crystallized at 25 °C for (a) 6 hours and (b) 1 week.

Fig. 11.6  Polarized light microscopy images hard stearin (HS) of high stearic high oleic sunflower oil (HSHO‐SFO) isothermally crystallized at 26 °C for 90 min. HS of HSHO‐SFO isothermally crystallized at 25 °C for (a) 6 hours and (b) 1 week.

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

(Loisel et al. 1998; Narine & Marangoni 1999; Braipson‐Danthine & Deroanne, 2004). Figure 11.6 shows that at 26 °C HS did not crystallize in β2 form. Thus, this processing condition will not be useful in chocolate manufacture. To crystallize HS and SS in a desired polymorphic form, different strategies may be used. Three of them, cooling, agitation, and sonification, will be described. 11.2.3.1  Effect of Processing Conditions

It has been well understood that cooling rate can affect nucleation by either accelerating or delaying crystallization when slow cooling rates are used (see Chapter 6). For nonpolymorphic systems such as hydrogenated sunflower seed oil (Herrera 1994) and milk fat/sunflower oil blends (Martini et al. 2001), the reported effect was shorter induction times of nucleation for slow cooling. These systems showed only one polymorphic form under the crystallization conditions used. When a fat is crystallized using either fast or slow cooling, the thermodynamic driving force for crystallization is the same because it depends on the difference between melting and crystallization temperature (Martini et al. 2001). In addition to thermodynamic factors, kinetic factors might play an important role in lipid crystallization. That is, even when the thermodynamic force for crystallization is reached, molecules must diffuse in the bulk lipid and must organize and align with their neighbors to form stable nuclei. In the studies reported by Herrera (1994) and Martini et al. (2001), TAG reorganization started during the slow cooling process and therefore the sample needed less time at crystallization temperature to nucleate resulting in an overall shorter induction time of nucleation (Martini et al. 2001). It was reported that when a fat is crystallized at fast cooling rate, the α polymorph can be expected, whereas at slow cooling rate, the more stable forms, β′ or β polymorphs are more often obtained (Sato 1988). In general, for the three main polymorphic forms of fats, induction times increase in the order α, β′, and β (Sato 1988). For polymorphic systems, the usual effect for slow cooling is a delay in nucleation, that is, an elongation in induction times since stable polymorphic forms nucleate at longer times (Sato 1988). In milk fat and lard, for example, more stable ­polymorphs were obtained for slow cooling (Campos et al. 2002). Goat’s milk fat showed a similar behavior (Amara‐Dali et al. 2007). In SS and HS, induction times as measured by NMR, increased with slow cooling (Herrera et al. 2015) indicating that the effect of cooling rate on crystallization of HSHO‐SFO stearins was closely related to changes in polymorphic behavior. Figure 11.7 shows the effect of cooling rate on polymorphism of HS crystallized at 23 °C. Cooling rate affected crystallization kinetics and the type of polymorphic form ­present in selected conditions. As was expected, fast cooling rate promoted crystallization of unstable forms. At 10 °C/min, α (Fig. 11.7a) and β′2 (Fig. 11.7b) forms were present in greater amount than at 1 °C/min (Fig. 11.7c and d). On the contrary, slow cooling promoted crystallization of β′1 (Fig. 11.7d). These results suggest that a particular polymorphic form may be obtained by choosing the appropriate cooling rate or specific temperature cycles (Herrera et al. 2015). Most of the studies related to crystallization behavior of HSHO‐SFO stearins were performed under static conditions where no agitation was applied to the system. Martini et al. (2013) has shown that agitation affects the crystallization, polymorphic behavior, and physical properties of SSs and HSs. In particular, agitation promoted the crystallization of most stable polymorphic form. For example, SS fractions crystallized under

333

334

Crystallization of Lipids

(a)

(b)

(c)

(d)

Fig. 11.7  Polarized light microscopy images of hard stearin (HS) of high stearic high oleic sunflower oil (HSHO‐SFO) crystallized at 23 °C. (a, b) 10 °C/min; (c, d) 1 °C/min. a, c, for 10 min, and b, d, for 35 min.

static conditions showed β’2 at low Tc and β2 crystals at high Tc, whereas mainly β2 and β1 crystals were obtained at low and high Tc, respectively, when the sample was crystallized under agitation. Similarly, HS samples crystallized under static conditions mainly showed β’1 crystals at low Tc and α crystals at high Tc. When HS was crystallized with agitation mainly β2 crystals were obtained at low Tc, and β1 crystals were obtained at high Tc. Agitation also affected the kinetics of crystallization of the stearins. As expected, a faster crystallization was observed in samples crystallized under agitation and fewer differences in SFC were observed as a function of Tc, especially for the SS samples. In addition to a faster crystallization, higher SFC were observed in the samples crystallized under agitation, especially for samples crystallized at high Tc. However, lower SFC was observed in SS and HS samples crystallized under agitation at low temperatures such as 16° and 24 °C, respectively, compared to samples crystallized under static conditions. Lastly, agitation resulted in softer crystalline networks for SS samples and harder networks for HS samples crystallized at high Tc. It is evident that agitation significantly affected the polymorphic behavior of SS and HS and these in turn affected physical properties of the materials such as final SFC and hardness (Martini et al. 2013). High intensity ultrasound (HIU) can be used as a novel technology to change the crystallization behavior (Higaki et  al. 2001; Ueno et  al. 2003a, 2003b) and physical

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

properties (Martini 2013) of lipids as discussed in more detail in Chapter 8. This technology has been used to evaluate changes in crystallization behavior in SSs and HSs (Rincón‐Cardona et al. 2015). Results from this study showed that the use of HIU in SSs induced its crystallization and increased the crystallization rate, while promoting the formation of the most stable polymorphic form (β1). Crystallization with HIU also resulted in a crystalline network with significantly higher melting enthalpy, which is probably because of the higher SFC and the polymorphic form obtained under these conditions. In addition, sonicated samples resulted in a lower elastic modulus suggesting a less solid‐like behavior. In summary, SSs and HSs are highly polymorphic in nature and processing conditions such as cooling rate, agitation, and the use of sonication can be used to obtain a specific polymorphic form. 11.2.3.2  Effect of Addition of Sucrose Esters

Emulsifiers can also be used to change the crystallization behavior of lipids. The effect of stearic sucrose ester (S‐170) on the crystallization behavior of SS and HS was evaluated by measuring SFC as a function of time. Results showed that the crystallization of both stearins was either accelerated or delayed by S‐170 depending on crystallization temperature (Rincón Cardona et al. 2014). At lower temperatures, the effect was acceleration of nucleation, whereas at higher temperatures it was delay of crystallization. For SS, S‐170 accelerated crystallization below 17 °C and retarded crystal formation above this temperature. For HS, the acceleration effect occurred below 23 °C and the delay of crystallization took place above this temperature. Figure 11.8 shows the effect of S‐170 on SS crystallization behavior at 16 °C. At this temperature, the first polymorphic form that appeared in SS crystallized without S‐170 after 4 minutes of crystallization was the α form. Then, after 29 minutes at 16 °C, the β’1 form was present (Rincón Cardona et al. 2013). When S‐170 was added to SS, the α form was present from the first pattern and β’1 form appeared after 13 minutes. It is clear from Figure 11.8 that S‐170 accelerated crystallization at 16 °C. Intensities values also showed that SFC was higher when emulsifier was added. This was confirmed by NMR studies (Rincón Cardona et al. 2014). Figure 11.9 shows the effect of S‐170 on crystallization kinetics of HS at 24 °C. At this temperature, the effect on β’1 form crystallization was a delayed in nucleation. Without additives, β’1 appeared after 44 minutes, whereas when S‐170 was added to HS it crystallized after 53 minutes. The delay in nucleation and overall crystallization caused by addition of S‐170 was confirmed by NMR experiments (Rincón Cardona et al. 2014). In addition to the kinetics effects, S‐170 also had an effect on the type of polymorphic form that crystallized in selected conditions. Sucrose ester S‐170 promoted the formation of the β′1 form at temperatures at which β′2 form was the polymorph expected to crystallize (i.e., 5 °C). This result is also important from the technological point of view. β′1 is the most stable of β′ forms and addition of S‐170 increased its life and stability. If the β′ form is required for a product, addition of S‐170 might increase product life (Rincón Cardona et al. 2014). When SS crystallized at 17 °C, SAXS and WAXS patterns showed that at the moment sample reached crystallization temperature the pattern obtained corresponded to an amorphous system. Then, the α form crystallized followed by the β’1 form that was the main polymorph after 36 minutes. Traces amounts of β2 form also crystallized after 40 minutes at 17 °C (Rincón Cardona et al. 2014). Figure 11.10 shows PLM images of SS

335

Crystallization of Lipids

(c) 10

8

8

6 4 2

6 4 2

e

Intensity [arb. u.]

10

1

2

3

4

0

5

Ti m

0

Ti m

e

Intensity [arb. u.]

(a)

1

2

q [nm–1]

3

4

5

4

5

q [nm–1]

(d)

(b) 10

10

8

8

Intensity [arb. u.]

6 4

6 4 2

1

2

3 q

[nm–1]

4

5

0

Ti

0

Ti

m

m

e

2

e

Intensity [arb. u.]

336

1

2

3 q

[nm–1]

Fig. 11.8  X‐ray diffraction patterns of soft stearin (SS) of high stearic high oleic sunflower oil (HSHO‐ SFO) crystallized at 16 °C. (a) 0 to 20 min and (b) 21 to 40 min of pure samples; (c) 0 to 20 min and (d) 21 to 40 min with addition of stearic sucrose ester S‐170.

isothermally crystallized at 17 °C without (Fig. 11.10, left column) and with (Fig. 11.10, right column) addition of stearic sucrose ester S‐170. In agreement to X‐ray studies, no crystallization occurred for SS without additives. After 30 minutes, β’ crystals were noticeable without additives, whereas β2 crystals were present when S‐170 was added. For SS without additives, a mixed of polymorphic forms were observed after 45 minutes at 17 °C. Then, β2 crystals crystallized together with β1 form. After 120 minutes, the β1 form was the main form. When S‐170 was added, β2 form grew, new β2 crystals appeared and finally the morphology after 120 minutes was the one of β2 form. It is clear from Fig. 11.10 that S‐170 promoted crystallization of β2 form under the selected conditions. This is the polymorphic form required for chocolate and confections. Thus, addition of S‐170 may be useful in chocolate production (Rincón Cardona et al. 2014). Depending on crystallization temperature and storage time, the β′1 or the β2 form could be the main form obtained. These forms are required forms for bakery and ­confectionary applications. Therefore, the use of SE S‐170 could improve stearins ­functionality which will be helpful in product manufacturing.

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

(a)

(c)

8

8 Intensity [arb. u.]

10

Intensity [arb. u.]

10

6 4

4 2

0 1

2

3 q

4

0

5

1

2

3

4

5

q [nm–1]

[nm–1]

(b)

(d) 10

8

8

β′1 (44 min)

2

2

1

2

α

m

e

α

Ti

0

β′1 (53 min)

4

3

4

5

0

Ti

4

6

e

6

m

Intensity [arb. u.]

10

Intensity [arb. u.]

Ti

Ti m

e

m

e

2

6

1

q [nm–1]

2

3

4

5

q [nm–1]

Fig. 11.9  X‐ray diffraction patterns of soft stearin (SS) of high stearic high oleic sunflower oil (HSHO‐SFO) crystallized at 24 °C. (a) 0 to 40 min and (b) 41 to 80 min of pure samples; (c) 0 to 40 min and (d) 41 to 40 min with addition of stearic sucrose ester S‐170.

11.3 ­Blends of Sunflower Oil and Milk Fat As discussed in Chapter  10, milk fat contains the most complex TAG composition among many natural fats. Furthermore, its composition changes with season, region, and cow feeding. To extend the use of milk fat in food, pharmaceutical, and cosmetic applications, fractionation may be performed to produce fractions with specific TAG composition and therefore specific properties (e.g., melting point). Milk fat fractions have found application in a variety of foods products including the use of high‐melting point stearins for puffy pastry or for the reduction of blooming in chocolate, the use of mid‐fractions in Danish cookies, and the use of different milk fat fractions in the production of cold‐spreadable butter and other dairy‐based spreads (Kaylegian & Lindsay 1995). Milk fat fractions are also blended with other fats and oils to provide manufacturers with greater flexibility and to tailor specific functional requirements than could not be accomplished by fractionation alone. Milk fat stearins’ fractions can be blended

337

338

Crystallization of Lipids

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 11.10  Polarized light microscopy images of soft stearin (SS) of high stearic high oleic sunflower oil (HSHO‐SFO) crystallized at 17 °C without (left column) and with (right column) addition of stearic sucrose ester S‐170. (a,b) 0, (c,d) 30, (e,f ) 45, (g,h) 60, and (i,j) 120 min.

with liquid oils such as sunflower oil (SFO) and soybean oil (SBO) in different proportions to offer nutritional properties including higher content of essential fatty acids and almost zero trans‐fatty acid content (Pal et al. 2001). Food manufacturers have faced many challenges during the development of new trans‐fat alternatives. First, any functional ingredient used as a trans‐fat replacer must

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

(g)

(h)

(i)

(j)

Fig. 11.10 (Cont’d)

provide appropriate functional characteristics such as appearance, texture, and shelf life. A second challenge faced by food producers is the assurance that ingredients used to replace trans‐fats will be available in adequate commercial quantities. Among trans‐fat alternatives, modification of high‐melting point stearins by blending with vegetable oil is becoming important because these fats result in shortenings with good nutritional properties and good functionality (List & Reeves 2005). Physical properties of crystalline networks formed during the processing of edible fats provide specific functionality to the material. In general, these functional properties can be modified by changing the crystallization behavior of the fat. Chapter  4 has already shown that crystallization is a complicated process in which molecules must first come in contact, orient, and then interact to form highly ordered structures known as nuclei. Nucleus formation can be promoted by stirring or by seeding the supercooled liquid with tiny crystals similar to the ones ultimately desired. Following nucleation, crystal growth occurs at a rate dependent on operating parameters such as temperature, agitation rate, and cooling rate (Hartel 2001). Addition of emulsifiers may also modify crystallization process (Martini et  al. 2004). Thus, the crystallization process can be controlled with selected processing conditions or with addition of emulsifiers. These changes in crystallization process will result in crystalline networks with a broad range of functionalities needed for food manufacturing.

339

340

Crystallization of Lipids

This section describes the use of different processing conditions to change the ­crystallization behavior of various milk fat/sunflower blends. 11.3.1  Chemical Composition Anhydrous milk fat (AMF) can be fractionated to obtain fractions with differing melting points and TAG chemical composition. In general, three main fractions can be obtained: high melting, medium melting, and low melting fractions. Herrera’s research group has thoroughly studied the crystallization behavior of a high melting fraction (HMF) of milk fat blended with SFO (Martini et al. 2001). The HMF used in these studies had a Mettler dropping point (MDP) of 40.2 °C and the SFO used was obtained from a commercial origin. SBO was blended with HMF in percentages of 10%, 20%, and 40%. The MDP values of these blends were 40.4°, 38.8°, and 37.4 °C, respectively. Their TAG compositions expressed as acyl carbon number are the following: SFO had two main TAGs, C52 (20.1%) and C54 (73.1%), whereas HMF had TAGs with carbon numbers from C30 to C54 containing C52 (9.2%) and C54 (4.3%). C52 and C54 carbon numbers increased in the SFO/HMF blends when SFO content increased because SFO provided a good source of oleic acid (C18:1) to the blend compared to HMF. Percentages of TAG with shorter carbon number diminished with increasing amounts of SFO. Therefore, SFO/HMF blends had a better TAG composition from the nutritional point of view because they had higher content of oleic acid and lower content of short and saturated fatty acid TAGs compared to HMF alone. 11.3.2  Physical Properties Martini et  al. (2001) evaluated the crystallization behavior of SFO/HMF blends as affected by crystallization temperature and cooling rate. These authors show that only one polymorphic form, β’‐form, was obtained for all the processing conditions tested when using a conventional X‐ray equipment. However, when a synchrotron source was used to study these systems it was found that the α‐form was present at some crystallization temperatures but only for a few minutes (Huck Iriart et al. 2009). Table  11.5 summarizes induction times for crystallization for HMF and its blends with SFO crystallized at fast and slow cooling rates. Because these samples were not polymorphic in nature the nucleation process was faster when samples were crystallized at slow cooling rates (0.1 °C/min). Faster nucleation is usually observed in slow cooled samples that did not reveal a strong polymorphic behavior because their nucleation mechanism mainly depends on the time that molecules need to reorganize and align to form a crystalline network rather than on thermodynamic factors driven by specific polymorphic behavior. When the samples were crystallized using a fast cooling rate (5.5 °C/min), it took longer for the sample to nucleate because molecular organization took place primarily at crystallization temperature, whereas a slow cooling allowed reorganization to occur at higher temperatures as the sample was cooled to reach crystallization temperature (Martini et al. 2001). Therefore, induction times of crystallization were shorter for slow cooled samples indicating that a slow cooling rate promoted crystallization in the blends (Table 11.5). In these systems, calculated values of activation free energies of nucleation were 10 times lower than values obtained for palm oil indicating that nucleation in

Crystallization Behavior of Sunflower Oil–Based Fats for Edible Applications

Table 11.5  Induction times of crystallization for HMF and its blends with SFO at two different temperatures for samples crystallized at 5.5 °C/min and 0.1 °C/min. Induction times of crystallization (min) 5.5 °C/min

0.1 °C/min

Sample

36 °C

38 °C

36 °C

38 °C

HMF

18.3 ± 1.0

46.5 ± 2.3

2.0 ± 1.2

18.4 ± 7.9

90% HMF

21.9 ± 1.7

78.7 ± 4.5

5.3 ± 2.0

23.5 ± 1.8

80% HMF

58.6 ± 3.1

126.7 ± 5.4

17.9 ± 0.8

64.3 ± 2.6

60% HMF

118.3 ± 7.1

244.9 ± 20.3

60.7 ± 2.9

141.4 ± 8.3

For the same sample and crystallization temperature, values of induction times were significantly different between cooling rates (p 99% pure) solutions in triolein and mineral oil (a). The arrows indicate the temperature for G’ onset. Cooling thermograms in triolein (b) and mineral oil (c). Martinez‐Avila and Toro‐Vazquez, unpublished results.

group esterified to choline (the polar “head). In an apolar environment, like in triolein or mineral oil, the PC develops a three‐dimensional network formed by aggregates of reverse microstructures like micelles or lamellas. The concentration of PC used in this study (35%) was well above the critical micelle concentration determined in nonaqueous systems (Manjula et  al. 2011). Therefore, the decrease in temperature of the

365

366

Crystallization of Lipids

(a)

(b)

50 μm

50 μm

Fig. 12.6  Polarized light microphotographs obtained at 15 ° C of 35% di‐palmitoyl phosphatidylcholine (>99% pure) organogels developed in triolein (a) and mineral oil (b). Martinez‐ Avila and Toro‐Vazquez, unpublished results.

PC‐solvent systems resulted in the formation of lamellar structures developed, first through the interaction between the polar “head” of the PC and then, as cooling proceeds, by the crystallization of the aliphatic tails in the bilayer structure. X‐ray studies of the PC organogels formed at 15 ° C in both solvents did confirm the presence of lamellar structures (results not shown). Although the PC used was more than 99% pure and it was maintained under desiccant conditions at all time, the presence of a small amount of bound water and its effect on the self‐assembly process of PC could not be excluded. However, in this study the effect of bound water on the self‐assembly of PC was considered negligible. The rheograms during cooling of the PC solutions showed that the formation of the inverse lamellar structures through the “head‐to‐head” interaction of the PC, results in a significant increase in elasticity. The onset in G’ occurred at higher temperature in triolein (≈130 ° C) than in mineral oil (≈100 ° C; Fig. 12.5a). In previous investigations using 25:75 blends of tripalmitin with different vegetable oils as solvents, including triolein, we showed that tripalmitin and triacylglycerides with palmitic acid interact in the liquid phase developing mixed lamellar microstructures at temperatures well above TC (Dibildox‐Alvarado et  al. 2010). The formation of these microstructures delayed the tripalmitin crystallization until a sufficient low temperature promoted the tripalmitin segregation from the mixed lamellar liquid structure. This resulted in a higher activation free energy (i.e., lower TC) for tripalmitin crystallization in vegetable oils with triacylglycerides with palmitic acid than in triolein (Dibildox‐Alvarado et  al. 2010). Similarly, the palmitic acid molecules of PC might develop mixed lamellar structures with the saturated n‐alkanes of the mineral oil. Should this molecular interaction occur, the self‐assembly of PC in the mineral oil would require of lower temperatures (i.e., higher supercooling) than in triolein. In lipid systems, such as triacylglyceride crystallization and formation of organogels, the use of high supercooling conditions results in the development of smaller and higher number of microstructures than those developed under low supercooling (Wang et al. 2006; Morales‐Rueda et al. 2009; Lam et al. 2010). At low supercooling, the crystal growth rate is favored over the nucleation rate, and the opposite occurs at high supercooling. Within this context, the PLM showed

Physical Properties of Organogels Developed with Selected Low-Molecular-Weight Gelators

that the organogels of PC developed in triolein were structured by larger ellipsoidal inverse micelles with higher extent of agglomeration (Fig. 12.6a). In contrast, the microstructures developed in the mineral oil were smaller, in higher number and with low extent of agglomeration (Fig. 12.6b). Consequently, the PC organogels in triolein had higher elasticity (G’ = 2 × 106 Pa ± 2.6 × 103) than the organogels developed in mineral oil (G’ ≈ 6.6 × 105 Pa ± 3.3 × 103; Martinez‐Avila and Toro‐Vazquez unpublished results). On the other hand, the corresponding cooling thermograms (Fig. 12.5b and c) of the PC solutions showed that the initial self‐assembly of PC molecules does not generate a significant amount of heat, particularly in the mineral oil. In triolein, the initial self‐ assembly process of PC was evidenced just by two small exotherms observed at temperatures below the G’ onset (Fig. 12.5b and c). In contrast, the crystallization of the hydrocarbon “tails” of the PC was associated with a major exotherm (Fig. 12.5b and c) that occurred at the same temperature (70.2 ° C ± 3.7 ° C) in both solvents used. We concluded that once the “head‐to‐head” interaction of the PC occurs, the TC of the aliphatic tails is independent of the solvent used to develop the organogel (i.e., vegetable oil or mineral oil). Then, as in the self‐assembly of monoacylglycerides, the TC of the aliphatic tails of PC depend mainly on the length and unsaturation extent of the hydrocarbon chains. Additionally, the crystallization of the aliphatic tails in the lamellar PC microstructures evidently do not result in a significant increment in the elasticity of the organogel. Then, most of the elastic properties of the PC organogels are associated with the size, shape, and extent of agglomeration of the self‐assembled microstructures developed by the PC. 12.3.2  Relationship between Molecular Structure of LMOGs and Physical Properties of Organogels Our group has investigated the rheological properties of organogels developed in safflower oil (high in triolein) at two cooling rates (1 ° C/min and 20 ° C/min) and their relationship with molecular structure, solid content, and the microstructures developed by several LMOGs after 75 min at 25 ° C (Toro‐Vazquez et al. 2010, 2013a). The LMOGs studied include primary and secondary amides synthesized from (R)‐12‐HSA, particularly: 1‐octadecanamide (OA), (R)‐12‐hydroxyoctadecanamide (HOA), (R)‐N‐propyl‐12‐hydroxyoctadecanamide (PHOA), and OHOA (Fig. 12.1). To establish a more comprehensive evaluation of the tentative relationship between the molecular structure and the physical properties of the organogels, we included the SA and the 12‐HSA in the study. The amides were synthesized through a well‐established methodology, their characterization and relative purity was determined through elemental analysis, pNMR analysis, and the melting temperature (Mallia et al. 2009). The approach used was to introduce small and systematic changes in the functional groups of 12‐HSA. We aimed at understanding the effect of the chemical change in the gelator’s ability to self‐assemble and develop a three‐dimensional network with particular rheological properties. The rationale of the synthesis was the following. The carboxyl group in the SA and 12‐HSA develops just intermolecular interactions in head‐to‐head arrangements through cyclic carboxylic dimers. When the secondary 12‐hydroxyl group is present in the molecule, like in 12‐HSA and its corresponding primary and secondary amide derivatives, the molecular polarity increases and allows the occurrence of molecular self‐assembly along the secondary axis through hydrogen bonds (Abraham et al. 2012a,

367

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Crystallization of Lipids

2012b). The introduction of an amide group into the SA and 12‐HSA molecules result in the formation of the corresponding primary amides without and with the secondary 12‐hydroxyl group (Fig.  12.1). Given the large dipole moment of the primary amide group, this chemical modification results in an increase in the polarity of the molecules and in stronger attractive intermolecular forces than in their parent molecules. The primary amide group is able to develop hydrogen bonds on both the oxygen and nitrogen atoms of the amide group. Finally, the secondary amides have lower capacity to develop hydrogen bonds than the primary amide. However, the propyl and the octadecyl chains present in the PHOA and OHOA (Fig. 12.1), increase the involvement of the London dispersion forces in the intermolecular interactions associated with the molecular self‐assembly. The main conclusions of this study were the following: 1) Independent of the type of LMOG, a higher cooling rate results in organogels structured by smaller crystals. This behavior has been observed in many crystallizing systems, including organogels (Rogers & Marangoni 2008; Morales‐Rueda et  al. 2009; Pal et al. 2013). For a given LMOG in solution at a given concentration, the molecular self‐assembly and subsequent gelation depends mainly on the gel setting temperature, Tset, where Tset must be lower than the saturation temperature of the gelator in the solvent (i.e., TC). The difference between the Tm of the gelator in the solvent and Tset establishes the supercooling (i.e., ΔT = Tm ‐ Tset), the thermodynamic driving force for microphase separation. Thus, it is apparent that during cooling of the LMOG solutions to achieve Tset, in this study 25 ° C (Toro‐Vazquez et al. 2013a), the supercooling increased faster as the cooling rate increased. Under this condition the nucleation rates are accelerated at the expense of growth rates, resulting in organogels structured by smaller crystals but in higher number than the organogels obtained at low cooling rate (i.e., 1 ° C/min). Additionally, at low cooling rates, the aggregating molecules have sufficient time to diffuse and to add selectively to an already nucleated crystallite surface. This favors crystal growth rate over the nucleation rate (Lam et al. 2010). 2) The comparisons between the organogels developed by gelators with and without the 12‐hydroxyl group showed that the presence of this polar group increases the solid content in the organogels, particularly in those organogels developed at 20 ° C/min (Fig.  12.7). Rogers and Marangoni (2008) showed that in organogels developed by 12‐HSA in canola oil, the constant rates of nucleation and crystal growth are smaller at cooling rates lower than 5 ° C/min than at higher cooling rates. This behavior seems to apply also to the amide derivatives of 12‐HSA, and would explain the lower mass of solids formed at 1 ° C/min by the LMOG in comparison with the higher mass of crystals present in the organogels developed at 20 ° C/min. 3) The presence of a chiral center along the molecule capable of establishing short‐ range, noncovalent interactions (i.e., the secondary 12‐hydroxyl group), seems essential for the development of stronger thixotropic organogels. The SA and OA, the only gelators without a secondary 12‐hydroxyl group, crystallize as microplatelets following a similar lamellar organization as hydrocarbons stabilized mainly through London forces throughout the hydrocarbon chain. The corresponding organogels are weak (Fig. 12.7) and after applying a small stress (i.e., 17.5 Pa) suffer permanent deformation with no recovery (Toro‐Vazquez et al. 2013a). In contrast,

Physical Properties of Organogels Developed with Selected Low-Molecular-Weight Gelators

Storage modulus, G′ (Pa)

(a)

5E5 2.5E5

OHOA

0.84% SOLIDS, σ = 223.48 Pa

75000 50000

PHOA

0.98% SOLIDS, σ = 200.79 Pa

HOA

1.21% SOLIDS, σ = 172.04 Pa

25000 7500 5000

1.49% SOLIDS, σ = 65.06 Pa

12-HSA

2500 750 500

OA

0.45% SOLIDS, σ = 1.29 Pa

SA

0.62% SOLIDS, σ = 1.04 Pa

250 0

10

20

30

40

50

60

70

Time (min) under isothermal conditions (25° C)

Storage Modulus, G′ (Pa)

(b)

5E5 2.5E5

OHOA

1.87% SOLIDS, σ = 182.20 Pa

75000 50000

HOA

1.40% SOLIDS, σ = 174.90 Pa

25000

PHOA

1.50% SOLIDS, σ = 88.10 Pa

12-HSA

1.71% SOLIDS, σ = 49.73 Pa

OA

0.60% SOLIDS, σ = 18.08 Pa

7500 5000 2500 750 500 250 0

10

20

30

40

50

60

70

Time (min) under isothermal conditions (25° C)

Fig. 12.7  Elasticity (G’) of organogels formed by the LMOG show in Fig. 12.1. The organogels (2%) were developed at 25 ° C in safflower oil high in triolein using a cooling rate of 1 ° C/min (a) and 20 ° C (b). After 15 minutes at 25 ° C the G’ was measured under isothermal conditions during 60 minutes (unpublished results). For each organogel the yield stress (σ) and the percentage of solid it is indicated. From Toro‐Vazquez et al. (2013a), published with permission of the American Chemical Society.

the presence of the secondary 12‐hydroxyl group in 12‐HSA and HOA, increases the molecular polarity and the capability of establishing hydrogen bonds along the secondary axis. Thus, in comparison with SA and OA the 12‐HSA and HOA molecules crystallize as fibers (Fig.  12.8), developing organogels with higher elasticity, yield stress (σ), and thixotropic behavior (Fig. 12.8; Toro‐Vazquez et al., 2013a). This conclusion applies to (R)‐12‐HSA, the enantiopure form because racemic 12‐HSA crystallizes as microplatelets (Rogers & Marangoni 2008; Abraham et  al. 2012a).

369

370

Crystallization of Lipids

(a)

(b)

200 μm

200 μm

(c)

(d)

200 μm

(e)

200 μm

(f)

200 μm

200 μm

Fig. 12.8  Polarized light microphotographs of organogels formed by 2% HOA (a, b), 2% PHOA (c, d), and 2% OHOA (e, f ) at 25 ° C using cooling rates of 1 ° C/min (a, c, e) and 20 ° C/min (b, d, f ). From Toro‐Vazquez et al. (2013a), published with permission of the American Chemical Society.

Evidently, as previously stated by Abraham et al. (2012b), the homochiral interactions among OH groups of chiral n‐HSA (i.e., [R]-12-HSA) lead to an extended secondary crystal growth resulting in fibers. In contrast, in racemic n‐HSA (where n represent the position of the OH group in the molecule) the orientation of the OH groups and the hydrogen‐bonded acyclic carboxylic dimers prevent longitudinal growth with the subsequent formation of microplatelets. As additional information, results obtained modifying the position of the keto group in ketooctadecanoic acid (n‐KSA; where n represent the position of the keto group in the molecule), showed

Physical Properties of Organogels Developed with Selected Low-Molecular-Weight Gelators

that the mechanical strength of the gels is enhanced when the functional group is far from the carboxylic head group (Pal et al. 2013). The same effect applies also for the position of the secondary hydroxyl group in n‐HSA (Abraham et al., 2012a). However, it is important to note that the n‐KSA has no chiral center in the molecule and that the study with n‐HSA (Abraham et al., 2012a) did not use the enantipure form but racemic mixtures. 4) The addition to 12‐HSA of a primary or a secondary amide, this last one bonded to an alkyl group, results in gelator molecules (HOA, PHOA, and OHOA, Fig.  12.1) that, independent of the cooling rate, crystallize as fibrillar spherulites (Fig. 12.8). Thus, HOA, PHOA, and OHOA developed organogels with cooling rate‐dependent rheological behavior (Fig.  12.7) and remarkable thixotropic properties (Toro‐ Vazquez et al. 2013a). Within this context, independent of the cooling rate the corresponding organogels have higher elasticity than those obtained by the parent molecule (Fig. 12.7). However, the rheological behavior of PHOA and OHOA at the two cooling rates investigated, shows that microstructure plays a more important role in determining the viscoelastic behavior of the organogels than their solid content (see Section 12.3.1.2). Thus, the microphotographs of the HOA gels formed at 20 ° C/min showed vicinal spherulites with higher fiber interpenetration than the one occurring between the spherulites present in the gels formed at 1 ° C/min (Fig. 12.8; Toro‐Vazquez et al. 2013a). Because spherulite interpenetration increases the presence of transient junction zones between the microfibers, this resulted in larger elasticity of the HOA organogels developed at 20 ° C/min (Fig.  12.7). The opposite results occurred in the organogels formed by the secondary amides, PHOA and OHOA, where the organogels formed at 20 ° C/min showed a microstructure formed by mutually exclusive spherulites with lower fiber interpenetration than the one occurring between the spherulites present in the gels developed at 1 ° C/min. For the PHOA and OHOA organogels, despite the lower solid content of the organogels formed at 1 ° C/min, these gels had higher G’ and σ than the organogels developed at 20 ° C/min (Fig. 12.7). 5) The TC depends mainly on the polarity of the functional groups of the LMOG. Thus, independent of the cooling rate, the relative polarity of the gelators investigated followed the same order than the TC of the corresponding LMOG. Subsequently, the primary and secondary amides with the secondary –OH group had the highest relative polarity, and therefore, the higher TC. The OA (the primary amide without the secondary –OH group) had a lower TC, and finally the 12‐HSA and SA the lowest TC. The entropy for nucleation, a value associated with the loss of entropy for the system and calculated from the thermal parameters obtained by DSC, was the lowest for the 12‐HSA and SA and the highest for the primary and secondary amides, particularly for OHOA the secondary amide with the largest alkyl group (Fig. 12.9). These results indicate that the ability of the molecules to arrange in such a fashion to minimize the exposition of the polar groups from the contact with the apolar solvent, is essential for molecular self‐assembly, and therefore, in the formation of a stable nucleus. This process may be limited as the molecular weight of the gelator increases, and therefore the self‐assembly of OHOA would require of higher energy for nucleation, and overall to develop the organogel. Recent results obtained by De la Peña‐Gil et  al. (2017), indicated that in contrast with 12‐HSA and HOA, the application of shearing before achieving TC (i.e., pre‐shearing) overcomes the requirement of higher

371

Crystallization of Lipids –1.5 –2.0 Entrophy for nucleation, J/mol* K

372

Mean

–2.5

Mean ± SD

–3.0 –3.5 –4.0 –4.5 –5.0 –5.5 –6.0 –6.5

SA

12-HSA

OA

HOA Gelator

PHOA

OHOA

Fig. 12.9  Molar entropy for nucleation of different LMOG at 2% in safflower oil high in triolein. The values were calculated independent of the cooling rate (1 ° C/min or 20 ° C/min) used to develop the organogels (the molar entropy was calculated form thermal parameters reported by Toro‐Vazquez et al. 2013a).

energy of nucleation of OHOA. Additionally, the application of pre‐shearing also resulted in the development of OHOA organogels with higher elastic properties than those formed with PHOA and HOA (results not shown). 6) The intermolecular forces involved in the molecular self‐assembly were closely assessed through the ΔHC, Tm, and ΔHm values (Table 12.3). Overall, these thermal parameters are higher in the organogels developed using the lower cooling rate. As previously stated, under these conditions, supercooling changes at a lower rate than at 20 ° C/min, and thus, gelator molecules have enough time and greater selectivity to develop self‐assembled structures than at the high cooling rate. Thus, the fibrillar microstructure of 12‐HSA results from the carboxylic acid dimer motifs and hydrogen bonds developed by the carboxylic functional group and the secondary –OH, respectively. For the HOA, the microstructures are stabilized by the strong hydrogen bonds developed by the primary amide group on both the oxygen and nitrogen atoms, and therefore this organogel showed higher ΔHC, Tm, and ΔHm values than the 12‐HSA organogels (Table  12.3). The secondary amides (PHOA and OHOA) have lower capacity to develop hydrogen bonds than the primary amide, but the propyl and the octadecyl chains present in the PHOA and OHOA (Fig. 12.1), increase the involvement of the London dispersion forces in the intermolecular interactions associated with the molecular self‐assembly (Toro‐Vazquez et al. 2013a). From here the higher ΔHC and ΔHm values observed in the PHOA and OHOA organogels in comparison with those obtained with the HOA organogels. This behavior was particularly evident for the OHOA, the secondary amide with the longer alkyl chain (i.e., octadecyl; Table 12.3).

Physical Properties of Organogels Developed with Selected Low-Molecular-Weight Gelators

Table 12.3  Thermal parameters (crystallization and melting) for 2% organogels developed at two cooling rates with different LMOG in safflower oil high in triolein. The ΔHC and ΔHm were normalized for the solid content in the organogels and are reported as kJ/mol. TCa (° C)

ΔHCa (kJ/molsolids)

Tma (° C)

ΔHma (kJ/molsolids)

1 ° C/min 20 ° C/min 1 ° C/min 20 ° C/min 1 ° C/min 20 ° C/min 1 ° C/min 20 ° C/min

12‐HSA

51.65 (1.24)

48.18 (0.43)

54.1 (7.5)

50.1 (2.9)

58.43 (2.06)

57.13 (0.02)

44.5 (2.2)

54.8 (3.0)

HOA

93.06 (1.44)

92.16 (2.12)

223.0 (38.3)

115.5 (11.8)

102.18 (2.12)

98.75 (1.00)

97.7 (1.2)

93.8 (6.4)

PHOA

77.44 (0.93)

74.85 (0.65)

139.9 (0.6)

87.8 (6.9)

91.56 (1.38)

91.22 (0.66)

123.3 (6.3)

86.2 (4.1)

OHOA

80.54 (0.67)

76.42 (0.91)

251.9 (21.1)

112.3 (4.8)

87.41 (1.40)

88.69 (1.20)

236.4 (42.0)

116.8 (0.5)

a  Mean and standard deviation of at least two independent determinations. From Toro‐Vazquez et al. 2013; published with permission of the American Chemical Society.

12.4 ­Organogels of Candelilla Wax The composition, physical and functional properties of organogels formed by CLW in vegetable oil have been extensively investigated by our group (Morales‐Rueda et  al. 2009; Toro‐Vazquez et al. 2007, 2010, 2013a, 2013b; Chopin‐Doroteo et al. 2011) and other research groups (Hong‐Sik et al. 2012; Barbosa Rocha et al. 2013; Hwang et al. 2013; Blake et al. 2014; Doan et al. 2014; Patel et al. 2015). The overall conclusions of these studies show that at 5 ° C, CLW develops organogels at concentrations lower than 2%. The CLW organogels have remarkably rheological (Toro‐Vazquez et al. 2007, 2013b; Patel et al., 2015) and oil‐binding (Blake et al, 2014) properties with long term stability to phase separation (i.e., no phase separation is observed even after 12 months of storage at 5° or 25 ° C; Toro‐Vazquez et al. 2007). All these studies have been done under static conditions, where supercooling is the main thermodynamic driving force for nucleation, crystal growth, and subsequent gelation. We had shown that shearing rate, the extent of its application as cooling proceeds, and cooling rate can be used as engineering variables to tailor the physical properties of CLW’s organogels (Chopin‐Doroteo et al. 2011; Alvarez‐Mitre et al. 2012, 2013). Through the use of these processing variables we might develop CLW organogels with the physical and functional properties required in different food products (e.g., bakery, shortenings, margarines, coatings). Here, we discuss some results obtained in this direction. 12.4.1  Rheological Properties of Candelilla Wax Organogels Developed Applying Shear Rate Our results of CLW organogels developed under shear (Chopin‐Doroteo et al. 2011), and similar results reported for monoglycerides (Ojijo et  al. 2004; Da Pieve et  al. 2010), 12‐HSA organogels (Co & Marangoni 2013) in vegetable oil, and for 2,

373

Crystallization of Lipids

3‐di‐n‐decyloxyanthracene in N, N‐dimethylformamide (Lescanne et al. 2002), suggested that the application of shearing during cooling of CLW solutions, applying the shear just under metastable conditions (i.e., above the TC of CLW), and then allowing the organogel to form under static conditions, might induce the alignment of gelator molecules present in the CLW. This indicates that under subsequent cooling the CLW crystallize developing a crystal network with transient and permanent junction zones throughout the microstructure of the organogels. Tentatively, this would result in CLW organogels with enhanced rheological properties compared with organogels developed under quiescent conditions or with the application of continuous shear (Chopin‐Doroteo et al. 2011). Within this context, we developed 3% CLW organogels in safflower oil high in triolein under static conditions (0 s−1) or applying different shear rates (30 to 600 s−1) continuously during cooling (6 ° C/min) until achieving 5 ° C. In an alternative set of conditions, the same shear rates were applied just until achieving 52 ° C (i.e., pre‐shearing), and then continuing cooling under quiescent conditions until achieving 5 ° C. Based on the results of Chopin‐Doroteo et al. (2011), for the 3% CLW in the vegetable oil solutions, metastable conditions prevail from 52 ° C until the TC (39.0° ± 0.08 ° C) of the CLW in the vegetable oil. Therefore, the pre‐shearing was applied before achieving nucleation (Alvarez‐Mitre et al. 2012). The elasticity of the 3% CLW organogels obtained under static conditions (0 s−1), constant shearing and applying pre‐shearing at different shear rates is shown in Fig. 12.10. It was evident that, independent of the shear rate applied, applying preshearing conditions resulted in organogels with higher G’ values than the gels developed statically or using constant shearing. The maximum value of G’ was obtained with the gels developed at 300 s−1 using preshearing conditions (Fig. 12.10). The PLM of the organogels 5.75 5.25 4.75 Log G′ (Pa)

374

2.75 2.25 1.75

Mean Mean ± SD

1.25 0 60

PS

00

3 PS

0 18

PS

60

PS

30 PS

0

0

60

0

30

0 18

60

30

Shear rate (s–1)

Fig. 12.10  Logarithm of the elastic modulus (G’) of organogels developed under constant shearing or pre‐shearing (PS) at the shear rates indicated. The G’ was measured after 60 minutes at 5 ° C. From Alvarez‐Mitre et al. (2012), published with permission of Elsevier.

Physical Properties of Organogels Developed with Selected Low-Molecular-Weight Gelators

(a)

(b)

50 µm

(c)

50 µm

(d)

50 µm

50 µm

Fig. 12.11  Polarized light microphotographs of 3% CLW organogels developed in safflower oil high in triolein under static conditions (a) and pre‐shearing at 180 s−1 (b), 300 s−1 (c), and 600 s−1 (d). The microphotographs were taken after 60 minutes at 5 ° C. From Alvarez‐Mitre et al. (2012), published with permission of Elsevier.

developed using pre‐shearing showed the characteristic microplatelets shape (i.e., fiber‐like or plate‐like) developed by CLW. However, these microplatelets were larger with an evident meshing organization (Fig. 12.11) in comparison with the microstructure observed in the organogels formed statically or with constant shearing (Fig. 12.12). These observations were more evident in the organogels formed with PS at 300 s−1 (Fig. 12.11 insert c), the system that revealed the higher elasticity (Fig. 12.10). We can conclude that the use of shearing at temperatures above TC but close to the metaestable conditions (i.e., 52 ° C), induces molecular alignment of the main components of CLW (i.e., n‐alkanes) developing mesophase structures before achieving nucleation (Alvarez‐ Mitre et al. 2012). On further cooling under static conditions, the mesophase structures crystallize, grow, and develop a three‐dimensional organization with higher extent of microplatelet‐microplatelet interaction, thus developing organogels with higher elasticity than those obtained under static conditions or constant shearing (Alvarez‐Mitre et al. 2012). As an additional note, the rheological behavior of the CLW organogels developed using preshearing conditions (Fig.  12.10) could not be explained by the additional

375

376

Crystallization of Lipids

(a)

(b)

50 µm

50 µm

(d)

(c)

50 µm

50 µm

Fig. 12.12  Polarized light microphotographs of 3% candelilla wax (CLW) organogels (5 ° C) developed in safflower oil high in triolein under static conditions (a) and applying constant shearing of 180 s−1 (b), 300 s−1 (c), and 600 s−1 (d). The microphotographs were taken after 60 minutes at 5 ° C. From Alvarez‐Mitre et al. (2012), published with permission of Elsevier.

formation of crystals (i.e., higher solid content) because of shearing. This is because, the mass of crystals in the 3% CLW organogels (measured by pNMR) was independent of the shearing rate applied or the way it was applied (i.e., continuously [2.59% ± 0.01% of solids] or pre‐shearing [2.52% ± 0.04% of solids]), showing just a slight higher value at 300 s−1 (2.77% ± 0.03% of solids; P 40–90 μm)

Crystal microstructure

10, 11

14

14

OPO β β polymorph Large crystal Solid fat content particle (~20 μm) β’ polymorph (SFC) α polymorph 12 13 8 8

9

Crystal properties

6

4 OOO Unsaturation Agitation SPO degree Saturation degree 2 (sn-1, 3) C18:0, C16:0, fatty-acid chain length

(sn-1, 3) C18:1, C18:2, PUFA, unsaturation index

Intrinsic factors

8

1,3 1,2

Time Temperature fluctuation

1

Fast cooling

2

Slow cooling

Extrinsic factors

Fig. 16.5  Factors influencing the macroscopic qualities of porcine fat. Arrows indicate increase or promote, and T‐bars indicate decrease or suppress. Arrows and T‐bars do not necessarily represent direct relationship. Source: 1Kalnin et al. 2005, 2Campos et al. 2002, 3Svenstrup et al. 2005, 4Davenel et al. 1999, 5 Bothma et al. 2014, 6Gandemer et al. 2002, 7Segura et al. 2015, 8Wang & Lin, 1995, 9Sasaki et al. 2015, 10de Man et al. 1991, 11Rousseau et al. 1998, 12Meng et al. 2013, 13Motoyama et al. 2013, 14Motoyama et al. 2016.

Lipid Crystals and Microstructures in Animal Meat Tissues

The effects of fatty‐acid positional distribution within TAG on the physical properties including firmness of adipose tissue were studied by Segura et al. (2015). The results of regression analysis of the firmness of subcutaneous adipose tissue of dry‐cured ham versus fatty‐acid compositions at each sn‐position are shown in Table 16.2. The increase in C18:0, saturated fatty acid, and average chain length in sn‐1 and ‐3 position increased the firmness, and the increase in C18:1, C18:2, polyunsaturated fatty acid, and unsaturation index in this position decrease the firmness. Agitation during the crystallization has the effect on the firmness of crystal fractions of porcine fats (Wang & Lin 1995). Higher agitation speed decreases the firmness and also the size of fat crystal particles. These changes were also related to the increase in unsaturated fatty acid and to their TAG compositions. In addition to firmness, stickiness, appearance and organoleptic quality have been investigated and the factors influencing them were reported (de Man et  al. 1991; Rousseau et al. 1998; Sasaki et al. 2015; Segura et al. 2015). In contrast to firmness, stickiness of porcine adipose tissue is related to fatty acids at the sn‐2 position, suggesting the effects of the fatty‐acids at the sn‐position on the fat physical properties (Segura et  al. 2015). Increase in C16:0, saturated fatty acids, and fatty‐acid chain length at sn‐2 position increase the stickiness, and C18:1, monounsaturated fatty acids, and unsaturation index decrease it (Table 16.2). Visual test showed that porcine fats becomes whiter when it is slowly cooled to 3° C compared to rapidly cooled samples (Sasaki et al. 2015). This result can be understood by taking into account of the quality‐factor relationship shown in Fig. 16.5 as follows: Rapid cooling decreases the size of fat crystal particles and increases SFC values. These changes probably modify the reflection of light at the surface and beneath the surface of the lard samples. Polymorphic forms of the fat crystals, which are changed by crystallization condition, may also have some effects on the appearance. Organoleptic quality of porcine fats is affected by the type of polymorphic forms of the fat crystals. The β crystals result in a dull and brittle product with a grainy texture, whereas β’ crystals give smooth and a better mouth feel (de Man et al. 1991; Rousseau et al. 1998; Fig. 16.5). The melting point and microstructures are different between the two polymorphs and cause the difference in mouth feel. Macroscopic quality of fat systems is formed directly by the crystal microstructures. However, it has not been fully understood the relationship between the macroscopic quality and the microstructures. Relationship among the microstructures and the factors influencing microstructures are also not well investigated. Looking at even a single fat system, a porcine fat, arrows showing the relationship are too simple to explain the scheme governing the macroscopic quality of the fat system (Fig. 16.5). New techniques, which can analyze the crystal microstructures with the information of crystal polymorph and crystallinity, compensate for the deficiency in the figure, as will be described in Section 16.4. 16.3.3  Application to Actual Meat and Meat Products Because the crystalline state of fats decides the macroscopic quality of meat fats, evaluation of the crystalline state at production sites is quite useful for quality management and authentication of meat products. Also from a view point of the limitation in reproducibility of the field condition within laboratory, monitoring the crystalline state under the real condition is ideal.

455

Table 16.2  Simple and multiple linear regression analysis of the firmness, and stickiness values versus fatty acid concentration (g/100 g) in sn‐2 or sn‐1,3 positions of the triglyceride. Dependent variable

R2

Firmness

0.62***

(N) 0.53**

SE

7.80

0.50***

0.37

C18:0, sn‐1,3 C18:2n‐6, sn‐1,3

13.28

t value

P value

8.51

1.09

0.2896

1.28

3.50

0.0026

−1.14

−2.35

0.0303

Constant

66.04

4.97

0.0001

−0.58

−2.49

0.0230

0.49

C18:2n − 6, sn‐1,3

−1.75

−3.53

0.0024

8.85

Constant

12.41

1.40

0.1778

0.21

SFA, sn‐1,3

0.52

PUFA, sn‐1,3

13.15 0.48

0.56

2.67

0.0156

−1.34

−2.58

0.0190

Constant

68.64

5.22

0.0001

MUFA, sn‐1,3

−0.56

−2.67

0.0155

PUFA, sn‐1,3

−1.90

−3.95

0.0009

Constant

76.31

6.44

0.0001

0.50***

11.84 16.18

UI, sn‐1,3

−73.25

−4.53

0.0002

0.20*

15.72

Constant

−11.38

−0.72

0.4781

0.54***

13.66

1.88 (10−2) (N · s)

0.35*

ACL, sn‐1,3 Constant

4.12

2.19

0.0415

−83.53

−6.12

0.0001

0.18

C16:0, sn‐2

0.92

5.16

0.0001

0.84

C18:0, sn‐2

2.88

3.42

0.0031

0.79

C18:2n − 6, sn‐2

1.49

1.88

0.7600

7.65

Constant

−5.83

−0.76

0.4548

0.27

C18:1, sn‐2

−1.01

−3.74

0.0014

1.03

C18:2n − 6, sn‐2

2.30

2.23

0.0382

0.36

C18:0, sn‐1,3 Constant

0.79

2.21

0.0393

−41.53

−4.01

0.0006

0.20*

10.36 0.18

SFA, sn‐2

0.45

2.50

0.2060

0.23*

7.79

Constant

3.81

0.49

0.6300

0.22

MUFA, sn‐2

−0.54

−2.52

0.0199

0.20*

6.64

Constant

0.20

0.03

0.9759

11.94

UI, sn‐2

−28.86

−2.42

0.0248

19.96

Constant

−58.78

−2.94

0.0077

1.67

ACL, sn‐2

3.62

2.16

0.0423

0.18* 2

Regression coefficient

C18:1, sn‐1,3

0.21

Stickiness

Constant

0.48 0.23 0.56***

Independent variable

P‐value for R was determined using F‐test. Reproduced from Segura et al. 2015. * P 98% pure) gels in ethylene glycol. Multidomain network with mutually exclusive spherulites (a, d), single‐fiber network with interpenetrated spherulites (b, e) or interconnecting fibrils (c, f ). From Yuan et al. 2011b; published with permission of The Royal Society of Chemistry.

(a)

(b)

(c)

Fig. 13.3  Photographs of (a) porous cryogel, (b) oil sorption by porous cryogel, and (c) oleogel prepared by shearing the oil‐sorbed cryogel.

0 mM

100 mM

200 mM

5 mm Fig. 13.6  Photographs of WPI hydrogels (top) and corresponding oleogels (bottom) prepared by solvent exchange. The hydrogels were prepared using 15 wt.% WPI with 0–200 mM NaCl. Reprinted with permission from de Vries et al. (2015). Copyright (2015) American Chemical Society.

Volume 7 Sponginess

6

Cell size

5 Oil Oleo Short

4

Crumbliness

Moistness

Stickiness

Fig. 13.10  Appearance and sensorial evaluation of cakes prepared using oil, oleogel, and commercial shortening.

Beef

(a)

Chicken

Pork

Pork

(a) 100 nm

(b)

(b)

>26%

0 w≤0

Fig. 16.7  Nondestructive detection of pork fat. (a) Optical images of the samples. (b) Raman spectrometric index corresponds to the ratio of β’‐crystal polymorph to the total crystal amount of the fat. (c) If the Raman spectrometric index is greater than threshold (discriminant w > 0), it determines as porcine fat (Motoyama et al. 2015).

Refrigeration duration: 0 hours

4 hours

10 days

2 months

(a) Optical image 20 μm

(b) Crystallinity: Ctrans

100% 28%

Merged image

(c) β′ crystal: αβ’

26% 10%

Merged image

(d) β crystal:

MCR-ALS resolved component

87% 0%

Merged image

Fig. 16.8  Simultaneous Raman microspectroscopic imaging of fat crystalline state of porcine adipose tissues refrigerated at 4 ° C for different durations. Scales are ranging from the minimum to the maximum values of the obtained result. (a) Optical microscopic images, (b) Raman index Ctrans corresponding to the fat crystallinity, (c) Raman index αβ’ corresponding to the amount of β’ crystal polymorph, (d) concentration profile of an MCR‐ALS‐resolved component corresponding to β crystal polymorph. Reproduced with permission from Motoyama et al. (2016) Food Chemistry. 196, 411–17. Copyright 2015 Elsevier Science Ltd.