Polymer Macro- and Micro-Gel Beads:Fundamentals and Applications

Amos Nussinovitch

Polymer Macro- andMicro-Gel Beads:Fundamentals andApplications

123

Amos NussinovitchInstitute of Biochemistry,

Food Science and Human NutritionThe Robert H. Smith Faculty of

Agriculture, Food and EnvironmentThe Hebrew University of JerusalemRehovotIsraelnussi@agri.huji.ac.il

ISBN 978-1-4419-6617-9 e-ISBN 978-1-4419-6618-6DOI 10.1007/978-1-4419-6618-6Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2010934122

© Springer Science+Business Media, LLC 2010All rights reserved. This work may not be translated or copied in whole or in part without the writtenpermission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use inconnection with any form of information storage and retrieval, electronic adaptation, computer software,or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they arenot identified as such, is not to be taken as an expression of opinion as to whether or not they are subjectto proprietary rights.

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Preface

Beads made from Egyptian faience have been excavated from grave deposits(c. 4000–3100 BC), together with beads of glazed steatite (a soft rock) and of semi-precious stones such as turquoise, carnelian, quartz, and lapis lazuli. Informationon these and many more ancient beads used for ornaments and jewelry, ritualceremonies, as art artifacts and gifts for amorous women throughout history, anddescriptions of the raw materials (e.g., glass, bone, precious and other stones)and manufacturing technologies used for their production can be located in manyreferences. Many books are devoted to the description of beads that are not ofwater-soluble polymer origin, techniques for their production, their art, value, anddistribution, reflecting the wealth of information existing in this field of science andart. On the other hand, there are no books fully devoted to the fascinating topic ofhydrocolloid (polymeric) beads and their unique applications. A few books con-tain scattered chapters and details on such topics, while emphasizing the possibilityof locating fragments of information elsewhere; however, again, there is no bookthat is solely devoted to hydrocolloid beads and their versatile applications. In themeantime, the use of water-soluble hydrocolloid beads is on the rise in many fields,making a book that covers both past and novel applications of such beads, as wellas their properties and ways in which to manipulate them, crucial. The aim in writ-ing this volume was to present, in an easy-to-follow sequence, a description of beadproduction methods and of techniques which can be used to estimate, and modify,their physical and chemical properties. This book offers a full description of not onlytraditional and recent developments and applications of beads in the fields of agri-culture, biotechnology, environmental studies, medicine, and food, but also topicswhich have never been covered in the literature, making it of the utmost importanceto industry and academia.

Chapter 1: Physical Properties of Beads and Their Estimation

In Chapter 1, the criteria used to describe the shape and size of beads are explained.In particular, sections on roundness, sphericity, measurement of axial dimensions,and resemblance to geometric bodies are included. A special section is devoted to

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the methods used to estimate average projected area, volume, and density, includ-ing specific gravity balance and pycnometric methods. Other sections are devotedto bead surface area and specific surface in porous media, e.g., dried beads. Alsocovered are image processing and its utilization for hydrocolloid beads. Finally,the chapter discusses the structure of hydrocolloid beads, their density, and theirporosity.

Chapter 2: Bead Formation, Strengthening, and Modification

This chapter begins with a brief overview of the typical polymeric materials used forbead creation and their limitations. A full description is then provided of proceduresto construct different bead forms, e.g., from cylindrical to almost perfectly spheri-cal, by changing both the molds and the media into which the molten or dissolvedhydrocolloid preparation is dropped or transferred. Also, some information on drop-ping methods, changing drop size and distribution, and liquid sprays is provided,affording a measure of control over bead size and distribution. The various water-soluble polymers that can be used for bead formation are discussed at length. Theproperties of gel beads prepared from agar/agarose κ-carrageenan, alginate, cellu-loses, chitosan, and to a lesser extent polyacrylamide and other synthetic polymers,among many others, are described. The use of crosslinking agents for both creationand strengthening of several bead types is thoroughly covered. Special methods tomodify the porosity of the formed beads are also described, as are methods of slowdissolution of crystals by acid to facilitate better growth of embedded cells via pHregulation. A special section is devoted to beads prepared from proteins, ways toincrease their stability (with, for example, glutaraldehyde), and their influence onthe cells embedded within them. Since a combination of alternative methods maywell provide a good means of overcoming the evident shortcomings of current bead-formation techniques, at the end of this chapter, a few approaches are presented,such as adding epoxy-resin reagent and curing agent to alginate for matrix stabi-lization, and other less known approaches for bead stabilization, as well as lesstraditional ways of producing and modifying beads.

Chapter 3: Methods and Mathematical Models for the Drying ofPolymeric Beads

Water-soluble polymer beads can be dried for a variety of purposes (described in fullin Chapter 6). In general, after drying, the texture is porous. In many cases, the beadis capable of retaining its integrity even after immersion in water for long periods.In addition, and in contrast to wet gel beads, porosity facilitates the liberation ofgases during fermentation without harming the dried bead’s integrity. This chaptercovers methods for drying polymeric beads, including air-drying, fluidized-bed and

Preface vii

microwave-assisted fluidized-bed drying, and freeze-drying, and freeze-dried bio-logical products are fully described. Sections also include drying of dosage formsmade of drug dispersed in a polymer, mathematical and numerical models to ana-lyze the drying, and a discussion of special cases such as drying a polymer beadwith shrinkage.

Chapter 4: Food and Biotechnological Applicationsfor Polymeric Beads and Carriers

The immobilization of microorganisms or cell suspensions in beads for a variety ofbiotechnological and food purposes is described—information which is hard to findin currently available books. Examples include amino acid (e.g., L-aspartic acid,L-alanine, and L-phenylalanine) production, organic acid (e.g., citric acid, malicacid, gluconic acid, lactic acid) fermentation and conversion, special uses in ethanol,wine, vinegar, and sake production such as malolactic fermentation, removal of ureafrom sake and wine by immobilized acid urease, beer brewing using an immobi-lized yeast bioreactor system, and uses in soy sauce production. Other uses relatedto miscellaneous flavor materials and aroma compounds are also discussed. Theseinclude, but are not limited to, biotransformation from geraniol to nerol, productionof limonin, β-ionone, naringin, blue cheese flavor, vanillin, and Japanese season-ing. Special beads that serve for immobilization and are used in the milk industry,e.g., for hydrolysis of lactose in milk, are also detailed. Miscellaneous applica-tions also include production of oligosaccharides, preservatives and bacteriocins,xylitol, carotenoids and leucrose, and cis,cis-muconic acid. Less known uses ofenzymes immobilized within beads for food applications are also described. Variousindustrial options such as fuel ethanol production, application of gels for sepa-ration matrices, bioartificial organs, and insect-cell immobilization are included.In general, the chapter attempts to touch upon all of the novel applications ofbead-immobilized cells for the food and biotechnology industries, such as the pro-duction of aroma compounds, the microbial production of bioflavors and theirbiotransformation.

Chapter 5: Medicinal Applications of Hydrocolloid Beads

This chapter gathers together information culled from many sources. It describesthe use of cells encapsulated in hydrogels, stem cells in bead environments, chargedhydrogel beads as new microcarriers for cell culture, as a potential support forendothelial cells, and for vaccine delivery. Other sections provide information oncrosslinked chitosan beads for different medicinal purposes: mucoadhesive beadsand their applications for eyes and the alimentary system and polyelectrolyte com-plexes. Additional sections describe novel approaches to cell encapsulation forimproved biocompatibility and immunoisolation. Emphasis is placed on methods

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using alginate–polylysine alginate for encapsulation, and a glimpse is providedof the art and science of artificial cells, encapsulated enzymes for the clinicallaboratory, and encapsulation of living cells and tissues for biomedical purposes.

Chapter 6: Dry Bead Formation, Structure, Properties,and Applications

The drying of hydrocolloid beads results in cellular moieties, and this chaptertherefore deals with cellular solids. A few manufacturing methods for hydrocol-loid cellular solids are described. They include, but are not limited to, dryingbicarbonate-containing gels after acid diffusion and cellular solids produced by fer-mentation and enzymatically. A special section deals with the inclusion of oils ingels and their influence on the properties of the resultant dried cellular solid. Severalmethods, e.g., compression studies, are described for evaluating the mechanicalproperties of the dried beads. The chapter also details the models used for describ-ing these beads’ stress–strain behavior. The structure and acoustic properties of suchcellular solids as a result of production method are also addressed. The applica-tions of dried beads have never been thoroughly reviewed. This chapter attempts toredress this by describing their use as carriers for vitamins, as study models, and forseparation and includes special dry beads for water treatment and matrices entrap-ping hydrocolloid cellular beads. Hydrocolloid cellular carriers for agricultural usesare also presented, e.g., the preservation of biocontrol agents in a viable form bydry cellular bead carriers and the carriers’ capacity to protect these agents againstUV radiation. The chapter ends with a discussion on the textural features of driedhydrocolloid beads.

Chapter 7: Liquid-Core Beads and Their Applications in Food,Biotechnology, and Other Fields

In 1971, Maddox’s patent on soft gelatin capsules was approved. In 1980, Lim andSun published their hallmark study in which microencapsulated islets were used asa bioartificial pancreas. In that manuscript, alginate–polylysine liquid-core capsuleswere produced and described. In the food area, Sneath’s patent (1975) and later ourgroup contributed to the manufacture and study of liquid-core hydrocolloid cap-sules. This chapter describes these liquid-core capsules, both natural and synthetic,and the procedures used to produce them. Methods for including oil within thesecapsules are also provided, along with an overview of their biotechnological andspecial food applications. Additional biotechnological applications of liquid-corecapsules include growth of microorganisms and activity of enzymes within them,and food applications include the manufacture of unique specialty foods and fruitproducts and the encapsulation of aroma and health compounds, among others. Thechapter also describes agricultural and environmental uses of liquid-core capsules

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and illustrates some special applications: aids to quitting smoking, in the beautyindustry for removal of body hair, and in the paper industry.

Chapter 8: Beads as Drug Carriers

Beads are often used as drug carriers in passive, as well as active drug targeting,making this a highly relevant topic in today’s research. Major general topics coveredin the first part of this chapter include controlled drug release, gels in drug-deliverysystems, dual drug-loaded beads, and drug release from the beads. Throughout,issues such as methods of drug incorporation, bead properties, extent and nature ofcrosslinking and the physicochemical properties of the drug, interactions betweenthe drug and the matrix material, concentration of the matrix material and releaseenvironment (e.g., the presence of enzymes) are discussed. Described beads includealbumin beads, alginate beads, alginate beads reinforced with chitosan, calciumalginate/PNIPAAM beads, different chitosan beads (e.g., chitosan–tripolyphosphatebeads, chitosan microspheres in treating rheumatoid arthritis, carboxymethyl chi-tosan beads), gelatin beads and those crosslinked with dextran, modified starchmicrospheres, dextran beads, gellan beads, guar beads, pectin beads for colon-specific drug delivery, pectin–chitosan beads, and modified poly(vinyl alcohol)microspheres. The chapter also summarizes information on achieving controlledproduction of the beads, such as preparation of biodegradable hydrogels based onpolyesters, hydrogels with degradable crosslinking agents and those crosslinkedwith small molecules, azo reagents or albumin, and hydrogels with biodegradablependant chains. The chapter finishes with a description of the more unique beads,such as those with floating ability and those made from xyloglucan.

Chapter 9: Beads and Special Applications of Polymersfor Agricultural Uses

The concept of bead encapsulation has become highly relevant to agriculture. Beadscan encapsulate microorganisms for use in the field of bacterial inoculation technol-ogy. Immobilized plant cell suspensions and single seed products have proven to beeasy to produce, store, and handle during industrial operation. This chapter describesthe goals of encapsulation in agriculture, e.g., to temporarily protect the encapsu-lated microorganisms from the soil environment and microbial competition and torelease them gradually for the colonization of plant roots. Special cases for enlarg-ing populations in which the entrapped bacterial biomass is low are described; othercases in which, for example, immobilized fungi are used as biocontrol agents againstsoil-borne pathogens are thoroughly detailed; survival of bead-entrapped popula-tions is compared with that of populations encapsulated in peat, and the influenceof special additives on bacterial survival is described. In addition, timing and meth-ods for the application of bacterial inoculants are delineated. In particular, topics

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such as carriers for the slow release of bacteria that affect plant growth, inoculationof seedlings and plants with beads containing fungal inoculum, joint immobiliza-tion of plant growth-promoting bacteria and green microalgae, cryopreservation byencapsulation/dehydration technique, and controlled release of agricultural chem-icals are discussed at length. The chapter also supports the reader with a list ofbiotechnological applications such as gene-delivery systems using beads, bioactivebead methods for obtaining transgenic plants and in synthetic seed technology anddescribes unique applications of polymers, including superabsorbent polymers andseed coating.

Chapter 10: Beads for Environmental Applications

This chapter focuses on the use of beads to immobilize microorganisms for pollu-tant biodegradation. Special emphasis is placed on chemically contaminated water,soil, and air. Water treatments are reviewed, and wastewater treatment by anaerobicfixed bed reactor or using immobilized microorganisms is discussed, along with themore specific examples of arsenic removal from water, chitosan and the removal ofheavy metal ions, and water denitrification. The chapter lists the advantages of usingencapsulated bacterial cells for soil applications, describes the preparation of suchbeads, and gives information on the protection of encapsulated cells from environ-mental stress. Another topic is the use of beads to protect against toxicity and therelated issues of soil treatments, agrochemicals, controlled release of pesticides intosoils, and sustained release of fungicide. Because these beads are introduced intosoils, a special section is devoted to release from these beads in the soil environment.In addition to discussing the advantages of beads for environmental applications, thechapter tries to account for the limitations of such technologies, such as the problemof substrate diffusion into immobilized preparations. Other covered issues includeair pollution and sampling and the determination of trace contaminants in air by con-centration on porous polymer beads. Finally, the chapter discusses miscellaneousapplications such as biodegradation and removal by microalgae.

In addition to covering the numerous types of hydrocolloid beads, this bookdescribes their many traditional and non-traditional uses, developed in many hydro-colloid R&D laboratories all over the world, including ours. The book addressesmany important industries and as such is designed to capture the interest of thosewho are looking for new applications in the fields of agriculture, food, environ-mental quality control, biotechnology, and medicine; it will be of interest to thepolysaccharide chemist, as well as to academic and other researchers and authoritiesin the fields of food, chemistry, medicine, and biotechnology.

My hope is that this book will assist all levels of readers. It is dedicated notonly to the academic community but also to the broader population of industrialistsand experimenters who will find this book to be not only a source of knowledgebut also a launching pad for novel ideas and inventions. In particular, this book

Preface xi

is expected to be of interest to personnel involved in food formulation, food sci-entists, food technologists, industrial chemists and engineers, pharmaceutical staffand medical doctors, and those who deal with drug delivery from beads. Potentialreaders also include both professional and dedicated non-professional environmen-talists, farmers, agriculturalists, and those working on the development of novelbeads and their applications. Finally, it is hoped that this book will find a prominentplace in the traditional university and research institute libraries where food science,chemistry, agriculture, environmental studies, and other theoretical and practicalindustrial topics are taught and studied.

Rehovot, Israel Amos Nussinovitch

Acknowledgments

This book was written over the course of 2 years. It contains a description of polymermacro- and micro-gel beads, their properties, and applications. It also includes theirmany traditional and non-traditional uses, developed in our and many other hydro-colloid laboratories worldwide. My hope is that this manuscript will assist readerswho are in search of comprehensive knowledge about the fascinating field of beads,as well as those seeking up-to-date information on the very different current andpast uses and applications of polymer beads in many areas. Comments and questionsfrom these readers will be very much appreciated.

I wish to thank the publishers for giving me the opportunity to write this book.Special thanks to David Parsons and Susan Safren for their efficient contribution tothe formation and processing of this manuscript. I wish to thank my editor, CamilleVainstein, for working shoulder to shoulder with me when time was getting short.Hanna Ben-Or’s help in locating and rectifying the many old and inaccurate refer-ences was above and beyond the call of duty. The permissions that we obtained fromdifferent publishers are warmly acknowledged. The many pictures adapted fromWikipedia are acknowledged in their turn, but I feel that it is equally appropriatehere to recognize the many who contributed to this gigantic educational achieve-ment. I particularly want to thank my family, Varda, Ya’ara, Eran, and Yoav, fortheir love, patience, and support during these last few difficult years, in which wewere under huge pressure from many different directions. Last, but not least, I wishto thank the Hebrew University of Jerusalem for being my home and refuge for thelast 20 years of very extensive research and teaching.

January 2010 Amos Nussinovitch

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Contents

1 Physical Properties of Beads and Their Estimation . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Bead Size and Shape . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2 Size of Drops and Beads . . . . . . . . . . . . . . . . . . 11.2.3 Bead Shape . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.4 Bead Volume and Surface Area . . . . . . . . . . . . . . 16

1.3 Bead Density, Porosity, and Structure . . . . . . . . . . . . . . . 18References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2 Bead Formation, Strengthening, and Modification . . . . . . . . . 272.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.2 Entrapment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.3 Single-Step Methods . . . . . . . . . . . . . . . . . . . . . . . . 28

2.3.1 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.3.2 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . 302.3.3 �-Carrageenan . . . . . . . . . . . . . . . . . . . . . . . 312.3.4 Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . 332.3.5 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . 362.3.6 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . 372.3.7 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 382.3.8 Synthetic Polymers . . . . . . . . . . . . . . . . . . . . 41

2.4 Two-Step Methods . . . . . . . . . . . . . . . . . . . . . . . . . 442.5 Cell Immobilization by Electrostatic Method . . . . . . . . . . . 45References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3 Methods and Mathematical Models for the Dryingof Polymeric Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.2 Methods for Drying Polymeric Gel Beads . . . . . . . . . . . . . 54

3.2.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.2.2 Air-Drying . . . . . . . . . . . . . . . . . . . . . . . . . 553.2.3 Fluidized-Bed and Microwave-Assisted

Fluidized-Bed Drying . . . . . . . . . . . . . . . . . . . 58

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3.2.4 Freeze-Drying . . . . . . . . . . . . . . . . . . . . . . . 613.2.5 Freeze-Dried Biological Products . . . . . . . . . . . . . 63

3.3 Drying of Dosage Forms Made of Drug Dispersed in a Polymer . 643.3.1 Mathematical Model . . . . . . . . . . . . . . . . . . . . 643.3.2 Numerical Model . . . . . . . . . . . . . . . . . . . . . 683.3.3 Drying a Polymer Bead with Shrinkage . . . . . . . . . . 70

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4 Food and Biotechnological Applications for PolymericBeads and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.2 Amino Acid Production . . . . . . . . . . . . . . . . . . . . . . 75

4.2.1 L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . 764.2.2 L-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . 774.2.3 L-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . 78

4.3 Organic Acid Fermentation and Conversion . . . . . . . . . . . . 794.3.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 794.3.2 Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . 804.3.3 Malic Acid . . . . . . . . . . . . . . . . . . . . . . . . . 814.3.4 Gluconic Acid . . . . . . . . . . . . . . . . . . . . . . . 814.3.5 Lactic Acid . . . . . . . . . . . . . . . . . . . . . . . . . 82

4.4 Ethanol, Wine, Vinegar, Sake, and the Like . . . . . . . . . . . . 854.4.1 Vinegar . . . . . . . . . . . . . . . . . . . . . . . . . . . 854.4.2 Soft Sake . . . . . . . . . . . . . . . . . . . . . . . . . . 864.4.3 Malolactic Fermentation . . . . . . . . . . . . . . . . . . 884.4.4 Removal of Urea from Sake and Wine by

Immobilized Acid Urease . . . . . . . . . . . . . . . . . 884.4.5 Beer Brewing Using an Immobilized Yeast

Bioreactor System . . . . . . . . . . . . . . . . . . . . . 894.5 Soy Sauce . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914.6 The Milk Industry . . . . . . . . . . . . . . . . . . . . . . . . . 92

4.6.1 Immobilization in the Milk Industry . . . . . . . . . . . . 924.6.2 Hydrolysis of Lactose in Milk . . . . . . . . . . . . . . . 934.6.3 Antibiotic Residues in Milk . . . . . . . . . . . . . . . . 94

4.7 Miscellaneous Flavor Materials and Aroma Compounds . . . . . 954.7.1 Biotransformation from Geraniol to Nerol . . . . . . . . 954.7.2 Limonin . . . . . . . . . . . . . . . . . . . . . . . . . . 954.7.3 β-Ionone . . . . . . . . . . . . . . . . . . . . . . . . . . 964.7.4 Naringin . . . . . . . . . . . . . . . . . . . . . . . . . . 964.7.5 Methyl Ketone (Blue Cheese Flavor) as a Flavor

Molecule from Higher Fungi . . . . . . . . . . . . . . . 974.7.6 Capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . 974.7.7 Vanillin . . . . . . . . . . . . . . . . . . . . . . . . . . . 984.7.8 Japanese Seasoning . . . . . . . . . . . . . . . . . . . . 99

4.8 Miscellaneous Applications . . . . . . . . . . . . . . . . . . . . 99

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4.8.1 Production of Oligosaccharides . . . . . . . . . . . . . . 994.8.2 Preservatives and Bacteriocins . . . . . . . . . . . . . . . 1004.8.3 Xylitol Production . . . . . . . . . . . . . . . . . . . . . 1014.8.4 Carotenoids and Leucrose . . . . . . . . . . . . . . . . . 1014.8.5 cis,cis-Muconic Acid (MA) . . . . . . . . . . . . . . . . 102

4.9 Various Industrial Options . . . . . . . . . . . . . . . . . . . . . 1024.9.1 Fuel Ethanol Production . . . . . . . . . . . . . . . . . . 1024.9.2 Application of Gels for Separation Matrices . . . . . . . 1034.9.3 Bioartificial Organs . . . . . . . . . . . . . . . . . . . . 1044.9.4 Insect Cell Immobilization . . . . . . . . . . . . . . . . . 104

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

5 Medicinal Applications of Hydrocolloid Beads . . . . . . . . . . . . 1175.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175.2 Encapsulation of Cells in Hydrogels . . . . . . . . . . . . . . . . 1175.3 Stem Cells in Bead Environments . . . . . . . . . . . . . . . . . 1205.4 Charged Hydrogel Beads as New Microcarriers for Cell Culture . 1225.5 Potential Support for Endothelial Cells . . . . . . . . . . . . . . 1245.6 Vaccine Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . 1255.7 Crosslinked Chitosan Beads: Different Medicinal Functions . . . 1265.8 Mucoadhesive Beads and Their Applications . . . . . . . . . . . 127

5.8.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 1275.8.2 Eyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285.8.3 Alimentary System . . . . . . . . . . . . . . . . . . . . . 128

5.9 Polyelectrolyte Complexes . . . . . . . . . . . . . . . . . . . . 1305.10 Soft Tissue Regeneration . . . . . . . . . . . . . . . . . . . . . 131References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

6 Dry Bead Formation, Structure, Properties, and Applications . . . 1376.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1376.2 General Properties of Cellular Solids . . . . . . . . . . . . . . . 1376.3 Manufacturing Methods for Hydrocolloid Cellular Solids . . . . 138

6.3.1 Drying Bicarbonate-Containing Gels After AcidDiffusion . . . . . . . . . . . . . . . . . . . . . . . . . . 138

6.3.2 Cellular Solids Produced by Fermentation . . . . . . . . 1406.3.3 Enzymatically Produced Cellular Solids . . . . . . . . . 1416.3.4 Inclusion of Oil in Cellular Solids . . . . . . . . . . . . . 1426.3.5 Porosity Control in Cellular Solids . . . . . . . . . . . . 143

6.4 Structure of Cellular Solids . . . . . . . . . . . . . . . . . . . . 1446.5 Mechanical Properties of Cellular Solids . . . . . . . . . . . . . 144

6.5.1 Compression of Cellular Solids . . . . . . . . . . . . . . 1446.5.2 Models for Describing Stress–Strain Behavior . . . . . . 1456.5.3 Elastic Properties of Cellular Materials . . . . . . . . . . 1476.5.4 Layered Cellular Solids and Compressibility

of Cellular Particulates . . . . . . . . . . . . . . . . . . . 1476.5.5 Acoustic Properties of Cellular Solids . . . . . . . . . . . 148

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6.6 Applications of Cellular Solids . . . . . . . . . . . . . . . . . . 1496.6.1 Hydrocolloid Cellular Solids as a Carrier for Vitamins . . 1496.6.2 Dried Gel Beads as Study Models and for Separation . . . 1506.6.3 Special Dry Beads for Water Treatment . . . . . . . . . . 1516.6.4 Matrices Entrapping Hydrocolloid Cellular Beads . . . . 151

6.7 Hydrocolloid Cellular Carriers for Agricultural Uses . . . . . . . 1546.7.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 1546.7.2 Preservation of Biocontrol Agents in a Viable

Form by Dry Cellular Bead Carriers . . . . . . . . . . . . 1546.7.3 Dry Carriers’ Capacity to Protect Biocontrol

Agents Against UV Light . . . . . . . . . . . . . . . . . 1566.7.4 Textural Features of Dried Hydrocolloid Beads . . . . . . 157

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

7 Liquid-Core Beads and Their Applications in Food,Biotechnology, and Other Fields . . . . . . . . . . . . . . . . . . . . 1637.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637.2 General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647.3 Soft Gelatin Capsules . . . . . . . . . . . . . . . . . . . . . . . 1647.4 Liquid-Core Capsules . . . . . . . . . . . . . . . . . . . . . . . 165

7.4.1 Liquid-Core Hydrocolloid Capsules . . . . . . . . . . . . 1657.4.2 Synthetic and Additional Liquid-Core Capsules . . . . . 168

7.5 Oil-Core Hydrocolloid Capsules . . . . . . . . . . . . . . . . . . 1717.6 Biotechnological Applications of Liquid-Core Capsules . . . . . 174

7.6.1 Growth of Microorganisms in Liquid-Core Capsules . . . 1747.6.2 Activity of Enzymes Within Liquid-Core Capsules . . . . 177

7.7 Special Food Applications . . . . . . . . . . . . . . . . . . . . . 1787.7.1 Jelly-Like Foods . . . . . . . . . . . . . . . . . . . . . . 1787.7.2 Fruit Products . . . . . . . . . . . . . . . . . . . . . . . 1797.7.3 Encapsulating Aroma and Health Compounds . . . . . . 1797.7.4 Other Foods . . . . . . . . . . . . . . . . . . . . . . . . 180

7.8 Agricultural Uses of Liquid-Core Capsules . . . . . . . . . . . . 1817.9 Environmental Uses of Liquid-Core Capsules . . . . . . . . . . . 1827.10 Special Applications of Liquid-Core Capsules . . . . . . . . . . 183

7.10.1 Stop-Smoking Aids . . . . . . . . . . . . . . . . . . . . 1837.10.2 The Beauty Industry—Removal of Body Hair . . . . . . 1847.10.3 The Paper Industry . . . . . . . . . . . . . . . . . . . . . 185

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

8 Beads as Drug Carriers . . . . . . . . . . . . . . . . . . . . . . . . 1918.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1918.2 Controlled Drug Release . . . . . . . . . . . . . . . . . . . . . . 1918.3 Gels in Drug-Delivery Systems . . . . . . . . . . . . . . . . . . 1928.4 Dual Drug-Loaded Beads . . . . . . . . . . . . . . . . . . . . . 1938.5 Drug Release from Beads . . . . . . . . . . . . . . . . . . . . . 194

8.5.1 Albumin Beads . . . . . . . . . . . . . . . . . . . . . . . 194

Contents xix

8.5.2 Alginate Beads . . . . . . . . . . . . . . . . . . . . . . . 1988.5.3 Chitosan Beads . . . . . . . . . . . . . . . . . . . . . . . 2008.5.4 Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . 2058.5.5 Modified Starch Microspheres . . . . . . . . . . . . . . . 2068.5.6 Dextran Beads . . . . . . . . . . . . . . . . . . . . . . . 2078.5.7 Cellulose Hydrogels . . . . . . . . . . . . . . . . . . . . 2098.5.8 Gellan Beads . . . . . . . . . . . . . . . . . . . . . . . . 2108.5.9 Guar Beads . . . . . . . . . . . . . . . . . . . . . . . . . 2108.5.10 Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2118.5.11 Modified Poly(Vinyl Alcohol) Microspheres . . . . . . . 2138.5.12 Biodegradable Hydrogels Based on Polyesters . . . . . . 2148.5.13 Hydrogels with Degradable Crosslinking Agents . . . . . 2158.5.14 Floating Beads . . . . . . . . . . . . . . . . . . . . . . . 2168.5.15 Xyloglucan Beads . . . . . . . . . . . . . . . . . . . . . 217

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

9 Beads and Special Applications of Polymersfor Agricultural Uses . . . . . . . . . . . . . . . . . . . . . . . . . . 2319.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2319.2 Immobilization of Plant Cell Suspensions and Single Seeds . . . 2319.3 Carriers for Slow Release of Bacteria that Affect Plant Growth . 2349.4 Inoculation of Seedlings and Plants with Beads

Containing Fungal Inoculum . . . . . . . . . . . . . . . . . . . 2359.5 Joint Immobilization of Plant Growth-Promoting Bacteria

and Green Microalgae . . . . . . . . . . . . . . . . . . . . . . . 2399.6 Cryopreservation by Encapsulation/Dehydration Technique . . . 2409.7 Controlled Release of Agricultural Chemicals . . . . . . . . . . 2419.8 Biotechnological Applications . . . . . . . . . . . . . . . . . . . 242

9.8.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 2429.8.2 Gene-Delivery Systems Using Beads . . . . . . . . . . . 2439.8.3 Bioactive Bead Method for Obtaining Transgenic Plants . 2439.8.4 Synthetic Seed Technology . . . . . . . . . . . . . . . . 244

9.9 Unique Applications of Polymers . . . . . . . . . . . . . . . . . 2459.9.1 Superabsorbent Polymers . . . . . . . . . . . . . . . . . 2459.9.2 Seed Coating . . . . . . . . . . . . . . . . . . . . . . . . 246

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

10 Beads for Environmental Applications . . . . . . . . . . . . . . . . 25510.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25510.2 Water Treatments . . . . . . . . . . . . . . . . . . . . . . . . . 255

10.2.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 25510.2.2 Wastewater Treatment by Anaerobic Fixed Bed Reactor . 25610.2.3 Wastewater Treatment Using Immobilized

Microorganisms . . . . . . . . . . . . . . . . . . . . . . 25810.2.4 Arsenic Removal from Water . . . . . . . . . . . . . . . 25910.2.5 Chitosan and Removal of Heavy Metal Ions . . . . . . . 261

xx Contents

10.2.6 Water Denitrification . . . . . . . . . . . . . . . . . . . . 26310.2.7 Anaerobic Ammonium Oxidation . . . . . . . . . . . . . 265

10.3 Soil Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . 26510.3.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 26510.3.2 Agrochemicals . . . . . . . . . . . . . . . . . . . . . . . 26510.3.3 Controlled Release of Pesticides into Soils . . . . . . . . 26610.3.4 Sustained Release of a Fungicide . . . . . . . . . . . . . 268

10.4 Air Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26810.4.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . . 26810.4.2 Sampling Air . . . . . . . . . . . . . . . . . . . . . . . . 26910.4.3 Determination of Trace Contaminants in Air

by Concentration on Porous Polymer Beads . . . . . . . 26910.5 Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

10.5.1 Biodegradation . . . . . . . . . . . . . . . . . . . . . . . 26910.5.2 Carbon Nanotubes . . . . . . . . . . . . . . . . . . . . . 27010.5.3 Removal by Microalgae . . . . . . . . . . . . . . . . . . 271

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

About the Author

Professor Amos Nussinovitch was born in Kibbutz Megiddo, Israel. He studiedChemistry at the University of Tel Aviv and Food Engineering and Biotechnologyat the Technion-Israel Institute of Technology. He has worked as an engineer at sev-eral companies and has been involved in a number of R&D projects in both theUnited States and Israel, focusing on the mechanical properties of liquids, semi-solids, solids, and powders. He is currently in the Biochemistry, Food Scienceand Human Nutrition Department of the Robert H. Smith Faculty of Agriculture,Food and Environment of the Hebrew University of Jerusalem, where he leads alarge group of researchers working on theoretical and practical aspects of hydrocol-loids. Prof. Nussinovitch is the sole author of the following books: HydrocolloidApplications: Gum Technology in the Food and Other Industries; Water-SolublePolymer Applications in Foods, and Plant Gum Exudates of the World: Sources,Distribution, Properties, and Applications. He is the author or co-author of numer-ous papers on hydrocolloids and on the physical properties of foods, and he hasmany patent applications. This book is devoted specifically to polymer macro- andmicro-gel beads. The author has been working in this area for many years and hasstudied the structure and texture of wet and dry beads; he has developed liquid-corecapsules, different polymeric beads for water denitrification, beads for biocontrolagent encapsulation, and beads as part of novel cellular solids, among many otherapplications.

xxi

List of Figures

1.1 (a) A caliper used especially for dry non-spherical beads.(b) Dry hydrocolloid bead . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 A sphere of radius r contained within a cylinder with radiusbase r and height 2r . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.3 (a) Ellipsoid, (b) oblate and (c) prolate spheroids . . . . . . . . . . 81.4 Structure of polyvinylpyrrolidone (PVP) . . . . . . . . . . . . . . . 201.5 Structure of polyethylene glycol . . . . . . . . . . . . . . . . . . . 202.1 Structure of an agarose polymer . . . . . . . . . . . . . . . . . . . 302.2 Structure of tricalcium phosphate . . . . . . . . . . . . . . . . . . . 322.3 Structure of patulin . . . . . . . . . . . . . . . . . . . . . . . . . . 332.4 Structure of alginic acid . . . . . . . . . . . . . . . . . . . . . . . . 342.5 Structure of cellulose (chain conformation) . . . . . . . . . . . . . . 372.6 Structure of polyacrylamide . . . . . . . . . . . . . . . . . . . . . . 423.1 Heat is transmitted by three distinct modes: conduction,

radiation, and convection . . . . . . . . . . . . . . . . . . . . . . . 543.2 (a) Atrazine formula and (b) three-dimensional structure . . . . . . 553.3 Light-dependent reactions of photosynthesis at the thylakoid

membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.4 Structure of nifedipine . . . . . . . . . . . . . . . . . . . . . . . . 573.5 Structure of sulfasalazine . . . . . . . . . . . . . . . . . . . . . . . 583.6 Diagram of a fluidized bed . . . . . . . . . . . . . . . . . . . . . . 593.7 A typical phase diagram . . . . . . . . . . . . . . . . . . . . . . . . 673.8 Space–time diagram for numerical analysis for a spherical

bead of radius R . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684.1 Structure of L-aspartic acid . . . . . . . . . . . . . . . . . . . . . . 764.2 Structure of L-alanine . . . . . . . . . . . . . . . . . . . . . . . . . 784.3 Structure of L-phenylalanine . . . . . . . . . . . . . . . . . . . . . 794.4 Structure of citric acid . . . . . . . . . . . . . . . . . . . . . . . . 804.5 Structure of malic acid . . . . . . . . . . . . . . . . . . . . . . . . 814.6 Structure of gluconic acid . . . . . . . . . . . . . . . . . . . . . . 824.7 Structure of lactic acid . . . . . . . . . . . . . . . . . . . . . . . . 834.8 Structure of urea . . . . . . . . . . . . . . . . . . . . . . . . . . . 884.9 Structure of the repeating unit of polyacrylonitrile . . . . . . . . . . 89

xxiii

xxiv List of Figures

4.10 Structure of chitosan: poly(β-1,4-D-glucosamine) . . . . . . . . . . 894.11 Skeletal structure of lactose . . . . . . . . . . . . . . . . . . . . . 944.12 Structure of geraniol . . . . . . . . . . . . . . . . . . . . . . . . . 954.13 Structure of nerol . . . . . . . . . . . . . . . . . . . . . . . . . . . 954.14 Structure of limonin . . . . . . . . . . . . . . . . . . . . . . . . . 964.15 Structure of β-ionone . . . . . . . . . . . . . . . . . . . . . . . . . 964.16 Structure of naringin . . . . . . . . . . . . . . . . . . . . . . . . . 974.17 Structure of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . 984.18 Structure of vanillin . . . . . . . . . . . . . . . . . . . . . . . . . . 994.19 Structure of nisin . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004.20 Structure of xylitol . . . . . . . . . . . . . . . . . . . . . . . . . . 1014.21 Structure of muconic acid . . . . . . . . . . . . . . . . . . . . . . 1025.1 (a) Section of dog pancreas. (b) Immunohistochemistry

of mouse pancreas for insulin . . . . . . . . . . . . . . . . . . . . . 1185.2 Structure of iopamidol . . . . . . . . . . . . . . . . . . . . . . . . 1195.3 Diagram of the human pelvis . . . . . . . . . . . . . . . . . . . . . 1215.4 Structure of hyaluronan . . . . . . . . . . . . . . . . . . . . . . . . 1215.5 TEM micrograph showing dengue virus virions . . . . . . . . . . . 1235.6 Compact bone and spongy bone . . . . . . . . . . . . . . . . . . . 1245.7 Gram-negative Bordetella bronchiseptica coccobacillus bacteria . . 1265.8 Gamma cyclodextrin cone shape showing arrangement of

glucose molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265.9 Skeletal formula of acyclovir . . . . . . . . . . . . . . . . . . . . . 1296.1 Sponge—a natural cellular solid . . . . . . . . . . . . . . . . . . . 1386.2 Typical appearance of a freeze-dried gel specimen . . . . . . . . . . 1406.3 (a) Schematic representation of the typical compressive

stress–strain relationship of a cellular solid after onecompression. (b) Typical stress–strain relationship of acellular solid after repeated compression cycles . . . . . . . . . . . 146

6.4 Cross section of hydrocolloid-based bead . . . . . . . . . . . . . . 1506.5 SEM micrograph of dry hydrocolloid cellular matrix,

including water-soluble polymer beads embedded within itsstructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

6.6 SEM micrograph of a cross section of cellular solid showingmicroorganisms on and/or entrapped within the solid walls . . . . . 155

7.1 A gelatin capsule for medicine . . . . . . . . . . . . . . . . . . . . 1647.2 Structure of polylysine . . . . . . . . . . . . . . . . . . . . . . . . 1657.3 A method for producing various liquid-core capsules . . . . . . . . 1667.4 Skeletal formula of the poly(methylmethacrylate) repeating

unit (C5O2H8)n . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1697.5 Production of liquid-core hydrocolloid oil capsules . . . . . . . . . 1727.6 Liquid-core hydrocolloid oil capsule consisting of glycerol

and 50% soybean oil before capsule shrinkage . . . . . . . . . . . . 1737.7 Plantago major . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1838.1 Structure of theophylline . . . . . . . . . . . . . . . . . . . . . . . 192

List of Figures xxv

8.2 Skeletal formula of cromoglicic acid . . . . . . . . . . . . . . . . . 1968.3 Progesterone: two-dimensional skeletal illustration . . . . . . . . . 1968.4 Structural formula of inulin (fructan) . . . . . . . . . . . . . . . . . 1978.5 Structure of chondroitin sulfate . . . . . . . . . . . . . . . . . . . . 1998.6 Structure of poly(N-isopropylacrylamide) (PNIPAAM) . . . . . . . 1998.7 Commercial chitosan is derived from the shells of shrimp

and other sea crustaceans, including Pandalus borealispictured here . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

8.8 Structure of sodium tripolyphosphate . . . . . . . . . . . . . . . . . 2018.9 Structure of riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . 2038.10 Skeletal formula of prednisolone . . . . . . . . . . . . . . . . . . . 2048.11 Structure of mitomycin C . . . . . . . . . . . . . . . . . . . . . . . 2088.12 Skeletal formula of poly(lactic-co-glycolic acid) (PLGA) . . . . . . 2088.13 Structural formula of a guaran unit . . . . . . . . . . . . . . . . . . 2118.14 Skeletal formula of indometacin (INN) . . . . . . . . . . . . . . . . 2118.15 Raman fluorescent microscopy depth profile of (a) an

unloaded bead and (b) a doxorubicin hydrochloride-loaded bead . . 2148.16 Structure of alloxan (2,4,5,6-tetraoxypyrimidine;

2,4,5,6-pyrimidinetetrone) . . . . . . . . . . . . . . . . . . . . . . 2158.17 Structure of flavin mononucleotide . . . . . . . . . . . . . . . . . . 2168.18 Two-dimensional structure of NSAID-class drug diclofenac . . . . . 2179.1 Structure of shikonin . . . . . . . . . . . . . . . . . . . . . . . . . 2329.2 Illustration of Nicotiana tabacum . . . . . . . . . . . . . . . . . . . 2339.3 Vermiculite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2369.4 Milled peat production in the Irish Midlands . . . . . . . . . . . . . 2379.5 Norway spruce . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2389.6 Structure of indole-3-acetic acid . . . . . . . . . . . . . . . . . . . 24010.1 Overview of the main health effects on humans of some

common types of pollution . . . . . . . . . . . . . . . . . . . . . . 25610.2 Raw sewage and industrial waste flows into the US from

Mexico as the New River passes from Mexicali in BajaCalifornia to Calexico, California . . . . . . . . . . . . . . . . . . . 257

10.3 A ceramic carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . 25710.4 Structure of acrylamide . . . . . . . . . . . . . . . . . . . . . . . . 25910.5 Arsenic production based on the US Mineral Commodity

Summaries Report 2007 . . . . . . . . . . . . . . . . . . . . . . . . 25910.6 Schematic drawing of the enzymatic synthesis of chitosan . . . . . . 26210.7 (a) Cross section of a bead filled with starch. (b) Bacterial

colonization on the starch granules . . . . . . . . . . . . . . . . . . 26410.8 Structure of alachlor . . . . . . . . . . . . . . . . . . . . . . . . . . 26710.9 Carbon nanotubes . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Chapter 1Physical Properties of Beads and TheirEstimation

1.1 Introduction

Accurate estimates of shape, size, volume, specific gravity, surface area, bulkdensity, and other physical characteristics are important in the study or considera-tion of hydrocolloid bead properties. There is not much information in the literatureon the physical properties of hydrocolloid beads, other than their mechanical prop-erties, which are discussed in Chapter 2. The aim of this chapter is to provide thereader with simple tools for the physical characterization of beads.

1.2 Bead Size and Shape

1.2.1 General

Most hydrocolloid beads are produced in a spherical shape. However, deviationsfrom sphericity occur, with spheroid and ellipsoid beads being quite common. Inaddition, beads that pass through a drying process for the manufacture of dried car-riers (see Chapters 3 and 6) may undergo a change in their spherical or roundedshape. Therefore, shape and size are essential parameters for adequately describinghydrocolloid beads. Moreover, the characterization of an object’s shape entails theneed to measure other dimensional parameters. Similarly, an increase in bead shapeirregularity entails an increase in the number of measurements needed to define itsform.

1.2.2 Size of Drops and Beads

Size is a measure of an object’s physical magnitude (how big or small it is). Whena size is measured, it is given a number. Size is an important attribute of beads inheat- and mass-transfer calculations, and when beads are transferred in fluid flow,their average size must be known. It is easier to specify the size of a regularly

1A. Nussinovitch, Polymer Macro- and Micro-Gel Beads: Fundamentalsand Applications, DOI 10.1007/978-1-4419-6618-6_1,C© Springer Science+Business Media, LLC 2010

2 1 Physical Properties of Beads and Their Estimation

shaped bead (i.e., spherical or ellipsoid); for irregularly shaped beads, the term sizeshould be arbitrarily defined. Beads can be formed in the millimeter, micrometer,or nanometer size ranges. Size can be determined using the projected-area method,in which three characteristic dimensions are defined: the major diameter (i.e., thelongest dimension of the maximum projected area), the intermediate diameter (i.e.,the minimum diameter of the maximum projected area or the maximum diameterof the minimum projected area), and the minor diameter (i.e., the shortest diameterof the minimum projected area) (Sahin and Sumnu 2006). The terms length, width,and thickness are commonly used for the major, intermediate, and minor diameters,respectively. They can be measured using a caliper or micrometer that is especiallydesigned for dry non-spherical beads (Fig. 1.1). In general, particle size can be deter-mined by sieve analysis, passage through an electrically charged orifice or settlingrate (Sahin and Sumnu 2006).

a b

Fig. 1.1 (a) A caliper used especially for dry non-spherical beads. (b) Dry hydrocolloid bead

Due to growing interest in the field of cell immobilization, numerous tech-niques have been developed for bead manufacture (see Chapters 9 and 10). Thesetechniques include dripping (Walsh et al. 1996; Romo and Perez-Martinez 1997),emulsification and coacervation (Poncelet et al. 1993, 1994; Green et al. 1996),rotating-disc atomization (Bégin et al. 1991; Ogbonna et al. 1991), air jet (Kleinet al. 1983; Levee et al. 1994), atomization (Siemann et al. 1990), electrostatic drip-ping (Bugarski et al. 1994; Halle et al. 1994; Poncelet et al. 1994), mechanicalcutting (Prüsse et al. 1998), and the vibrating nozzle technique (Ghosal et al. 1993;Brandenberger et al. 1997; Seifert and Phillips 1997). Some of these methods sufferfrom limitations, particularly with respect to difficulties in achieving the simultane-ous production of beads at a high production rate with a satisfactory level of materialutilization under mild and non-toxic conditions or under completely sterile condi-tions, in the ability to scale up the process and, in particular, in obtaining beads witha narrow size distribution.

To calculate the diameter of a fluid drop, prior to its transformation into a beadby further chemical/physical manipulation, a theory which was first developed to

1.2 Bead Size and Shape 3

explain the jet breakup of Newtonian fluids must be considered (Rayleigh 1879).This theory proposed an equation relating the wavelength of the perturbation to thejet diameter:

λopt = π√

2djet (1.1)

where λopt is the optimal wavelength for jet breakup and djet is the jet diameter. Thistheory was later developed by Weber (1931) to include the effect of fluid viscosity:

λopt = π√

2djet

√1 + 3η√

ρσdjet(1.2)

where η is the fluid viscosity (Pa s), ρ is the fluid density (kg/m3), and σ is thesurface tension (kg/s2). Since one droplet is produced by each Hertz of vibration,the drop diameter dd can be calculated via a straightforward mass balance equation:

dd = 3

√6

F

πν(1.3)

where F is the flow rate (m3/h) and ν is the frequency of the vibration (Hz) which isitself linked to the wavelength by

ν = F

λ

4

d2jetπ

(1.4)

As a consequence, the droplet diameter, as a function of wavelength and jetdiameter, is specified by

dd = 3

√3d2

jetλopt

2(1.5)

This suggests that the optimal frequency for the production of drops from a givennozzle diameter is given exclusively by the physicochemical properties and the flowrate of the extruded fluid (Serp et al. 2000).

However, when a non-Newtonian fluid, such as alginate or any other poly-mer commonly used for cell immobilization and encapsulation, is employed, theactual viscosity within the nozzle has to be calculated. In addition to being diffi-cult to estimate, djet also has to be determined through preliminary experimentation(Brandenberger and Widmer 1998). As already noted, this theoretical approach onlyyields the optimum frequency for drop generation, which does not necessarily corre-spond to the optimal frequency for bead production. Indeed, if this frequency is toohigh for an alginate solution, for instance, doublets appear due to contact betweenthe droplets and the beads in the hardening solution. On the other hand, when the

4 1 Physical Properties of Beads and Their Estimation

frequency is too low, satellites are observed. Thus only an empirical approach canbe used to determine the actual conditions for optimum bead production (Serp et al.2000).

The following is a brief description of a few techniques used to approximatea narrow bead-size distribution. A very popular and simple method for obtainingbeads is crosslinking of an alginate solution. It is therefore not surprising that itis the model system of choice for many encapsulations and for bead manufactur-ers. The properties of different alginates, their manufacture, and their chemicaland physical properties have been reviewed in many textbooks and manuscripts(among them Davidson 1980; Nussinovitch 1997; Phillips and Williams 2000)and the reader is referred to these for further details. The vibrating nozzle tech-nique is a suitable process for cell immobilization within the requested sizerange for alginate beads. It can be used to produce a mono-dispersion of beadslarger than 200 μm in diameter, depending on the application (Serp et al. 2000).Furthermore, their manufacture can be scaled up. In particular, reasonably highproduction rates, between 1 and 15 ml/min, depending upon the desired beadsize and polymer solution rheology can be achieved, producing an alginate bead-size distribution of less than 5% (Brandenberger et al. 1997; Seifert and Phillips,1997).

It is essential to distinguish between the size of an alginate drop formed by anencapsulation apparatus and the size of the alginate bead, that is, the size of thedrop once it comes into contact with the hardening/crosslinking solution (Serp et al.2000). In fact, using the jet breakup technique, it is not possible to form alginatebeads of the desired size and within the required size distribution, with any of thecommercially available alginates. Consequently, a detailed series of experiments tocharacterize the alginate is indispensable, before successful conditions for extrusioncan be defined (Serp et al. 2000). A 2% solution of sodium alginate was extruded,using an encapsulation device fitted with a range of nozzles (diameters 50–500 μm),into a hardening solution of 110 mM CaCl2 at 25◦C, beads were sampled from thehardening solution at 2 min intervals over a period of 60 min, and bead size andsize distribution were determined. The following observations were made: beaddiameter decreased progressively during the first 5 min following extrusion andthen remained relatively stable. However, irrespective of initial bead size (diame-ters within the range of 300–600 μm), this contraction represented less than 10%of the initial bead diameter. Size determinations over several days of beads incu-bated in a solution of 110 mM CaCl2 revealed further gradual shrinkage. However,this size decrease represented only 1–2% of the diameter measured after 5 min.These results indicated that the alginate beads are rapidly saturated with Ca2+ ionswithin the first few minutes of incubation and that a slow rearrangement of thealginate chains, inducing further shrinkage, then occurs (Serp et al. 2000). Anothermanuscript described the use of alginate beads as a vehicle for the controlled releaseof entrapped essential oil as an antiviral agent via oral administration. In this case,the freshly prepared calcium alginate beads exhibited a regular, spherical shape thatwas maintained after drying. The bead size did not vary significantly when the expo-sure time to CaCl2 was changed, possibly due to the relatively high concentration

1.2 Bead Size and Shape 5

of crosslinker. In particular, these freshly prepared formulations displayed a narrowsize range. A decrease in bead size was noted due to water loss during the dryingprocess (Lai et al. 2007).

Agar is another potent gelling agent composed of repeating units of D-galactoseand 3,6-anhydro-L-galactose. The gelling component of agar, agarose, is an essen-tially sulfate-free, neutral (non-ionic) polysaccharide (Nussinovitch 1997). Theeffect of processing conditions on the mean size and size distribution of porousagarose beads manufactured by emulsification in a single stirred vessel was inves-tigated. Agarose beads can be successfully manufactured in a single stirred vesselby emulsification of hot agarose solution and subsequent freezing/gelling of theaqueous drops into soft solid particles. The particle size and size distribution canbe controlled by selection of energy dissipation rate and/or surfactant concentration(Mu et al. 2005). Despite the fact that flow in a stirred vessel is transitional, therelationship between drop/particle mean size and energy dissipation rate is similarto that for turbulent flow (Baldyga et al. 2000).

Another common technique for producing spherical polymer beads that affordscontrol of particle size is suspension polymerization. In typical suspension polymer-ization processes, microscopic droplets of monomer stabilized by small amountsof surface-active polymers are suspended in a continuous non-solvent medium,most commonly water, and polymerization is carried out by the addition ofmonomer-soluble initiators. The advantages of suspension polymerization are goodtemperature control and ease of handling of the final product (O’Connor and Gehrke1997). Suspension crosslinking technique was used to produce thermally respon-sive gels of hydroxypropylmethylcellulose (HPMC) in a spherical form. Suspensioncrosslinking of HPMC with divinylsulfone was accomplished by dispersing aqueouspolymer droplets, containing all of the reactants, in a continuous organic phase. Thespherical beads had diameters ranging from 500 to 3000 μm. Bead size generallydecreased with the use of a larger impeller, swinging at high stirring speeds, or at alower phase ratio. As bead size decreased, the size distribution also narrowed. Thegel beads exhibited the same swelling properties and crosslinked network formationas bulk HPMC gels (O’Connor and Gehrke 1997).

1.2.3 Bead Shape

The shape of an object located in a space is the part of that space occupied bythe object, as determined by its external boundary—abstracting from other proper-ties such as color, content, and material composition, as well as from the object’sother spatial properties (position and orientation in space, size). Another defini-tion of shape is the geometrical information from an object that remains whenlocation, scale, and rotational effects are filtered out (Kaye 1993). Simple two-dimensional shapes can be described by basic geometry, such as points, lines,curves, and planes. A shape whose points all belong to the same plane is calleda plane figure. Most shapes occurring in the physical world are complex. Some,such as plant structures and coastlines, may be so arbitrary as to defy traditional

6 1 Physical Properties of Beads and Their Estimation

mathematical description—in which case they may be analyzed by differential orfractal geometry.

Describing the shape of an object is difficult in a three-dimensional space: we areoften obliged to be satisfied with a two-dimensional description. A shape index is adimensionless number that is used to characterize a shape. The word index comesfrom the Latin word “indicare” meaning “to show,” in a way similar to a book’sindex which shows one where to find a particular topic. To create a shape index fora non-circular shape, it is necessary to specify the diameters of the profile (Kaye1993). The word diameter originates from the Greek, “dia” meaning through and“metron” a measure. The diameter of a circle is measured and defined as the linemoving through the center of the circle. An ellipse is always defined by its majorand minor profile diameters. Shape indices of irregular profiles can be obtained byconstructing an ellipse of equal area and then calculating the ratio of the minorand major axes of that equal area. However, this can sometimes be problematicdue to possible difficulties in measuring the area of the profile and the subsequentcalculation of the dimensions of the ellipse of equal area (Kaye 1993).

If a profile is irregular, it can be enveloped by a profile curve termed convex hullof the profile. Convex means “bulging outward” and hull means “an outer covering.”For such a profile, the shape of the convex hull is used to generate a shape indexusing the length and breadth of the profile. A smooth profile can be achieved bymodifying the concept used to generate π for a circle and denote the perimeterdivided by the profile’s maximum diameter. This shape factor is 3.14 for a circleand decreases to 2 for a very long fiber. However, this approach is not widely usedsince it is difficult to measure the perimeter of a profile, and for very rugged profiles,the real perimeter of the profile approaches infinity and the modified shape indexbecomes indeterminate and not a good indication of structure (Kaye 1993).

The sphere is one of the most commonly occurring shapes in nature (Musserand Burger 1997). Many sphere-like objects that are of interest in the life sciencescan be easily located, from stones (geology, mineralogy), to oranges, watermel-ons, and tomatoes (food science), from soap bubbles (children’s games and surfaceresearch), to frog eggs (biology and genetics), moving even further to the earth andmoon (astronomy and physics), not to mention other fields. The sphere’s ability toenclose the greatest volume for a given surface area is a good explanation for theconsiderable frequency of its appearance in nature. In other words, a sphere requiresthe least amount of material to surround a given volume. This may help explain whyanimals curl up in a ball when it is cold outside (Musser and Burger 1997).

Because most beads are produced in a sphere-like shape, a brief review of thebasic formulas related to their surface area and volume estimation is warranted.Archimedes (287–212 BC), considered to have been the greatest mathematician inantiquity and ranked by many with Sir Isaac Newton and Carl Friedrich Gauss,observed that the surface area (volume) of a sphere is two-thirds the surface area(volume) of the smallest cylinder containing that sphere. Figure 1.2 shows a sphereof radius r contained within a cylinder with radius base r and height 2r. The surfacearea of the cylinder is 2πr2 + 2πr(2r) = 6πr2. Based on Archimedes’ observation,the formula for the surface area of a sphere of radius r is 2/3(6 πr2) = 4πr2. The

1.2 Bead Size and Shape 7

Fig. 1.2 A sphere of radius r contained within a cylinder with radius base r andheight 2r (courtesy of André Karwath at http://commons.wikimedia.org/wiki/File:Archimedes_sphere_and_cylinder.png)

volume of the sphere is 2/3(2 πr2) = 4/3 πr3. Of course, the r in 4πr2 is squared,a unit of area, whereas the 4/3πr3 is cubed, a unit of volume (Musser and Burger1997).

The shape of a bead can be described by borrowing terms from differentfields. These include round—approaching spheroid; oblong—vertical diametergreater than the horizontal diameter, ovate—egg shaped, obvate—inverted ovate,elliptical—approaching ellipsoid; truncate—having both ends squared or flattened;ribbed—in cross section, sides are more or less angular; regular—horizontal crosssection approaches a circle; irregular—horizontal cross section departs considerablyfrom a circle (Mohsenin 1970).

Let us define some of the most commonly occurring bead shapes. A spheroid isan ellipsoid (Weisstein 2003). It is a quadric surface obtained by rotating an ellipseabout one of its principal axes; in other words, it is an ellipsoid with two equalsemi-diameters. If the ellipse is rotated about its major axis, the result is a prolate(elongated) spheroid, like a rugby ball. If the ellipse is rotated about its minor axis,the result is an oblate (flattened) spheroid, like a lentil (Fig. 1.3). If the generat-ing ellipse is a circle, the surface is a sphere. Images of cut surfaces of beads oftenresemble a circular shape. A circle is the set of all points in a plane that are at afixed distance from a given point (called the center). The distance from the centerto a point on the circle is called the radius. Any segment whose endpoints are thecenter and a point of the circle is also called a radius. The length of a line segmentwhose endpoints are on the circle and which crosses the center is called the diam-eter (Musser and Burger 1997). Many properties of a circle, including its area, areobtained by comparing the circle to regular polygons with increasingly large valuesof n. The perimeter of a circle, namely the length of the circle, is given the special

8 1 Physical Properties of Beads and Their Estimation

a

b

c

Fig. 1.3 (a) Ellipsoid (http://en.wikipedia.org/wiki/File:Ellipsoid_321.png), (b) oblate and(c) prolate spheroids

1.2 Bead Size and Shape 9

name of circumference. It is important to mention that the ratio between the circum-ference and the diameter is a constant called π , which is the Greek letter “pi.” Thecircumference of a circle is 2πr or πD, where r and D are the radius and diameter ofthe circle, respectively. The area of a circle, A = πr2, was first estimated by inscrib-ing a circle within a regular polygon and calculating its area. A rigorous verificationof this formula can be achieved using calculus (Musser and Burger 1997).

In view of the above knowledge, it is not surprising that many bead manufac-turers want to create beads in a spherical or sphere-like shape. For example, gellanbeads can be formed in a spherical shape and thus mathematical modeling studiesof their spherical geometry can be performed by solving Fick’s equation to computeconcentration profiles and diffusion coefficients. To produce such shapes, a disper-sion of gellan gum was extruded into a solution containing a mixture of calcium andzinc ions (counter ions). By changing the experimental variables, for instance pH,of the counter ion solution and the amount of drug (cephalexin) loading, processvariables could be optimized to achieve control over the final percentage of drugentrapment efficiency and release rates, as well as over bead size and morphology(Agnihotri et al. 2004). The average bead size ranged from 925 to 1183 pm as mea-sured by laser light scattering, and up to 69.3% cephalexin entrapment was achieved(Agnihotri et al. 2004). Different bead shapes are sometimes the result of ingredientsused in the manufacturing process, as well as of the entrapped moieties. Dropping a3.5% κ-carrageenan-containing papain solution into 0.5 M potassium chloride usingthe ionotropic gelation method, with a hardening time of 20 min, it was possible toobtain beads characterized by a spherical disc shape with a collapsed center and anabsence of aggregates (Sankalia et al. 2006a).

Bead manufacturers often need to consider whether loading a drug into a beador chemical changes (oxidation) in its hydrocolloid composition will influence thebead’s shape. In the case of ionic binding of drugs to beads made of cellulose and itsderivatives, the physical properties of the beads, such as spherical shape and poros-ity, were not significantly influenced by oxidation of cellulose or by drug loading(Wolf and Finke 1992). Controlling formulation and process variables in order toachieve a particular bead shape may also change the bead’s density, porosity, andfriability (Agrawal et al. 2004).

Different combinations of water-soluble polymers and production technologiescan lead to control of bead size and porosity (Agrawal et al. 2004). For instance,beads containing chitosan, fine-particle ethylcellulose, HPMC, and caffeine as themodel drug were manufactured. Bead size, yield, shape, friability, density, andporosity were determined, and release studies performed. Spherical beads with goodmechanical properties could be created without microcrystalline cellulose (Agrawalet al. 2004). Furthermore, beads with high percentage yield and high sphericitycould be obtained at high chitosan content, and low HPMC content, water content,spheronizer speed, and extruder speed. Less friable beads were obtained at highlevels of the studied formulation’s variables and low levels of the studied process’svariables. Beads of high density and low porosity could be manufactured at highlevels of the studied formulation’s and process’s variables (Agrawal, Howard andNeau 2004).