This thesis is dedicated, with love and gratitude, to my parents and teachers.

Most of them would never read it so, unless someone tells them, they will never know.

Thanks to Henri Chanzy and Laurent Heux for illuminating discussions.

Thanks to my friend Kim Anh Nguyen who forwarded me the information of the application of this thesis. I would not know about this PhD project without her help.

Also, there are so many other people that I would like to thank, but I won't mention them all by name, because someone would be forgotten. Huge thanks to all of the past and present members of Centre de Recherches sur les Macromolécules Végétales (CERMAV), more particularly the Glycomaterials group for the last few brilliant years! I've had great time and a lot of memories.

I have to say an extra special thanks to my family, especially my wife, Mai, who have always followed and supported me.

Finally, I would like to thank LabEx Arcane for funding my PhD. Without this, it would not have been possible to carry out this work. Also, thank you to the Société Française des Microscopies, the Glyco@Alps program and the Polysolvat-12 meeting for offering me scholarships to participating to national and international conferences. Résumé L'amylose est un homopolymère quasi-linéaire d'unités glucosyles liées en α(1,4) qui, extrait de l'amidon natif, possède la propriété remarquable de former des complexes cristallins avec une grande variété de petites molécules organiques. Ces complexes sont regroupés sous le terme générique d'amylose V. Nous avons testé la capacité de 121 composés à induire la cristallisation de l'amylose à partir de solutions aqueuses diluées. La morphologie et la structure des cristaux lamellaires formés ont été caractérisées par microscopie électronique en transmission ainsi que par diffraction des électrons et des rayons X. Les données révèlent que les structures de ces complexes peuvent être classées en 10 familles dont 5 sont décrites pour la première fois. Des spectres de résonnance magnétique nucléaire du solide du 13 C montrent clairement que l'hélicité de l'amylose V est corrélée à la résonnance du carbone C1 qui se déplace vers les champs faibles lorsque le nombre d'unités glucosyles par tour augmente. Des modèles géométriques préliminaires ont été proposés pour tous les allomorphes, la structure de cristaux de V1-butanol ayant été analysée en détail en combinant des calculs de conformation et d'énergie d'empilement avec un affinement de structure de polymère cristallin classique. Tous les allomorphes contiennent des simples hélices d'amylose d'ordre 6, 7 ou 8 et les molécules invitées peuvent être localisées dans ces hélices, entre elles ou les deux. Chaque type d'allomorphe peut être obtenu avec différents complexants et la conformation de l'hélice d'amylose dépend de la taille du complexant. De plus, un ligand donné est susceptible d'induire la formation de plusieurs allomorphes. Le polymorphisme cristallin de l'amylose serait donc une caractéristique plus générale que ce qui avait été rapporté auparavant. La propension au polymorphisme dépend non seulement de la nature du complexant mais aussi des conditions de cristallisation. Le degré de polymérisation de l'amylose, sa concentration et celle du complexant, la température de mélange ou de cristallisation et la composition du solvant ont un impact significatif sur la formation de cristaux et la structure de l'amylose V. Par ailleurs, nous avons utilisé les complexes avec l'ibuprofène comme modèle afin d'évaluer le potentiel de l'amylose V comme système de délivrance de principes actifs.

Différentes fractions d'ibuprofène, probablement corrélées aux positions possibles de la molécule dans le cristal, sont sélectivement relarguées en variant le pH du milieu de dissolution.

Puisque le relargage intervient principalement à pH élevé, ces complexes d'inclusion sont donc potentiellement intéressants pour cibler une libération intestinale et pourraient donc améliorer l'effet thérapeutique de l'ibuprofène en évitant les dommages à l'estomac.

Mots-clés: biopolymère, amylose, cristal, complexe d'inclusion, analyse structurale, relargage Summary Amylose, a mostly linear homopolymer of α(1,4)-linked glucosyl units extracted from native starch, has the remarkable property to form "V-amylose" crystalline complexes with a variety of small organic molecules. We have tested the ability of 121 compounds to induce the crystallization of amylose from dilute aqueous solutions. The morphology and structure of the resulting lamellar crystals were characterized by transmission electron microscopy as well as electron and X-ray diffraction. The data revealed that the structures of the complexes could be classified into 10 families, 5 of which were described for the first time. In addition, 13 C solidstate nuclear magnetic resonance spectra clearly showed that the helicity of V-amylose was correlated with the resonance of carbon C1 that was shifted downfield with increasing number of glucosyl units per turn. Tentative geometrical models were proposed for all allomorphs and the structure of V1-butanol was analyzed in more details by combining conformational and packing energy calculations with classical crystalline polymer structure refinement. All allomorphs contained 6-, 7-or 8-fold amylose single helices and the guest molecules could be located inside these helices, in-between, or both. Each allomorph could be obtained with different complexing agents and the helical conformation was found to depend on the size of the complexing agent.

In addition, a given ligand could induce the formation of several allomorphs, suggesting that the polymorphism of V-amylose crystals is a more general characteristic than what was previously reported. The propensity for polymorphism does not only depend on the nature of the complexing agent but also on the crystallization conditions. The degree of polymerization of amylose, its concentration and that of the complexing agent, the temperature of mixing and crystallization, and the solvent composition have a significant impact on the formation and crystal structure of V-amylose. In addition, crystalline complexes prepared with ibuprofen were used as a model to evaluate the potential of V-amylose as a delivery system of bioactive molecules. Distinct fractions of ibuprofen, likely correlated with the different locations of the guest in the crystal, were selectively released by varying the pH of the dissolution medium.

Since the release mainly occurred at high pH, these inclusion complexes appear to be potentially interesting for intestinal targeting and would thus improve the therapeutic effect of ibuprofen while avoiding stomach damage.

Keywords: biopolymer, amylose, crystal, inclusion complex, structural analysis, release Résumé des travaux de thèse L'amylose est un homopolymère quasi-linéaire d'unités α-D-glucosyles reliées par des liaisons glucosidiques α(1→4). Ce polysaccharide est l'un des principaux constituants des grains d'amidon natifs (environ 20-30% de sa masse). Il peut être aussi synthétisé in vitro par différentes enzymes. L'amylose possède la propriété remarquable de former des complexes d'inclusion lorsqu'il est recristallisé in vitro en présence d'une grande variété de petites molécules organiques.

Dans cette forme, appelée "V-amylose", le polymère adopte une conformation en hélice simple et les molécules invitées peuvent être localisées dans ces hélices, entre elles ou les deux.

Les complexes d'inclusion présentent plusieurs propriétés intéressantes et applications potentielles, par exemple dans l'industrie alimentaire et pharmaceutique. Des agents complexants tels que le 1-butanol, le thymol ou la menthone ont également été utilisés pour fractionner l'amidon natif en ses principaux constituants (amylopectine et amylose). De plus, l'amylose présentant des propriétés de complexation très similaires à celles des cyclodextrines, il a été exploité pour encapsuler des arômes et des molécules bioactives.

L'amylose V peut être préparée sous différentes formes (fibres, monocristaux lamellaires, sphérolites) selon les conditions de cristallisation. La diffraction des rayons X et celle des électrons ont permis d'identifier différents allomorphes. Des modèles moléculaires basés sur des simples hélices gauches ont été proposés, mais seul un petit nombre d'entre eux ont été validés par des méthodes cristallographiques. En particulier, plusieurs informations structurales restent à déterminer, telles que la conformation hélicoïdale ou la localisation des molécules invitées. De plus, le phénomène de complexation et les paramètres qui influent sur la formation d'une structure cristalline spécifique doivent être étudiés en détail.

Cette thèse visait à étudier la cristallisation de l'amylose V en présence de diverses complexants organiques afin de mieux comprendre l'interaction de l'amylose avec les molécules invitées et, si possible, prédire la structure qui résulterait de la cristallisation en présence d'un ligand donné. Comme c'est généralement le cas avec les polymères, il n'a pas été possible de faire croître des monocristaux d'amylose V d'une taille suffisante pour permettre leur analyse par cristallographie aux rayons X. Notre approche générale a donc consisté à préparer des cristaux lamellaires à partir de solutions aqueuses diluées. Nous avons caractérisé leur morphologie et leur structure en combinant microscopie électronique en transmission (MET) et diffraction électronique (DE) de ces monocristaux, avec des données de diffraction des rayons X de poudres hydratées. Le protocole général de cristallisation consistait à solubiliser l'amylose dans l'eau ou dans un mélange eau / DMSO à haute température, à y ajouter un agent complexant puis à maintenir la solution à une température prédéterminée pour induire la cristallisation.

La première partie de ce travail a donc été consacrée à l'optimisation des protocoles de cristallisation en étudiant l'impact de plusieurs paramètres (degré de polymérisation (DP) et concentration en amylose, nature du solvant, température et temps de cristallisation) sur la morphologie et la structure des cristaux. L'étude des complexes préparés avec une série d'acides gras linéaires saturés (de C3 à C20) a apporté un résultat nouveau et inattendu: un acide gras donné pouvait induire la formation d'allomorphes différents contenant des hélices d'ordre 6 ou 7, jusqu'à 3 allomorphes dans certains cas. Cette observation nous a incités à tester davantage de séries de molécules de structure chimique différente (diols, esters, amines, cétones, composés aromatiques, etc.).

Dans un second temps, nous avons caractérisé les nombreux cristaux préparés dans diverses conditions et analysé la quantité importante de données collectées à l'aide des différentes techniques d'imagerie, de diffraction et de spectroscopie. Plusieurs conclusions ont pu être tirées. Dans la gamme de conditions de cristallisation sélectionnées, sur les 121 agents testés, 28 n'ont conduit à aucune cristallisation et 43 ont induit la formation de 2 à 4 allomorphes.

Dix familles d'allomorphes de cristaux lamellaires de V-amylose contenant des hélices d'ordre 6, 7 ou 8 ont été identifiées, parmi lesquelles 5 correspondent à des structures précédemment publiées et 5 sont de nouveaux allomorphes décrits pour la première fois. Afin de distinguer ces structures, nous avons élargi la précédente nomenclature des formes d'amylose V. Les 10 allomorphes ont été nommés en fonction de la conformation de l'amylose et de la taille relative de la maille cristalline: V6I, V6II, V6III, V6IV, V7I, V7II, V7III, V7IV, V8I et V8II.

Des analyses spectroscopiques ont complété les données de cristallographie. Les spectres infrarouge montrent une augmentation de l'intensité de bandes vibrationnelles spécifiques par rapport à l'amylose amorphe ou aux amyloses cristallines de type A ou B. En outre, la bande proche de 1022 cm -1 pourrait être utilisée comme empreinte de l'amylose V. En nous appuyant sur des résultats de la littérature, nous avons observé une corrélation significative entre l'hélicité de l'amylose V et la résonance du carbone Cl dans les spectres de résonnance magnétique nucléaire à l'état solide du 13 C. La résonance est décalée vers les plus faibles champs lorsque le nombre d'unités glucosyles par tour d'hélice augmente. Trois déplacements chimiques du C1 ont été identifiés, correspondant probablement aux hélices d'ordre 6, 7 et 8.

Bien que nous ayons proposé des modèles géométriques pour chaque allomorphe, nous avons mené une étude plus détaillée des cristaux de V1-butanol. D'une part, ce complexe revêt une importance historique puisque le 1-butanol a été l'un des premiers complexants utilisés dans les années 1940 pour fractionner l'amidon natif par cristallisation sélective avec l'amylose. D'autre part, parmi les 5 formes cristallines connues auparavant, c'était la seule pour laquelle l'organisation des hélices était encore hypothétique. De plus, 43 de nos agents complexants testés ont donné l'allomorphe V6II obtenu avec le 1-butanol. Notre approche a reposé sur des analyses d'énergie de conformation et d'empilement, combinées à l'affinement classique de la structure des polymères cristallins. Le modèle avec le facteur d'accord le plus faible est décrit par un réseau orthorhombique P212121 de simples hélices gauches d'ordre 6 antiparallèles dans lesquelles les groupements hydroxyméthyles présentent un désordre conformationnel. La maille contient 4 molécules de 1-butanol et 16 molécules d'eau réparties dans 4 poches interstitielles allongées et 1 molécule de 1-butanol à l'intérieur de chaque hélice. Ce résultat confirme le modèle proposé par [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. Cependant, notre structure n'a été affinée qu'avec les données de DE du plan de base des cristaux. Par conséquent, les positions atomiques le long de l'axe c ne sont pas connues avec précision. Afin de déterminer la structure 3D, des clichés de DE doivent être enregistrés sur des cristaux inclinés autour des axes principaux de l'espace réciproque et l'intensité des tâches de diffraction analysée quantitativement.

En raison du temps limité et du grand nombre de ligands testés, nous n'avons pas pu effectuer le même type d'analyse pour les 5 nouveaux allomorphes, mais nous avons proposé des modèles géométriques basés sur les données cristallographiques et spectroscopiques.

L'empilement des hélices dans les structures V6I, V7I, V7III, V7IV et V8I est plutôt compact et les agents complexants ne seraient situés que dans les hélices. En revanche, il y a plus d'espace interstitiel dans V6II, V6III, V6IV, V7II et V8II pour accueillir les molécules invitées. De plus, les agents complexants peuvent éventuellement être situés à l'intérieur des hélices, à l'exception de V6IV. Puisque V6IV a été obtenu avec de l'acide 4-hydroxybenzoïque, dont la taille semble être incompatible avec la cavité d'une hélice d'ordre 6, le ligand cyclique ne devrait être localisé que dans les espaces interstitiels. Tous les modèles géométriques proposés doivent encore être validés par une analyse structurale détaillée associant données expérimentales et modélisation.

Nos résultats montrent que chaque allomorphe peut être obtenu avec différents agents complexants. Cependant, V6I, V6II et V7II sont les formes les plus répandues. De plus, la conformation hélicoïdale dépend de la taille du ligand. Des complexes de type V6 ont été obtenus avec des molécules à chaînes carbonées linéaires, tandis que les molécules à chaîne ramifiée ou cycliques ont tendance à induire des hélices d'ordre 7. Des complexes de type V8 ont été obtenus avec du 1-naphthol, de la quinoléine et de l'acide salicylique. Cependant, il reste toujours difficile de prédire la structure cristalline en fonction de la nature du ligand car un agent complexant est susceptible d'induire différentes structures cristallines.

Le polymorphisme de l'amylose V ayant été observé avec un grand nombre d'agents complexants, c'est donc une caractéristique plus générale que ce qui avait été précédemment rapporté dans la littérature. La propension d'un complexe au polymorphisme est non seulement liée à la nature de l'agent complexant, mais également aux conditions de cristallisation. Les ligands à chaîne linéaire sont plus susceptibles d'induire un polymorphisme que les composés à chaîne ramifiée ou cyclique. En outre, les molécules à chaîne linéaire, les alcools, les acides gras, les aldéhydes, les amides et les amines induiront probablement la formation d'allomorphes différents que les esters et les cétones.

Différents paramètres, notamment le DP de l'amylose, sa concentration et celle du ligand, la température de mélange et de cristallisation, et la composition du solvant ont un impact important sur la cristallisation et la structure de l'amylose V. Diverses conditions de cristallisation ont dû être explorées afin d'explorer le polymorphisme. Cependant, le mécanisme qui contrôle la structure finale n'est pas encore totalement éclairci.

Nous avons montré que l'eau jouait un rôle crucial sur la stabilité et la cristallinité des complexes. Lors du séchage, V6I, V6II, V6IV, V7I, V7II et V7IV ont été transformés en structures hexagonales plus compactes. Pour V7III, V8I et V8II, une perte significative de cristallinité a été observée, mais la structure cristalline est restée la même. V6III est la seule structure qui s'est montrée stable au séchage. De manière surprenante, dans de nombreux cas, après un séchage sous vide entraînant une perte de cristallinité, la structure d'origine a été restaurée de manière réversible par réhydratation en atmosphère humide. Dans de nombreuses études précédentes, l'accent était mis sur le piégeage et la libération de l'agent complexant, mais au vu de nos résultats, le rôle de l'eau doit être étudié de manière plus approfondie.

Nos résultats suggèrent également que d'autres allomorphes d'amylose V restent à découvrir. De nouveaux agents complexants doivent donc être testés. L'influence de facteurs tels que la composition du solvant et les additifs doit aussi être étudiée de manière plus approfondie. Dans notre étude, seul le DMSO a été utilisé en tant qu'additif. De plus, différentes techniques de préparation pourraient être testées comme par exemple l'insertion d'un ligand dans des hélices préformées ou le chauffage en ampoule scellée.

Pour évaluer le potentiel de l'amylose en tant que système de vectorisation de principes actifs, nous avons étudié des complexes modèles préparés en présence d'ibuprofène racémique.

Des fractions très cristallines de cristaux lamellaires ont été préparées par cristallisation en solution aqueuse ou un mélange eau / DMSO (0,1 à 1% en masse d'amylose). Le diagramme de diffraction électronique du plan de base des cristaux, le profil de DRX de poudre et les spectres de spectroscopie IR et de RMN du solide du 13 C ont montré que le complexe présentait un type allomorphique V7II. Des modèles moléculaires numériques avec ou sans ibuprofène interstitiel ont été construits et optimisés par dynamique moléculaire en se basant sur la maille isomorphe du complexe V2-propanol précédemment publiée. Le résultat suggère que la stabilité des complexes ne dépendrait pas de la présence d'ibuprofène dans les espaces interstitiels.

Toutefois, l'adéquation entre le diagramme de DE expérimental et ceux calculés à partir des modèles proposés n'est pas encore suffisamment satisfaisant. Il faudra donc poursuivre l'étude de simulation pour améliorer le modèle à l'aide d'une analyse de l'énergie d'empilement.

Notre étude de dissolution in vitro a révélé que les complexes de Vibuprofène présentaient différents profils de libération. En particulier, différentes fractions d'ibuprofène sont libérées sélectivement en fonction du pH du milieu. Ce résultat suggère que l'ibuprofène serait localisé à différents endroits dans la maille et aurait différentes interactions avec l'amylose. Par exemple, les molécules d'ibuprofène situées à l'intérieur de l'hélice seraient plus étroitement liées et joueraient un rôle plus important sur la stabilité des complexes que celles situées dans l'espace inter-hélice. La libération intervient principalement à pH élevé, les complexes d'inclusion semblent être un système de délivrance d'ibuprofène potentiellement intéressant pour le ciblage intestinal et permettraient donc d'améliorer son effet thérapeutique et d'éviter les dommages à l'estomac.

Introduction

Amylose is an almost linear homopolymer of α-D-glucosyl units bound through α(1→4) glucosidic linkages. This polysaccharide is one of main components of native starch granules, accounting for about 20-30% of its weight. In addition, it can be synthetized in vitro by using different enzymes. The linear nature of the chain and the type of glucosidic bond have a significant influence on the behavior of amylose in solution. An important property of amylose is its versatility to form inclusion complexes when it is crystallized in the presence of a large variety of inorganic and organic guests. In this so-called V-amylose form, the polymer adopts a single helical conformation.

The inclusion complexes present a number of interesting properties and potential applications, i.e. in food and pharmaceutical industry. For instance, the complexation of amylose with lipids modulates the rheological and hydrolysis properties of amylose/starch and they are used in the baking industry to delay the staling of bread. Organic complexing agents like 1-butanol, thymol or menthone, have also been used for the fractionation of native starch into its amylopectin and amylose constituents. In addition, amylose present complexation properties very similar to those of cyclodextrins. Therefore, it has been exploited to encapsulate flavors and bioactive molecules.

V-amylose can be prepared in different crystalline forms (fibers, lamellar single crystals, spherulites) depending on the crystallization conditions. X-ray and electron diffraction have allowed identifying different allomorphs. Molecular models based on left-handed single helices have been proposed but only a small number of them have been validated by crystallographic approaches. In particular, several structural details remain unresolved, like the helical conformation, packing arrangement and location of the guest molecules. Moreover, the complexation phenomenon and crystallization parameters affecting the crystal structure still remain to be investigated in details This thesis aimed at further elucidating the formation and crystallization behavior of V-amylose from dilute solutions. The complexation was systematically investigated by varying several crystallization parameters: degree of polymerization of amylose, nature of complexing agent, concentration of amylose and complexing agent, crystallization temperature, and solvent composition. A major part of this study was devoted to the structural characterization of the crystalline complexes in both hydrated and dry states. The collection of crystallographic data from different allomorphs, especially those that we have identified for the first time, allowed proposing a helical conformation and packing arrangement for each allomorphic family. In addition, in order to evaluate the potential of V-amylose as a delivery system of bioactive molecules, complexes with ibuprofen were prepared as a model to investigate the encapsulation and release properties in relation to the crystal structure.

This manuscript is divided into six chapters. Chapter I is a bibliographic review on amylose and its inclusion complexes under the generic name of V-amylose. Chapter II presents the materials, the experimental protocols to prepare the complexes and the characterization techniques that were used. In Chapters III to VI, we describe and discuss the results of our study. Chapter III presents the molecular and crystal structure of V1-butanol which is one of the first molecules that have been used to recrystallize amylose on a large scale but which model was still hypothetical. In Chapter IV, we describe the morphology and crystal structure of all allomorphic families determined in this thesis. Chapter V describes the effect of different crystallization parameters on the formation and crystal structure of V-amylose complexes prepared with different guest molecules. Finally, Chapter VI presents the crystal structure and release properties of the Vibuprofen complex.

Chapter I Bibliography

I.1. Amylose

Amylose is a polysaccharide that, together with amylopectin, is a main component of native starch granules, accounting for about 20-30% of its weight [START_REF] Hanashiro | Fine structure of amylose[END_REF]. In addition, amylose can be synthetized in vitro by using different enzymes [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF].

In contrast to highly branched amylopectin, amylose is generally considered as linear or slightly branched. The linear nature has an important effect on the physicochemical properties of amylose, i.e. the conformation in solution and the crystallization properties. In the starch granules, amylose contributes to the amorphous regions in alternation with the semi-crystalline layers of amylopectin. However, in terms of in vitro crystallization, amylose is more versatile than amylopectin.

I.1.1. Chemical structure

Amylose is a homopolymer of α-D-glucosyl units. Although the polysaccharide was once considered linear in which the glucose units bound to each other uniquely through α(1→4) glucosidic linkages, evidence revealed that the molecule may contain a few very long branches or multiple short branches linked with the main chain through α(1→6) linkages [START_REF] Hanashiro | Fine structure of amylose[END_REF][START_REF] Jane | Structural features of starch granules II[END_REF][START_REF] Takeda | Structures of sub-fractions of corn amylose[END_REF][START_REF] Tester | Starch-composition, fine structure and architecture[END_REF]. The chemical structure of a linear amylose is illustrated in short chains; Ø, reducing end [START_REF] Takeda | Structures of sub-fractions of corn amylose[END_REF].

The presence of branches in native amylose was first demonstrated by the incomplete degradation of amylose by β-amylase which is an exo-acting enzyme splitting selectively the α(1→4) bonds [START_REF] Peat | Enzymic synthesis and degradation of starch. Part XV. α-Amylase and the constitution of amylose[END_REF][START_REF] Peat | The amylolytic enzymes of soya bean[END_REF]. However, amylose is totally degraded into maltose by the successive action of a β-amylase and a debranching enzyme which specifically hydrolyzes the α(1→6) bonds [START_REF] Banks | The fine structure of amylose: the action of pullulanase as evidence of branching[END_REF][START_REF] Kjølberg | Studies on carbohydrate-metabolizing enzymes. 9. The action of isoamylase on amylose[END_REF].

Several other evidences for the existence of α(1→6) linkages were then reported from the differences in size distribution of amyloses with and without debranching by an isoamylase [START_REF] Colonna | Macromolecular structure of wrinkled-and smooth-pea starch components[END_REF][START_REF] Würsch | Structure of starch from mango seed[END_REF] or the differences in molecular size determined by osmotic pressure measurement and determination of the non-reducing terminal residues by periodate oxidation (Potter & Hassid, 1948a, 1948b). Recently, the advancement in atomic force microscopy has enabled imaging branched amylose, revealing the presence of single-branched molecules with a long side-chain and multiple-branched molecules with shorter side-chains [START_REF] Gunning | Surfactant-mediated solubilisation of amylose and visualisation by atomic force microscopy[END_REF].

Similarly to other native polysaccharides, amylose is polydisperse in molecular size [START_REF] Hanashiro | Fine structure of amylose[END_REF]. The size of the polymer is defined by its molecular weight (M) or more frequently its degree of polymerization given by DP = M/Mo, where Mo is the molecular weight of the monomer unit. Because of the polydispersity, the M or DP values are generally expressed as average values and can be either weight-or molar-based (denoted by subscript w for weight-average or n for number-average, respectively, as in Mw, DPw or Mn, DPn). Different methods are now available to characterize the chain length of amylose. However, each technique often only gives either the number or weight-average value. For example, the osmometry (Potter & Hassid, 1948b;[START_REF] Van Dijk | The determination of the molecular weight distribution of amylose by gel permeation chromatography[END_REF], the determination of the reducing residues such as a modified Park-Johnson method [START_REF] Hizukuri | Multi-branched nature of amylose and the action of debranching enzymes[END_REF]Potter & Hassid, 1948a), and a bicinchoninic acid method using a microtiter plate format [START_REF] Utsumi | Quantitative assay method for starch branching enzyme with bicinchoninic acid by measuring the reducing terminals of glucans[END_REF],

give a number-average value, while viscometry [START_REF] Banks | The hydrodynamic behaviour of native amylose in good solvents[END_REF] and light scattering measure a weight-average value [START_REF] Miles | Gelation of amylose[END_REF]. The most common technique for measuring the size of amylose is a variant of high-pressure liquid chromatography (HPLC)

in combination with a weight-and/or a molar-based detection method, known as gel-permeation chromatography (GPC) or high-performance size-exclusion chromatography (HPSEC) [START_REF] Ozcan | The impact of thermal events on amylose-fatty acid complexes[END_REF][START_REF] Takeda | Examination of the purity and structure of amylose by gel-permeation chromatography[END_REF][START_REF] Van Dijk | The determination of the molecular weight distribution of amylose by gel permeation chromatography[END_REF]. These techniques make the characterization of an amylose solution easier and the result can be expressed as a size distribution. There is no specified range for the molecular size of amylose. The DPn can vary from a few dozens to several thousands [START_REF] Bertoft | Analysing starch structure[END_REF].

I.1.2. Sources of amylose

I.1.2.1. Native amylose

Native amylose designates the molecules retrieved by fractionation of starch. The amylose content depends on the botanical as well as varietal sources of starches. So-called 'standard' starches contain 20-30 wt% amylose. In 'waxy' starches produced by certain mutant plants, the amount of amylose is nearly zero to a few percent, while 'high-amylose' starches can contain up to 80-100% amylose [START_REF] Bertoft | Analysing starch structure[END_REF][START_REF] Carciofi | Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules[END_REF][START_REF] Hanashiro | Fine structure of amylose[END_REF].

Native amylose contains a mixture of linear and branched molecules [START_REF] Hanashiro | Fine structure of amylose[END_REF].

So far, no effective method for the separation of linear and branched amyloses is known [START_REF] Buléon | Starch granules: structure and biosynthesis[END_REF]. The ratio of linear to branched molecules is usually measured by determining the β-amylase limit dextrin that is labeled at its reducing end prior to β-amylolysis [START_REF] Hanashiro | Branched structures of rice amylose examined by differential fluorescence detection of side-chain distribution[END_REF]Takeda et al., 1992a). On a molar basis, branched molecules are accounted for 15-70% with typical values of 20-50% , depending on the botanical and varietal sources [START_REF] Hanashiro | Fine structure of amylose[END_REF][START_REF] Jane | Structural features of starch granules II[END_REF]. The amount of branched amylose also varies between the different molecular weight fractions. For example, the fraction of large molecules (DP 2500) of maize and rice amyloses are mainly branched molecules (66% for maize, 61% for rice) while the fraction of small molecules (DP 400) contains lower amounts of branched chains (29% for maize, 25% for rice) [START_REF] Hanashiro | Fine structure of amylose[END_REF][START_REF] Jane | Structural features of starch granules II[END_REF].

The average chain length of native amylose considerably varies between the different botanical sources with DPn in the range of 5.10 2 to 6.10 3 [START_REF] Jane | Structural features of starch granules II[END_REF][START_REF] Ong | Simultaneous determinations of the molecular weight distributions of amyloses and the fine structures of amylopectins of native starches[END_REF].

Generally, cereal amyloses are shorter than amyloses from root and tuber starches, and branched amylose molecules are 1.5-3.0-fold larger than linear ones from the same preparation [START_REF] Hizukuri | Molecular structures of rice starch[END_REF][START_REF] Takeda | Structures of amylose subfractions with different molecular sizes[END_REF][START_REF] Takeda | Structures of indica rice starches (IR48 and IR64) having intermediate affinities for iodine[END_REF]. On a molar basis, long amyloses with DP > 1000 are predominant (75-90%) in potato and sweet potato starch, while short amyloses with DP < 1000 are predominant (50-75%) in amyloses from maize, wheat, barley, and rice [START_REF] Hanashiro | Examination of number-average degree of polymerization and molar-based distribution of amylose by fluorescent labeling with 2-aminopyridine[END_REF].

Many experimental methods are described in the literature for the fractionation of amylose from starch. Based on their underlying separation principles, [START_REF] Hanashiro | Fine structure of amylose[END_REF] divides them into four groups: (1) aqueous leaching (hot-water extraction) of amylose [START_REF] Banks | The properties of amylose as related to the fractionation and subfractionation of starch[END_REF][START_REF] Greenwood | Physicochemical studies on starches. Part XXIV. The fractionation and characterization of starches of various plant origins[END_REF][START_REF] Higginbotham | 12-The fractionation of starch. Part II-The separation of amylose and amylopectin[END_REF][START_REF] Hizukuri | Properties of hot-water-extractable amylose[END_REF]Meyer et al., 1940a;Meyer et al., 1940b;[START_REF] Mua | Fractionation of regular corn starch: a comparison of aqueous leaching and aqueous dispersion methods[END_REF][START_REF] Young | Fractionation of starch[END_REF], (2) precipitation of an insoluble complex of amylose with complexing agents [START_REF] Bourne | Thymol and cyclohexanol as fractionating agents for starch[END_REF][START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF][START_REF] Lansky | Properties of the fractions and linear subfractions from various starches[END_REF][START_REF] Leiser | Process for separating amylaceous material into amylose and amylopectin[END_REF][START_REF] Marotta | Method for the preparation of dustless starches[END_REF][START_REF] Schoch | Physical aspects of starch[END_REF],

(3) chromatography [START_REF] Karve | Separation of starch components by affinity chromatography[END_REF][START_REF] Kennedy | Fractionation of starch amylopectin and amylose by high performance gel filtration chromatography[END_REF][START_REF] Ulmann | Fragen der papierchromatographischen Untersuchung von Amylopektin und Amylose[END_REF][START_REF] Yamada | Fractionation of maize starch by gel-chromatography[END_REF], and (4) separation of soluble amylose from an insoluble complex of amylopectin with a lectin, concanavalin A [START_REF] Matheson | The chemical structure of amylose and amylopectin fractions of starch from tobacco leaves during development and diurnally-nocturnally[END_REF][START_REF] Matheson | Estimation and fractionation of the essentially unbranched (amylose) and branched (amylopectin) components of starches with concanavalin A[END_REF]. Other techniques such as selective retrogradation [START_REF] Etheridge | Starch fractionation[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Kurimoto | Methods for the separation and purification of amyloses[END_REF][START_REF] Young | Fractionation of starch[END_REF] or ultracentrifugation [START_REF] Takeda | Purification and structure of amylose from rice starch[END_REF] have also been reported. Each method has advantages and disadvantages in terms of its requirement of starch amount, yield and purity of amylose, and throughput. Methods 3 and 4 require less samples and are suitable for high-throughput analysis, but they are not suitable for large-scale production due to low yields. With aqueous leaching, the yield and composition of leached materials depend on several factors, i.e., extraction temperature, heating rate, starch concentration, stirring speed, defatting and other treatments of starch granules before leaching. However, the leached product always contains a certain amount of amylopectin. The fractionation by formation of insoluble complexes of amylose (method 2) allows a higher purity of amylose and is usually used to further purify amylose obtained from other methods, e.g. aqueous leaching.

However, the complexing agents, among which 1-butanol is widely used, should be carefully chosen.

I.1.2.2. In vitro biosynthesis of amylose

Amylose can be synthesized in vitro using various enzymes, as listed in Table I.1 [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF]. Unlike native amylose, synthetic amylose has a narrower molecular weight distribution and an unbranched, linear structure. In addition, while the fractionated native amylose is usually contaminated by amylopectin, the in vitro synthesis of amylose allows obtaining pure fractions. However, the chain length of amylose and yield depend on the enzymes that are used.

Glucan phosphorylase (GP) and its substrate, glucose-1-phosphate (G-1-P), constitute an excellent system to produce amylose since the molecular size of amylose can be controlled precisely with high yield (>60%) [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF][START_REF] Yanase | Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L α-glucan phosphorylase[END_REF]. However, the problem of this system is that G-1-P is too expensive for industrial purposes. Similarly, starch (glycogen) synthases [START_REF] Leloir | Starch and oligosaccharide synthesis from uridine diphosphate glucose[END_REF][START_REF] Recondo | Adenosine diphosphate glucose and starch synthesis[END_REF][START_REF] Rongine | Mechanism of starch biosynthesis[END_REF][START_REF] Tanaka | Enzymic mechanism of starch synthesis in ripening rice grains VI. Isozymes of starch synthetase[END_REF] are not suitable for the mass production since their substrates (UDP-and ADP-glucose) are not available on an industrial scale.

Isoamylases [START_REF] Akai | Complete hydrolysis of branching linkages in glycogen by Pseudomonas isoamylase: distribution of linear chains[END_REF][START_REF] Harada | Characterization of Pseudomonas isoamylase by its actions on amylopectin and glycogen: comparison with Aerobacter pullulanase[END_REF]Masashi & Kaname, 1975), CGTase [START_REF] Niemann | Enzymatic synthesis of low molecular weight amyloses with modified terminal groups[END_REF][START_REF] Shibuya | The formation of amylose-granules from cyclomaltodextrin glucanotransferase by αcyclodextrin[END_REF], D-enzyme [START_REF] Walker | Synthesis of amylose by potato D-enzyme[END_REF], amylosucrases [START_REF] De Montalk | Amylosucrase from Neisseria polysaccharea: novel catalytic properties[END_REF][START_REF] Potocki-Veronese | Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties[END_REF][START_REF] Roblin | SAXS conformational tracking of amylose synthesized by amylosucrases[END_REF] require cheaper substrates and produce amylose with high yields. However, it is not possible to obtain amylose with a desired chain length using these methods since the DP of the products is about 10-100 glucose units.

Recently, the combination of a GP with sucrose phosphorylase and cellobiose phosphorylase allowed the production of synthetic amylose using cheaper substrates [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF][START_REF] Ohdan | Phosphorylase coupling as a tool to convert cellobiose into amylose[END_REF][START_REF] Waldmann | The enzymic utilization of sucrose in the synthesis of amylose and derivatives of amylose, using phosphorylases[END_REF][START_REF] Yanase | α-Glucan phosphorylase and its use in carbohydrate engineering[END_REF]. Sucrose phosphorylase and cellobiose phosphorylase catalyze, respectively, the phosphorolysis of sucrose and cellobiose and produce G-1-P which can be used by the GP for amylose synthesis. These methods yield amylose with a desired chain length similar to the amylose production using GP and G-1-P. The dissolution of amylose is essential for many applications. Amylose can easily be dissolved in several solvents such as DMSO, urea solutions (6-10 M), DMSO/urea mixtures, formamide, aqueous alkaline solutions, acidic conditions and DMAc/LiCl, etc. (Banks & Greenwood, 1971a;[START_REF] Bertoft | Analysing starch structure[END_REF][START_REF] Buléon | Starch granules: structure and biosynthesis[END_REF]. The solutions of amylose in these solvents are usually used for separation, purification and characterization of the structure of amylose. However, due to the solvent toxicity, water is better choice, for instance in food or pharmaceutical industries. The solubility of amylose in water is generally lower and depends on different factors. In addition to conventional factors such as temperature, pressure and molecular weight, the crystallinity or the organization of amylose chains strongly affects the solubility of the polymer. Amorphous amylose can be cold-water soluble [START_REF] Protzman | Process of casting amylose films[END_REF][START_REF] Sarko | Cold water soluble amylose[END_REF] while that occurring in natural starch is easily leached into warm water (70-90 °C) [START_REF] Green | Which starch fraction is water-soluble, amylose or amylopectin[END_REF][START_REF] Mitchell | Starch solubility[END_REF].

On the other hand, amylose obtained by retrogradation or recrystallization is in an organized stable crystalline state which is no longer soluble in cold or warm water [START_REF] Green | Which starch fraction is water-soluble, amylose or amylopectin[END_REF][START_REF] Mitchell | Starch solubility[END_REF][START_REF] Protzman | Process of casting amylose films[END_REF][START_REF] Sarko | Cold water soluble amylose[END_REF]. Commercial retrograded amylose is insoluble in water unless heated above 120 °C, preferably 140 to 170 °C (autoclave)

to destroy hydrogen bonding (Johannes, 1958;[START_REF] Protzman | Process of casting amylose films[END_REF][START_REF] Young | Fractionation of starch[END_REF]. Oxidation and degradation may occur at high temperatures [START_REF] Protzman | Process of casting amylose films[END_REF], but the problem can be avoided by removing oxygen by nitrogen bubbling or addition of a reducing agent such as bisulfites. Furthermore, an alternative method for preparing aqueous solutions of amylose is to dissolve amylose in a good solvent such as DMSO or alkaline solution and then dilute with water to the desired concentration. The advantage of this method is that the conventional autoclaving procedure is avoided.

It is important to note that a solution of amylose in water is metastable at room temperature. With time, a significant precipitation (or retrogradation) of amylose is observed, the rate of which depends upon amylose concentration, ionic strength, and particularly upon the molecular weight of amylose [START_REF] Foster | A light scattering investigation of the retrogradation of amylose[END_REF][START_REF] Loewus | A potentiometric study of the change in iodine binding capacity of amylose while retrograding in dilute solution[END_REF][START_REF] Whistler | Effect of acid hydrolysis on the retrogradation of amylose[END_REF]. The dependence on molecular weight is particularly striking, as low molecular weight amylose alkaline solution (DP of about 100) shows signs of turbidity within minutes of neutralization while high molecular weight amylose solution (DP of about 1000 and higher) remain stable for days or weeks, under comparable conditions. [START_REF] Pfannemüller | Conformation of amylose in aqueous solution: optical rotatory dispersion and circular dichroism of amylose-iodine complexes and dependence on chain length of retrogradation of amylose[END_REF] reported that the retrogradation rate of amyloses in water containing 5% of DMSO exhibits a sharp maximum at DPn of 80. A solution of shorter and longer molecules is more stable. c) helix with the overall form of a random coil. Adapted from Banks and Greenwood (1971a).

I.1.3.2. Conformation of amylose in solution

The conformation of amylose in solution has been the subject of many studies (Banks & Greenwood, 1971a;[START_REF] Everett | The conformation of amylose in solution[END_REF][START_REF] Hayashi | The conformation of amylose in solution[END_REF][START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF][START_REF] St | Nuclear magnetic resonance and conformational studies on amylose and model compounds in dimethyl sulfoxide solution[END_REF]. Different techniques (viscometry, optical rotation, fluorescence spectroscopy, depolarization, NMR spectroscopy) have been employed but the results are still controversial. The models vary from a random coil, interrupted helix to a rigid helix, as shown in Figure I.3 (Banks & Greenwood, 1971a) Several studies has shown that amylose exists as a helix in neutral aqueous solution.

Foster and Zucker (1952) using streaming dichroism concluded that the uncomplexed amylose has the same conformation in aqueous solution as amylose complexed with iodine. Later studies showed a remarkable difference in optical rotation and the intrinsic viscosity between neutral aqueous solution and the alkaline solution of amylose [START_REF] Hayashi | The conformation of amylose in solution[END_REF][START_REF] Rao | Studies of the conformation of amylose in solution[END_REF]. The authors suggested that amylose adopts a helical state stabilized by hydrogen bonds in neutral solution. In alkaline solution, the hydrogen bonds collapses and the helix transforms into a negatively charged expanded coil, consistent with a drop in intrinsic viscosity. The helixcoil transition occurs at about pH 12 [START_REF] Erlander | The polyelectrolyte behavior of amylose and its helixto-coil transition in aqueous alkaline solutions[END_REF]. [START_REF] Holló | Die struktur der amylose-moleküle in wässrigen lösungen[END_REF] showed that the intrinsic viscosity of an amylose aqueous solution was almost unaltered by the addition of iodine up to the point of equivalence for formation of the complex. A similar behavior was also observed by addition of sodium dodecyl sulfate into an amylose solution [START_REF] Rao | Studies of the conformation of amylose in solution[END_REF].

These results supported the fact that amylose must predominantly contain helical segments in the absence of complexing agent. The drop in viscosity at higher complexing agent concentration probably represents an increase in helix content and in perfection of the helical structure. [START_REF] Szejtli | Über die konfiguration der amylosemoleküle in wäßriger lösung[END_REF] concluded that, in aqueous solution at room temperature, the rotation about the α(1→4) glucosidic bonds was hindered by steric factors and only the helix, or the interrupted helix structure, may exist. [START_REF] Erlander | The stability of the helix of amylose and amylopectin in DMSO and water solutions[END_REF] suggested that the helical conformation of amylose in DMSO/water solution is stabilized by means of the hydrogen bonds between the hydroxyl group of adjacent glucose (the C2 and C3'). [START_REF] St | Nuclear magnetic resonance and conformational studies on amylose and model compounds in dimethyl sulfoxide solution[END_REF] also came to the same conclusion after observing the upfield shift of signals attributed

to HO(2) and HO(3') hydroxyl protons in the NMR spectra of amylose in DMSO solution.

In contrast, a number of studies have brought evidence which favors the concept that amylose in aqueous solution and in the absence of a complexing agent behaves as a random coil. The investigation of the viscosity of amylose as a function of Mw in water and neutral aqueous potassium chloride solution gave the relation 𝜂 = 0.115 M w 0.5 [START_REF] Banks | Hydrodynamic properties and dimensions of linear potato amylose molecules in dilute aqueous salt solution[END_REF], 1968a;[START_REF] Banks | Viscosity and sedimentation studies on amylose in aqueous solution-further evidence for non-helical character[END_REF][START_REF] Cowie | Studies on amylose and its derivatives. Part I. Molecular size and configuration of amylose molecules in various solvents[END_REF][START_REF] Cowie | Studies on amylose and its derivatives. Part IV. The viscosity behaviour of amylose in mixed solvents[END_REF][START_REF] Everett | The conformation of amylose in solution[END_REF]. The exponent value of Mw is that expected for a random coil. If amylose possesses a helical, rodlike structure, an exponent of 1.8 would be expected [START_REF] Young | Fractionation of starch[END_REF]. Related investigations showed that amylose in "good" solvents such as DMSO, alkaline solution and formamide gave an exponent values of Mw in range of 0.62-0.89, indicating that the random coil was expanded as a result of long-range interactions with the solvents [START_REF] Banks | The hydrodynamic behaviour of native amylose in good solvents[END_REF][START_REF] Banks | Viscosity and sedimentation studies on amylose in aqueous solution-further evidence for non-helical character[END_REF]Banks & Greenwood, 1971a;[START_REF] Burchard | Das viskositätsverhalten von amylose in verschiedenen lösungsmitteln[END_REF][START_REF] Cowie | Studies on amylose and its derivatives. Part I. Molecular size and configuration of amylose molecules in various solvents[END_REF][START_REF] Everett | The conformation of amylose in solution[END_REF][START_REF] Nakanishi | Conformation of amylose in dimethyl sulfoxide[END_REF]. [START_REF] Banks | The hydrodynamic behaviour of native amylose in good solvents[END_REF] believe that amylose in solution behaves like a random coil and, while there may be some helical character present, it is a very loose in structure.

In contradiction with the previous evidence in favor of a helical conformation of amylose in aqueous solution [START_REF] Holló | Die struktur der amylose-moleküle in wässrigen lösungen[END_REF][START_REF] Rao | Studies of the conformation of amylose in solution[END_REF], [START_REF] Banks | Amylose: a non-helical biopolymer in aqueous solution[END_REF] showed that the viscosity decreased upon addition of complexing agents.

The authors also pointed out the errors that the previous works suffered from. The ionic strength varied during the experiment, and, more importantly, an incorrect viscosity function was used.

This result unambiguously confirmed that amylose existed as random coils in aqueous solution and adopted a helical structure upon the addition of complexing agents. They also suggested that the helicity resulted not only from intramolecular hydrogen bonds but also as a consequence of the geometry of the α(1→4) glucosidic linkages. In agreement with this conclusion, [START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF] observed the marked downfield shift of the signals of C1 and C4 carbons for amylose complexes formed by addition of DMSO, triiodide, alcohols, etc., into aqueous amylodextrin solutions. The changes in the 13 C NMR spectra were shown to be reversed by the chemical destruction of the complexing agent, confirming the role of complexing agent in the helix→coil transition. From the review of its behavior in diluted solution, amylose is likely to have the conformations indicated in Figure I.4 (Banks & Greenwood, 1971a).

I.1.4. Crystalline structure of recrystallized amylose: A-and B-amylose

Native starch granules exhibit two allomorphic types (A-and B-type) that can be readily identified by X-ray diffraction (XRD) and solid-state 13 C NMR spectroscopy (Figure I.5) [START_REF] Lourdin | Crystalline structure in starch[END_REF]. However, starch granules of many different sources exhibit a third XRD pattern referred to as C-type but it has been shown to correspond to a mixture of A-and B-types. It is generally accepted that, in starch granules, amylose forms amorphous layers separated by the layers of semicrystalline amylopectin. [START_REF] Lourdin | Crystalline structure in starch[END_REF][START_REF] Tester | Starch-composition, fine structure and architecture[END_REF]. The extraction of amylose from starch granules by leaching in appropriate conditions does not significantly affect the crystallinity or disrupt the granules [START_REF] Montgomery | Separation of amylose from amylopectin of starch by an extraction-sedimentation procedure[END_REF]. Recently, amylose-only starch granules were successfully produced with high yield in barley by suppression of amylopectin synthesis via silencing the entire complement of genes encoding starch branching enzymes [START_REF] Carciofi | Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules[END_REF]. The amylose-only starch exhibited a relatively low crystallinity (25%), and contained a mixture of B (55%) and V (45%) allomorphs. The V-type was thought to correspond to the crystalline complexes of amylose with the lipids present in the starch granules (see Section I.2.). A similar composition was also observed for high-amylose starches [START_REF] Morell | Barley sex6 mutants lack starch synthase IIa activity and contain a starch with novel properties[END_REF].

Various studies have shown that of both A-type and B-type can be prepared in vitro in the form of single crystals or spherocrystals, as shown in Figures I.6 and I.7, by recrystallization of amylose, especially short-chain amylose or limit dextrins [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]Creek et al [START_REF] Gidley | Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices[END_REF][START_REF] Helbert | Morphological and structural features of amylose spherocrystals of A-type[END_REF][START_REF] Imberty | New three-dimensional structure for A-type starch[END_REF][START_REF] Montesanti | Système modèle de cristallisation de l'amylose A[END_REF][START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF][START_REF] Pfannemüller | Influence of chain length of short monodisperse amyloses on the formation of A-and B-type X-ray diffraction patterns[END_REF][START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF][START_REF] Popov | Aamylose single crystals: unit cell refinement from synchrotron radiation microdiffraction data[END_REF]Putaux et al., 2011a;[START_REF] Ring | Spherulitic crystallization of short chain amylose[END_REF]. The solvent, temperature, concentration of amylose and DP of amylose are the main decisive parameters that control the morphology and the crystal structure [START_REF] Buléon | Self-association and crystallization of amylose[END_REF]. A general rule is that long chains and low crystallization temperatures favor B-type, whereas short chains, high temperatures and high concentrations induce A-type [START_REF] Buléon | Self-association and crystallization of amylose[END_REF]. In addition, a high concentration of amylose, at least 5 wt%, is required for the formation of spherocrystals while single crystals are prepared at lower concentrations (ca. 0.05 wt%) [START_REF] Buléon | Self-association and crystallization of amylose[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Ring | Spherulitic crystallization of short chain amylose[END_REF]. Precipitants such as ethanol and acetone can be added to promote the crystallization of A-amylose while this is not essential for B-amylose [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF][START_REF] Imberty | New three-dimensional structure for A-type starch[END_REF][START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF]. [START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF]; c) scheme describing the orientation of double helices in the crystal [START_REF] Putaux | Helical conformation in crystalline inclusion complexes of V-amylose: a historical perspective[END_REF]; d) SEM image of A-amylose single crystals radially organized in a spherical aggregate [START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF]; e) SEM image of A-amylose spherulites. The radial organization is well revealed in the fracture of one of them, as indicated by the arrow [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

Micrometer-size single crystals of A-amylose with a facetted morphology, as shown in Figure I.6, have been prepared in dilute solutions of short-chain amylose [START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF]. The electron diffraction (ED) analysis showed that the chain axis was parallel but in an opposite direction to that of the crystal growth (Figure I.6). In addition, the crystals exhibited a suitable size and sufficient perfection to allow the collection of ED as well as XRD datasets up to the resolution of 0.151 nm [START_REF] Montesanti | Système modèle de cristallisation de l'amylose A[END_REF][START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF]. On the other hand, lamellar B-amylose crystals with a similar size and perfection have never been prepared from dilute solution. The only lamellar B-type crystals reported in the literature, so far, were aggregated (Figure I.7a) but monocrystalline zones could be identified, giving single crystal ED patterns (Figure I.7b) [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]. [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]; b) base-plane ED pattern recorded on a single crystal [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]; c) SEM image of B-amylose spherocrystals [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. [START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF][START_REF] Takahashi | Crystal structure of B-amylose[END_REF].

By comparison to the starch granules, which exhibit a relatively low crystallinity and a complex ultrastructure, model crystals of recrystallized amylose are more suitable for studying the morphology and crystal structure of allomorphs A and B [START_REF] Imberty | New three-dimensional structure for A-type starch[END_REF]Imberty & Perez, 1988). In the most recent models, based on diffraction data of single crystals and fibers, amylose are organized in parallel-stranded 6-fold left-handed double helices with pitches of 2.12 and 2.11 nm for A and B-type, respectively [START_REF] Montesanti | Système modèle de cristallisation de l'amylose A[END_REF][START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF][START_REF] Takahashi | Crystal structure of B-amylose[END_REF]). In the A-type, these double helices are packed with the B2 space group in a monoclinic unit cell (a = 2.083 nm, b = 1.145 nm, c = 1.058 nm, γ = 122°) with 8 water molecules per unit cell (Figure I.8a) [START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF]. In the B structure, double helices are packed with the P61 space group in a hexagonal unit cell (a = b = 1.852 nm, c = 1.057 nm, γ = 120°) with 36 water molecules per unit cell (Figure I.8b) [START_REF] Takahashi | Crystal structure of B-amylose[END_REF]. On the basis of the space group, the repeating unit is a maltotriosyl unit in the A-form and a maltosyl unit in the B-form. This is supported by the solid-state 13 C NMR data: the C1 peak in the spectra of the A-form is a triplet while it is a doublet in the spectra of the B-form (Figure I.5b).

I.2. Crystalline inclusion complexes of amylose: V-amylose

I.2.1. Definition

V-amylose is the generic term to describe crystalline helical inclusion complexes of amylose with small guest molecules [START_REF] Putseys | Amylose-inclusion complexes: formation, identity and physico-chemical properties[END_REF]Obiro et al., 2012). V-amylose was first reported by [START_REF] Katz | Alle Starkearten haben das gleiche retrogradationsspektrum[END_REF] who studied the baking of bread and characterized samples of starch precipitated with alcohols [START_REF] Katz | Physical chemistry of starch and bread making. XII. The conversion of starch preparations with the potato starch spectrum (B-spectrum) into those with the wheat starch spectrum (A-spectrum) and the reverse[END_REF][START_REF] Bear | The significance of the "V" X-ray diffraction patterns of starches[END_REF]. By studying complexes with iodine, [START_REF] Freudenberg | Neue ansichten über die stärke[END_REF] proposed that V-amylose contained single helices. Later, XRD studies confirmed without ambiguity the helical nature of V-amylose [START_REF] Rundle | On the nature of the starch-iodine complex[END_REF][START_REF] Rundle | The configuration of starch in the starch-iodine complex. IV. An X-ray diffraction investigation of butanol-precipitated amylose[END_REF]. The structure was also characterized and supported by NMR spectroscopy [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF][START_REF] Veregin | Investigation of the crystalline" V" amylose complexes by high-resolution 13 C CP/MAS NMR spectroscopy[END_REF]. The flexibility of the linear chain and the geometry of the α(1→4) glucosidic bonds [START_REF] French | Allowed and preferred shapes of amylose[END_REF][START_REF] French | Intramolecular changes during polymorphic transformations of amylose[END_REF]) are thought to promote helical trajectories, generating a central cavity that can host complexing molecules. A large variety of inorganic and organic molecules have the ability to form the complexes with amylose (Tomasik & Schilling, 1998a, 1998b). The most common are iodine, fatty acids, alcohols, ketones, esters as well as many flavor compounds. Recently, the inclusion complexes of amylose with several polymers [START_REF] Gotanda | Amylose stereoselectively includes poly (d-alanine) to form inclusion complex in vine-twining polymerization: a novel saccharide-peptide supramolecular conjugate[END_REF][START_REF] Kadokawa | Formation of an amylosepolyester inclusion complex by means of phosphorylase-catalyzed enzymatic polymerization of α-D-glucose 1-phosphate monomer in the presence of poly (ε-caprolactone)[END_REF][START_REF] Kadokawa | Vine-twining polymerization: amylose twines around polyethers to form amylose-polyether inclusion complexes[END_REF] and carbon nanotubes [START_REF] Lii | Single-walled carbon nanotubepotato amylose complex[END_REF][START_REF] Yang | In situ synthesis of amylose/single-walled carbon nanotubes supramolecular assembly[END_REF] have also been reported. V-amylose complexes with lipids have been detected in native starch granules [START_REF] Carciofi | Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules[END_REF][START_REF] Gernat | Crystalline parts of three different conformations detected in native and enzymatically degraded starches[END_REF][START_REF] Morell | Barley sex6 mutants lack starch synthase IIa activity and contain a starch with novel properties[END_REF][START_REF] Morrison | Lipids in cereal starches: a review[END_REF]. However, the structure of V-amylose has generally been studied from samples prepared by in vitro crystallization.

I.2.2. Preparation methods

I.2.2.1. Crystallization of amylose in aqueous solution in the presence of a complexing agent

As previously mentioned, aqueous amylose solutions are metastable with the chains adopting a random coil conformation. In the presence of suitable complexing agents, amylose forms helical inclusion complexes and, at low amylose concentration, crystallizes in the form of lamellar crystals. The procedure generally involves dissolving amylose in an appropriate solvent, adding the complexing agent at an elevated temperature and incubating the mixture at a suitable temperature or cooling down to allow the crystallization to occur. The most frequently used solvents are water [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]Nuessli et al., 2003;[START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF], alkaline solution [START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], and DMSO/water mixtures (Biliaderis & Galloway, 1989;Godet et al., 1993). In water, high temperatures (120-170 °C) and high pressures (autoclave) are required for a complete solubilization. Prior to heating, oxygen must be removed from the system by nitrogen bubbling in order to avoid degradation and oxidation. In alkaline solution and DMSO, the dissolution of amylose occurs at room temperature in about 24 h but the process can be accelerated by heating to a higher temperature (90-100 °C). Then, the solution is diluted with water to reach the desired concentration. Besides, a neutralization step is required for the complex formation in alkaline medium [START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF].

Several specific techniques such as homogenization [START_REF] Lesmes | Continuous dual feed homogenization for the production of starch inclusion complexes for controlled release of nutrients[END_REF][START_REF] Meng | Properties of starch-palmitic acid complexes prepared by high pressure homogenization[END_REF], extrusion-cooking [START_REF] Colonna | Macromolecular modifications of manioc starch components by extrusion-cooking with and without lipids[END_REF][START_REF] De Pilli | Study on formation of starch-lipid complexes during extrusion-cooking of almond flour[END_REF][START_REF] Mercier | Formation of amylose-lipid complexes by twin-screw extrusion cooking of manioc starch[END_REF][START_REF] Raphaelides | Effect of processing history on the functional and structural characteristics of starch-fatty acid extrudates[END_REF][START_REF] Raphaelides | A process designed for the continuous production of starch inclusion complexes on an industrial scale[END_REF], steam jet-cooking [START_REF] Byars | The effect of cooling conditions on jet-cooked normal corn starch dispersions[END_REF][START_REF] Fanta | Steam jet cooking of high-amylose starch-fatty acid mixtures. An investigation of complex formation[END_REF][START_REF] Fanta | Formation of crystalline aggregates in slowly-cooled starch solutions prepared by steam jet cooking[END_REF][START_REF] Fanta | Preparation of spherulites from jet cooked mixtures of high amylose starch and fatty acids. Effect of preparative conditions on spherulite morphology and yield[END_REF] and microwave heating [START_REF] Felker | Comparison of microwave processing and excess steam jet cooking for spherulite production from amylose-fatty acid inclusion complexes[END_REF] have been applied to prepare V-amylose complexes. They are thought to increase the solubility of both amylose and ligand under high-shearing and high-heating temperature conditions, and thus enhance the complexation. In addition, these techniques allow to manipulate high starch / amylose concentrations (10-20 wt%), and are thus suitable for a large-scale production.

Besides, the preparation of V-amylose complexes in differential scanning (DSC) calorimetry pans [START_REF] Cieśla | DSC studies of gamma irradiation influence on gelatinisation and amylose-lipid complex transition occurring in wheat starch[END_REF][START_REF] Creek | Potential sources of error in the calorimetric evaluation of amylose content of starches[END_REF][START_REF] Sievert | Determination of amylose by differential scanning calorimetry[END_REF], visco-analyzers and rheometers (D 'Silva et al., 2011;[START_REF] Nelles | Maize starch biphasic pasting curves[END_REF]Obiro et al., 2012) have also been reported.

Recently, [START_REF] Rangelov | Novel mechanochemical approach for wheat starch-LPC complex formation[END_REF] reported a new method of preparation of V-amylose complexes with lysophosphatidylcholine by mechanical milling of aqueous suspension of starch (10 wt%) in the presence of complexing agent. In addition, Le [START_REF] Bail | Formation and stability of amylose ligand complexes formed by high pressure treatment[END_REF] reported the formation of V-amylose complexes under high pressure treatment. The advantage of the use of high pressure and mechanical milling is that they allow producing a large amount of compound in a mild condition (low temperature, i.e. 40 °C).

I.2.2.2. Crystallization of amylose in the dry state (sealed-heating method)

The procedure consists in sealing the physical mixture of amylose and complexing agent in a glass container and then heating at 100-150 °C to allow the formation of complexes [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF]Tozuka et al., 2006;[START_REF] Uchino | The change of the structure of amylose during the inclusion of 2-naphthol in sealed-heating process[END_REF][START_REF] Uchino | Inclusion compound formation of amylose by sealed-heating with salicylic acid analogues[END_REF]. The predissolution of amylose in a solvent is not necessary. Despite being prepared in the dry state, the resulting V-amylose appears to be significantly crystalline, as indicated by rather well defined powder XRD patterns.

I.2.2.3. Crystallization of amylose by solvent removal

This method has been used to prepare the complexes of amylose in the presence of solvent molecules such as DMSO and ethylenediamine (French & Zobel, 1967;[START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF][START_REF] Zobel | X-Ray diffraction of oriented amylose fibers. II. Structure of V amyloses[END_REF]. First, concentrated amylose solutions (20-25 wt%) in the solvent are prepared, then casted in the form of a film on a glass plate. After evaporation of the excess solvent, the crystalline films are uniaxially stretched so that fiber XRD patterns of the resulting V-amylose can be recorded.

I.2.2.4. Vine-twinning enzymatic polymerization

This method uses glucan phosphorylase (GP) and glucose-1-phosphate (G-1-P) to enzymatically construct an amylose helix around appropriate hydrophobic ligands such as lipids, single-wall carbon nanotubes, ibuprofens and many polymers (Gelders et al., 2005b;[START_REF] Kadokawa | Preparation and applications of amylose supramolecules by means of phosphorylase-catalyzed enzymatic polymerization[END_REF][START_REF] Yang | In situ synthesis of amylose/single-walled carbon nanotubes supramolecular assembly[END_REF][START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF]. In particular, for amylose-polymer inclusion complexes, the method has been referred as 'vine-twinning polymerization'.

This biotechnology approach shows several advantages over other preparation methods of V-amylose. First, it allows preparing inclusion complexes with polymers or carbon nanotubes which are hardly obtained by other methods. Second, the DP of amylose can be controlled by varying the amount of reacted G-1-P (Gelders et al., 2005b;[START_REF] Kaneko | Amylose selectively includes a specific range of molecular weights in poly (tetrahydrofuran) s in vine-twining polymerization[END_REF]Kaneko et al., 2008b;[START_REF] Putseys | Production of tailor made short chain amylose-lipid complexes using varying reaction conditions[END_REF]. Thirdly, it allows selective inclusions: i) of one from a mixture of two resembling guest polymers [START_REF] Kaneko | Amylose selectively includes one from a mixture of two resemblant polyethers in vine-twining polymerization[END_REF]Kaneko et al., 2008a); ii) of one from a mixture of two stereoisomers [START_REF] Gotanda | Amylose stereoselectively includes poly (d-alanine) to form inclusion complex in vine-twining polymerization: a novel saccharide-peptide supramolecular conjugate[END_REF]; and iii) of a specific range of molecular weights of guest polymers [START_REF] Kaneko | Amylose selectively includes a specific range of molecular weights in poly (tetrahydrofuran) s in vine-twining polymerization[END_REF]. On the other hand, the disadvantages of this method are the use of expensive substrate (G-1-P) and extended reaction times for preparing small amounts of amylose complexes.

I.2.2.5. Insertion of a complexing agent in preformed V-amylose

This method uses a preformed V-amylose to produce inclusion complexes of a desired complexing agent. One of the approaches is to exchange the complexing agent present in the preformed V-amylose by the new one. This can be done by dispersing or successive washing the preformed V-amylose crystals with the new complexing agents (solvents) or with hydroalcoholic solutions of the new complexing agents [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF][START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Zobel | X-Ray diffraction of oriented amylose fibers. II. Structure of V amyloses[END_REF]. It is interesting to note that the exchange of complexing agents can result in a change in crystal structure and helical conformation but the initial morphology of the crystal is maintained.

Recently, [START_REF] Kong | Molecular encapsulation of ascorbyl palmitate in preformed V-type starch and amylose[END_REF] proposed another approach for preparing amylose complexes in which the guest molecules were inserted into preformed "empty" V-amylose helices. The "empty" V-amylose was prepared by thoroughly drying V-amylose complexed with highly volatile ethanol. The preformed "empty" V-amylose and the guest molecules were then mixed together in an acetone/ethanol/water mixture to induce the formation of complexes.

The acetone/ethanol/water mixture was used to increase the diffusibility of the guest molecules.

The advantage of the above techniques is that the insertion of the desired complexing agent can be conducted at room temperature and without solubilizing amylose. However, the results would depend on the affinity of the complexing agents with the preformed V-amylose.

I.2.3. V-amylose polymorphism

In the literature, V-amylose complexed with more than 150 different complexing agents have been prepared in the form of crystalline fibers or lamellar crystals [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. They

have been grouped into 8 allomorphic families based on XRD or ED patterns, as listed in Table I.2. The origin of the polymorphism has been accounted for by the flexibility of the linear amylose chains that adapt their arrangements and helical conformation to adjust to the molecular shape and chemical nature of the complexing agents [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] French | Allowed and preferred shapes of amylose[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

Although the knowledge of the molecular structure is important to understand the interaction between amylose and complexing agents, the conformation and packing of amylose are difficult to determine, with a limited number of ED or XRD reflections recorded from fiber and lamellar crystals. Therefore, only a small number of structures have been resolved and several models are still hypothetical [START_REF] Putaux | Helical conformation in crystalline inclusion complexes of V-amylose: a historical perspective[END_REF].

It is interesting to note that all the models proposed for V-amylose are based on lefthanded single helices with different conformations (Figure I.9). In the following, the V-amylose allomorphic families will be presented in two groups of amylose helical conformations: compact-helix and extended-helix. [START_REF] Sarko | Crystal structure of the KOH-amylose complex[END_REF].

I.2.3.1. Compact-helix V-amylose

The majority of examples of V-amylose complexed with iodine and small organic molecules contain compact helices whose cavity can host the complexing agents. Depending on the nature and size of the guest molecule, helices with 6, 7 and 8 glucosyl residues per turn have been reported [START_REF] Putaux | Helical conformation in crystalline inclusion complexes of V-amylose: a historical perspective[END_REF]. All of them have a similar pitch of about 0.8 nm, indicating a compact arrangement of glucosyl residues (Figure I.9). The characterization of compact-helix complexes having different helical conformations by solid-state 13 C NMR revealed that all the V-amylose single helices give only one signal for each carbons of the glucosyl residues and are thus more symmetric than that of the double-stranded A-and B-type [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF][START_REF] Veregin | Investigation of the crystalline" V" amylose complexes by high-resolution 13 C CP/MAS NMR spectroscopy[END_REF]. The helices are stabilized by intramolecular hydrogen bonds between O-2 and O-3(2) of each pair of contiguous glucose residues and between atoms O-6(i+7) and O-2(i+1) of each pair of contiguous helix loops [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF][START_REF] Sarko | Crystal structures of amylose and its derivatives: a review[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF].

Five allomorphic families of compact-helix V-amylose have been reported, in which three contain 6-fold helix, one contains 7-fold helix and one contains 8-fold helix. [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] proposed a nomenclature system for the compact-helix V-amylose based on the number of residues per turn and volume of inter-helical space. Consequently, the three 6-fold V-amylose families can be called as V6I, V6II as V6III as a function of the volume of inter-helical space. The 7-and 8-fold V-amylose families can be called V7 and V8, respectively. Some of the compact-helix V-amylose samples have been prepared as fibers yielding wellresolved X-ray fiber diffraction diagram [START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF]French & Zobel, 1967;[START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF][START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF][START_REF] Winter | The crystal structure of amylose-DMSO complex[END_REF][START_REF] Zaslow | The V amylose-H2O system: structural changes resulting from hydration[END_REF][START_REF] Zobel | X-Ray diffraction of oriented amylose fibers. II. Structure of V amyloses[END_REF]. However, when crystallized from dilute solution, amylose can form micrometer-size lamellar single crystals that can be analyzed by electron diffraction [START_REF] Buléon | Self-association and crystallization of amylose[END_REF][START_REF] Putaux | Helical conformation in crystalline inclusion complexes of V-amylose: a historical perspective[END_REF]. The crystals are about 10 nm-thick and the chain axis is perpendicular to the lamella surface. This implies that when chains are much longer than the lamella thickness, chain folding occurs, favored by the so-called 'flip' between two adjacent glucosyl residues which leads to a reversal of molecular trajectory [START_REF] Jacob | Strain-induced "band flips" in cyclodecaamylose and higher homologues[END_REF].

I.2.3.1.1. V6I
V6I, also frequently called Vh, is the best documented crystalline V-amylose family in the literature [START_REF] Buléon | Self-association and crystallization of amylose[END_REF]. It can be prepared with iodine as well as many organic molecules having a linear aliphatic chain such as fatty acids, alcohols, emulsifiers [START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF][START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. In addition, some bulky molecules such as quinoline, (-)-borneol, trans-decalin, p-aminobenzoic acid and analogues were also reported to yield this structure, although their size are not compatible with the 6-fold helical cavity [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]Tozuka et al., 2006). [START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF] first proposed a model determined from XRD data of crystalline fibers (Figure I.10d), which consists of a pseudo-hexagonal orthorhombic unit cell, space group P212121, with a = 1.365 nm, b = 2.370 nm, and c = 0.805 nm. The unit cell contains two 6-fold left-handed helices and 16 water molecules (eight inside and eight between the helices). Within the model, the helices are packed on a hexagonal network but with a regularly alternating arrangement of up and down chains. [START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF] also proposed a similar model from fibers of V6I obtained with iodine.

On the other hand, lamellar single crystals prepared in dilute solution exhibit a characteristic hexagonal shape and give a sharp ED diagram with a hexagonal symmetry (Figure I.10a anh I.10b) [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Based on the ED data, [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] proposed a another model based on a hexagonal unit cell, space group P6522 with a = b=1.365 nm, and c = 0.805 nm (Figure I.10c). The unit cell contains one 6-fold left-handed helix and 18 water molecules (6 inside and 12 between the helices). The helices are thus packed on a hexagonal network with statistically random up/down chain disorder.

The two models are essentially similar in terms of helical conformation and hexagonal arrangement of helix. The center-to-center helix distance (helix packing diameter) is equal to the a-parameter (1.37 nm). In the compact arrangement, the complexing agent can only reside inside the helix since the interstitial space is limited [START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF]Godet et al., 1993).

Upon thorough drying, V6I is transformed into the so-called Va with a smaller unit cell (a = b = 1.30 nm and c = 0.79 nm for the hexagonal unit cell, and a = 1.30 nm, b = 2.25 nm and c = 0.79 nm for the orthorhombic one) [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Murphy | The structure of V amylose dehydrate: a combined X-ray and stereochemical approach[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]Winter & Sarko, 1974b). The hexagonal arrangement remains the same but the helices get closer, probably due to the elimination of interstitial water molecules. Indeed, [START_REF] Murphy | The structure of V amylose dehydrate: a combined X-ray and stereochemical approach[END_REF] showed that the dry complex was nearly free of water. [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]; c,d) projections on the (a,b) plane of the molecular models based on hexagonal [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] and orthorhombic unit cells [START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF], respectively.

I.2.3.1.2. V6II

V6II complexes, also called Vn-butanol type, can be obtained by crystallization of amylose in the presence of n-butanol as well as other linear aliphatic alcohols, ketones having 6-8 carbons, hexanal, ethyl hexanoate, hexanoic acid and some dicarboxylic acids [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF][START_REF] Schoch | Fractionation of starch by selective precipitation with butanol[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF]). These crystals are very sensitive to desolvation.

Air or vacuum drying the crystals results in a transition from V6II to V6I and finally to Va.

Lamellar crystals prepared from dilute solution have a rectangular shape (Figure I.11a).

Base-plane ED patterns recorded on frozen-hydrated crystals at low temperature exhibit a rectangular pattern (Figure I.11b) that can be indexed on the basis of an orthorhombic unit cell, space group P212121, with a = 2.74 nm, b = 2.65 nm and c = 0.80 nm [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. The same result is obtained from XRD powder data of hydrated crystals [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF]. Since V6II can easily be transformed into V6I upon drying, the crystals have been thought to contain 6-fold left handed-helices. [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF] proposed that amylose helices were organized in antiparallel pairs with some solvent molecules located between the helices and occupying about 10% of the cell volume (Figure I.11c).

Although crystalline fibers of V6II prepared with n-butanol have also been studied [START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF], there is still no conclusive data regarding the helical conformation and packing arrangement of the amylose chains.

I.2.3.1.3. V6III

The term V6III was once used to refer the V2-propanol crystals and isomorphous complexes [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. However, the structure of V2-propanol has recently been solved by refinement against ED data combined with packing analyses,

showing that the complexes contain 7-fold helices [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF] rather than the previously proposed 6-fold helices [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF].

The term V6III is now used to name the 6-fold allomorphic family of VDMSO (French & Zobel, 1967) and isomorphous structures (French & Murphy, 1977a;[START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF][START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF]. The crystals have been prepared in the form of fibers and characterized by XRD.

Lamellar single crystals were also prepared from dilute solutions in the presence of glycerol [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF]. They have a square shape with lateral dimensions of several micrometers with a = b = 1.917 nm and c = 2.439 nm. It contains two antiparallel 6-fold left-handed helices with three turns per crystallographic repeat. DMSO is located both inside and between the helix along with some water molecules. The interstitial DMSO is thought to be the source for additional layer lines and the higher crystallographic repeat which is three times of the 0.81-nm helix repeat distance. Similar lattices constants have been found for Vglycerol (French & Murphy, 1977a) and Vethylenediamine [START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF] by XRD but the c-parameter was reported to be equal to the helix repeat. On the other hand, [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF] found a pseudo-tetragonal unit cell with a slightly different from b, based on ED data (a = 1.93 nm, b = 1.86 nm and c = 0.83 nm).

Unlike other V6 complexes, the V6III is stable under vacuum, suggesting that water and complexing agents are bound very tightly. The removal of the complexing agent and water from V6III complexes requires drastic conditions such as washing with alcoholic solutions. This results in a transition into V6I (French & Zobel, 1967;[START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF].

I.2.3.1.4. V7

This 7-fold allomorphic family includes V2-propanol and isomorphous complexes prepared with DMSO, branched alcohols, ketones with 3-5 carbons, short-chain fatty acids and many cyclic compounds [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF][START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]Nuessli et al., 2003;[START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF][START_REF] Shogren | X-ray diffraction study of crystal transformations in spherulitic amylose/lipid complexes from jet-cooked starch[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF]. The crystals prepared from dilute solution are rectangular lamellae, radiating from the same nucleation site, forming characteristic flower-like aggregates (Figure I.13a) [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF]Nuessli et al., 2003;[START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF][START_REF] Yamashita | An electron microscopie study on crystals of amylose V complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]. They yield a sharp base-plane ED pattern exhibiting an orthorhombic symmetry (Figure I.13b).

The existence of a 7-fold helical conformation was first proposed by [START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF] for complexes with tert-butanol and was then supported by [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]. However, [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] observed that the isomorphous complexes prepared with 2-propanol or acetone could be transformed into V6I after washing with alcohols and drying without changing the crystal morphology. The authors thus proposed an alternate model based on 6-fold helices and the "V6III" denomination was then given for this allomorphic type by [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

Recently, the crystallographic study by [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF] has demonstrated that the constituting helices were more likely 7-fold. Consequently, we will use the notation "V7" to name this allomorphic family. Upon thorough drying, V7 complexes are transformed into a new structure with pseudohexagonal unit cell with a = b = 1.47 nm [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF]. The 7-fold helical conformation remains unchanged, but the packing arrangement is reverted from orthorhombic to hexagonal, probably due the removal of interstitial waters and complexing agent. This behavior is similar to that of V6II complexes. At present, there is still no name proposed for this structure. Since it is an anhydrous form of V7, we will refer it as V7a.

Similarly, the Va-type obtained by drying V6I and V6II can be called V6a. In addition, the extraction of complexing agents from V7 complexes using n-aliphatic alcohols miscible with water (methanol, ethanol and n-propanol) or their aqueous solution results in a transition from 7-to 6-fold helices [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF].

I.2.3.1.5. V8

The V8 family includes V1-naphthol and the isomorphous Vquinoline [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF]. So far, no third complex with a similar structure has been identified. Lamellar single crystals of V1-naphthol prepared in dilute solution have a squarish shape and yield a base-plane ED pattern with a square symmetry (Figure I.14a and I.14b) [START_REF] Cardoso | From rice starch to amylose crystals: alkaline extraction of rice starch, solution properties of amylose and crystal structure of V-amylose inclusion complexes[END_REF]. The most recent molecular model proposed by [START_REF] Cardoso | From rice starch to amylose crystals: alkaline extraction of rice starch, solution properties of amylose and crystal structure of V-amylose inclusion complexes[END_REF] [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF].

Lamellar crystals of Vquinoline exhibit a morphology similar to that of V1-naphthol [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] reported that the a and b parameters (4.66 nm) was twice larger than those of V1-naphthol. However, a recent study by [START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF] found similar unit cell parameters for the two complexes. The V8 complexes were found to be stable upon drying. In addition, the transformation from 8-fold helix into 6-fold helix has not been reported by solvent exchange [START_REF] Cardoso | From rice starch to amylose crystals: alkaline extraction of rice starch, solution properties of amylose and crystal structure of V-amylose inclusion complexes[END_REF].

I.2.3.2. Extended-helix V-amylose

Unlike compact amylose helices, the extended helices exhibit a much longer pitch which varies depending on the number of glucosyl residues per turn (Table I.2). In addition, the intramolecular hydrogen bonds, which are responsible for the conformation of compact helices, are not possible in these extended conformation [START_REF] Sarko | Crystal structure of the KOH-amylose complex[END_REF][START_REF] Sarko | Crystal structures of amylose and its derivatives: a review[END_REF]. The helices assume much more extended shapes, losing the inner cavity found in the compact helix (Figure I.9). Therefore, the amylose chains have been called linear and the extended-helix amylose complexes are not considered as V-amylose by some authors (French & Murphy, 1977a;[START_REF] Sarko | Crystal structures of amylose and its derivatives: a review[END_REF]. However, these forms are still mathematically helices and may be described with the same parameters. Therefore, the extended-helix amylose complexes generally meet all the criteria of V-amylose. They have been induced when complexing amylose with several alkali and inorganic salts [START_REF] Miller | Accurate fiber X-ray diffraction data from films[END_REF][START_REF] Sarko | Crystal structure of the KOH-amylose complex[END_REF][START_REF] Senti | Structure of alkali amylose[END_REF][START_REF] Senti | Oriented filaments of amylose and alkali amylose[END_REF][START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF]. The complexes can be classified into several allomorphic families based on their fiber XRD pattern.

However, there is still no nomenclature system for them, so far. [START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF].

I.2.3.2.1. VKOH and isomorphous structures

V-amylose complexed with a series of alkali including lithium (LiOH), sodium (NaOH), potassium (KOH), ammonium (NH4OH), cesium (CsOH), guanidinium hydroxides can be obtained during the solid-state deacetylation of amylose triacetate by alkali solutions in 75% ethanol, as first reported by [START_REF] Senti | Structure of alkali amylose[END_REF]. All of the alkali-amylose complexes are isomorphous, exhibiting similar fiber XRD patterns and unit cell parameters.

The detailed structure of VKOH has been determined by [START_REF] Sarko | Crystal structure of the KOH-amylose complex[END_REF] In contrast, extraction with 75% MeOH or EtOH results in the characteristic compact-helix V structure. Soaking in 75% alcohol containing 2% alkali restores the original pattern.

I.2.3.2.2. VKBr and isomorphous structures

A series of isomorphous V-amylose complexes with salts such as iodide, bromide, acetate, formate and bicarbonate of potassium can be obtained from VKOH fibers by exchange the salts with alkali in a hydroalcoholic solution [START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF]. The molecular and crystal structure of VKBr, shown in Figure I.15b, has been determined based on fiber XRD data [START_REF] Miller | Accurate fiber X-ray diffraction data from films[END_REF][START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF]. In this structure, two left-handed 4-fold helix, together with four KBr molecules are packed into a tetragonal unit cell, space group P43212, with a = b = 1.07 nm, and c (fiber axis) =1.61 nm. The adjacent amylose are bonded alternately through primary hydroxyl groups and potassium ions while bonding through anions contacts is less important. Water was not included in the model although the water content was measured to be about 10.6%. Drying the complex led to a smaller unit cell, with a = b = 1.02 nm, and c (fiber axis) =1.64 nm.

I.2.3.3. Other unresolved structures and towards new structures of V-amylose

Several other complexes have been reported to exhibit powder or fiber XRD patterns that are distinct from those of the previously described allomorphic families. However, their crystal structures still remain unknown. First are the complexes of amylose with salicylic acid and some analogues prepared by sealed-heating method [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Uchino | Inclusion compound formation of amylose by sealed-heating with salicylic acid analogues[END_REF]. These complexes gave similar powder XRD patterns, and thus appeared to be isomorphous. In addition, there are some similarities in their powder XRD patterns with that of polyisobutylene-γ-cyclodextrin (CD) complex. Therefore, the authors claimed that the complexes contained 8-fold helices similar to that of V8 obtained with 1-naphthol [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Uchino | Inclusion compound formation of amylose by sealed-heating with salicylic acid analogues[END_REF]. However, there is no confirming data for this suggestion and the unit cell parameters as well as space group are still unknown.

Second, [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF] obtained orthorhombic unit cells by swelling

Vn-butanol crystals in different solvents or mixtures of solvents. Although the molecular structure of Vn-butanol (V6II) and the swollen crystals have not been resolved, the authors believed that they had a similar organization of 6-fold left-handed helices, with the exception that the inter-helical space and shape varied depending on the swelling agents.

The VK acetate and VK propionate complexes can be obtained from VKOH fiber by exchange method, similar to the previous method used to prepare VKBr [START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF]. They are orthorhombic structures and appear to be isomorphous, with the unit cell having a=1.10 nm, b=1.81 nm, c (fiber axis) =1.79 nm for VK acetate and a=1.14 nm, b=1.80 nm, c=1.76 nm for VK propionate [START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF]. The molecular structure of these complexes is still unknown. However, since K acetate also yields an isomorphous tetragonal structure to that of VKBr and the c-parameter is similar between the orthorhombic and tetragonal structures, the orthorhombic structure is expected to have an extended helical conformation.

Finally, [START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF] reported that the amylose complexes with ammonium fluoride had an orthorhombic unit cell with c = 1.89 nm without any further information about the crystal structure. French and Murphy (1977a) noted that the c-parameter of 1.89 nm is similar with the 5-fold left-handed extended helix of the so-called intermediate amylose.

From the above examples, it is likely that other forms of V-amylose remain to be discovered. In addition, modeling works suggest allowed and disallowed helical conformations from an n-h map, in which n is the number of monomers per turn and h is the raise per monomer [START_REF] French | Accessible conformations of amylose[END_REF]. The allowed values of n range from 2-10 and h ranges from 0.08-0.44 nm.

This indicated that many V-amylose structures may possibly exist in addition to the wide variety of already observed forms. New complexing agents as well as suitable preparation methods with a variation of conditions would be required to allow these new forms to appear.

I.2.4. Origin of polymorphism

The formation of V-amylose results from the interaction between amylose and complexing agents in solution or in solid state. However, the structure, yield and crystallinity of complexes with a given agent may vary with the conditions of crystallization. Different parameters have been shown to have significant effects on the formation of V-amylose, such as the nature of complexing agent, degree of polymerization of amylose, nature and composition of the solvent, concentration of complexing agent, concentration of amylose and temperature.

I.2.4.1. Complexing agent

The complexing agents of amylose were once described as molecules having a polar group in addition to a dominant nonpolar portion [START_REF] Bear | Complex formation between starch and organic molecules[END_REF]. However, several hydrophobic compounds such as hydrocarbons or halogenated hydrocarbons are also effective complexing agents while some of them are not [START_REF] French | Starch fractionation by hydrophobic complex formation[END_REF][START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF]). In addition, molecules such as 1-naphthol and quinoline form complexes with amylose while their analogues like 8-quinolinol do not. Therefore, no clear rule is now available for predicting the possibility for a molecule to form a complex with amylose.

In addition, the nature of the complexing agent does not allow predicting the crystal structures of the resulting complexes. Indeed, an allomorphic type such as V7 can be obtained with a variety of molecules having very different chemical natures (propanoic acid, 2-propanol, acetone, thymol, etc.). In addition, a few complexing agents such as hexanoic acid, some dicarboxylic acids and n-alcohols can produce crystals with both V6I and V6II structures depending on the crystallization condition [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF].

On the other hand, several studies suggested that the dimension of the complexing agent was closely related to the induced helical conformation of amylose [START_REF] Rutschmann | Formation of inclusion complexes of starch with different organic compounds. IV, ligand binding and variability in helical conformations of V amylose complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Yamashita | An electron microscopie study on crystals of amylose V complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF].

More particularly, linear molecules such as aliphatic fatty acids and n-alcohols whose diameter in cross-section is about 0.3 nm yield 6-fold helical complexes while those having branched chain or ring structures with a dimeter in cross-section of 0.45-0.65 nm induce V7. V8 can be obtained with bulkier molecules such as 1-naphthol and quinoline. While most of the known complexing agents followed the above rule, some exceptions have been reported. For example, molecules such as DMSO [START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF], propionic and butyric acids [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], 2-propanol, acetone and quinoline [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], and salicylic acid [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF] are capable to induce two different helical conformations. Quinoline, (-)-borneol, trans-decalin were reported to form V6I complexes although their size is not compatible with either the helix cavity or inter-helix space [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. The above results suggest that the mechanism of complex formation is complicated and it seems impossible to predict the allomorphic structure of the complexes using a simple rule.

I.2.4.2. Degree of polymerization of amylose

The flexibility of amylose and the geometry of the α(1→4) glucosidic bonds allow the folding of polymer chain and the formation of the lamellar crystals of V-amylose with a wide range of DP. However, there exists a minimum DP of amylose essential for the formation of stable helical complexes [START_REF] Dvonch | Behavior of low molecular weight amylose with complexing agents[END_REF][START_REF] Gelders | Amylose-lipid complexation: a new fractionation method[END_REF]Godet et al., 1995). Shorter chain lengths favor the formation of A-and B-amylose [START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]. The minimum DP depends on the complexing agents. Godet et al. (1995) corresponding to the length needed to include two ligand molecules [START_REF] Gelders | Amylose-lipid complexation: a new fractionation method[END_REF]). In addition, Godet et al. (1995) noted that the yield of complexes increased with increasing DP, which could be explained by the lower solubility of amylose and its ability to form complexes that can precipitate. Furthermore, the DP of amylose can have a significant effect on the morphology and perfection of the crystals. [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF] suggested that DP 100 amylose formed crystals of V1-naphthol with an optimum perfection due to the absence of chain-folding.

I.2.4.3. Concentration of amylose

The concentration of amylose has a significant impact on the morphology and crystallinity of the complexes. The crystallization from dilute solution (0.05-0.1 wt%) usually produces lamellar single crystals whereas high amylose concentrations (> 0.5 wt%) results in spherulites or polycrystalline aggregates [START_REF] Conde-Petit | Chapter 8 Self-assembly of starch spherulites as induced by inclusion complexation with small ligands[END_REF][START_REF] Fanta | Preparation of spherulites from jet cooked mixtures of high amylose starch and fatty acids. Effect of preparative conditions on spherulite morphology and yield[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]Nuessli et al., 2003). In addition, when the concentration of amylose is higher than a certain value, the retrogradation of amylose is more favored over the complexation with the complexing agent, resulting in the formation of semicrystalline B-amylose (Nuessli et al., 2003).

I.2.4.4. Concentration of complexing agent

According to [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], there exists a critical concentration of each complexing agent necessary for the formation of the complexes. For poorly water-soluble complexing agents such as long-chain fatty acids, the critical concentration is very low. The complexing agents are usually added at saturation to ensure the complete complexation. For water-miscible complexing agents such as acetic and propionic acids, a concentration of complexing agent up to 40-50 vol% is required [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF].

In addition, some complexing agents such as n-propanol, as well as propionic and butyric acids induce more than one type of allomorph, depending on their concentration [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. At critical concentration, the complexes adopt an orthorhombic structure (V6II or V7) with a large inter-helix space for the complexing agents to reside. In contrast, at high concentration, the crystals adopt a compact V6I structure. This variation in crystal structure can be attributed to the change of hydration state of amylose as a function of the complexing agent concentration. According to [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], when the complexing agent is added at a low critical concentration, it is totally solvated and forms orthorhombic complexes with amylose. On the other hand, at high concentration, it may capture all solvent molecules leading to the desolvation of amylose. Consequently, the association between the complexing agent and amylose mediated by water is no longer possible and the crystallization simply results from a desolvation of the polymer [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

I.2.4.5. Temperature of crystallization

Previous studies have shown that the crystallinity of V-amylose complexed with lipids was controlled by the temperature of crystallization (Biliaderis & Galloway, 1989;[START_REF] Gelders | Amylose-lipid complexation: a new fractionation method[END_REF][START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF]. In particular, an incubation at ≤ 60 °C yields amorphous complexes due to a high nucleation rate whereas the crystallinity significantly increases when crystallization is carried out at ≥ 90 °C due to a slow nucleation. Moreover, the crystallization temperature varies for different complexing agents. For example, complexes with 1-naphthol crystallize at room temperature [START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF] whereas those with fatty acids can be formed within a wide range of temperatures (30-90 °C) [START_REF] Zabar | Studying different dimensions of amylose-long chain fatty acid complexes: molecular, nano and micro level characteristics[END_REF]. This difference in crystallization behavior could be attributed to a difference in solubility. Complexes with a higher solubility would crystallize at higher supercooling [START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF].

I.2.4.6. Solvent

V-amylose has frequently been prepared by crystallization from aqueous solutions, neutralized alkali solutions or DMSO/water mixtures. This does not generally have an influence on the nature of the complexes, but rather on the quality of the crystals. However, [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] reported that the crystal structure of amylose complexes with some molecules varied in the presence of DMSO. The complexation with quinoline resulted in a V8 structure only in the presence of 15% DMSO, while without DMSO, V6I crystals were obtained. For n-butanol and n-pentanol, the complexation in water yielded V6II but in DMSO/water mixture, V6I crystals were formed instead. The origin of the effect of DMSO on the formation of V-amylose complexes is still unclear but previous studies have shown that DMSO affected the conformation of amylose in solution [START_REF] Erlander | The stability of the helix of amylose and amylopectin in DMSO and water solutions[END_REF][START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF][START_REF] St | Nuclear magnetic resonance and conformational studies on amylose and model compounds in dimethyl sulfoxide solution[END_REF]. DMSO also affects the solubility of amylose, complexing agent and probably V-amylose.

I.2.5. Properties and potential applications of V-amylose

I.2.5.1. Identification, quantification and fractionation of amylose from native starch

The formation of complexes with iodine has commonly been used to identify amylose in starchy products and to measure the amylose content by differential scanning calorimetry [START_REF] Creek | Potential sources of error in the calorimetric evaluation of amylose content of starches[END_REF][START_REF] Sievert | Determination of amylose by differential scanning calorimetry[END_REF]. Moreover, the formation of V-amylose has been applied for selective fractionation of amylose from starch [START_REF] Banks | The fractionation of labortory-isolated cereal gereal starches using dimethyl sulphoxide[END_REF][START_REF] Bourne | Thymol and cyclohexanol as fractionating agents for starch[END_REF][START_REF] Cantor | Alkaline earth hydroxide fractionation of starch[END_REF][START_REF] French | Starch fractionation by hydrophobic complex formation[END_REF][START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF][START_REF] Schoch | Fractionation of starch by selective precipitation with butanol[END_REF][START_REF] Schoch | The fractionation of starch[END_REF][START_REF] Whistler | Separation of amylose and amylopectin by certain nitroparaffins[END_REF]. Upon addition of complexing agents into the starch solution, amylose forms inclusion complexes and precipitates, whereas amylopectin remains in solution. Amylose can then be separated and the process can be repeated on the recovered amylose to increase its purity.

I.2.5.2. Modulation of starch digestibility and glucose response

The formation of V-amylose with fatty acids have been shown to reduce the digestibility of starch and increase the resistance to enzymatic hydrolysis [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Cui | The effect of amylose-lipid complex formation on enzyme susceptibility of sago starch[END_REF][START_REF] Eliasson | Physical properties of amylose-monoglyceride complexes[END_REF]Gelders et al., 2005a;[START_REF] Godet | Crystalline amylose-fatty acid complexes: morphology and crystal thickness[END_REF][START_REF] Guraya | Effect of rice starch-lipid complexes on in vitro digestibility, complexing index, and viscosity[END_REF][START_REF] Holm | Digestibility of amylose-lipid complexes in-vitro and in-vivo[END_REF][START_REF] Sievert | Thermal behavior of potato amylose and enzyme-resistant starch from maize[END_REF]. The resistance to enzymatic hydrolysis increases with increasing crystallinity of V-amylose [START_REF] Seneviratne | Action of α-amylases on amylose-lipid complex superstructures[END_REF]. During hydrolysis the amorphous regions are degraded first, followed by the more crystalline regions [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF]. Therefore, there is an increase in crystallinity with hydrolysis to an optimum, beyond which it decreases due to degradation of crystalline regions [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Godet | Crystalline amylose-fatty acid complexes: morphology and crystal thickness[END_REF]. In lamellar crystals of V-amylose, the amorphous component is made of the folded section of the chains. As a consequence, if amylose with appropriate DP is used, chain-folding would not occurs and the resulting complexes would exhibit a higher crystallinity and stability.

Starch with induced or added V-amylose would modulate the glycemic response due to an increased resistance to hydrolysis resulting in a slower digestion (Obiro et al., 2012). Recent studies in humans reported that the consumption of bread containing amylose-lipids complexes resulted in lower postprandial plasma glucose and insulin levels compared to regular bread [START_REF] Hasjim | Characterization of a novel resistant-starch and its effects on postprandial plasma-glucose and insulin responses[END_REF][START_REF] Lau | Effect of fat type in baked bread on amylose-lipid complex formation and glycaemic response[END_REF]. Therefore, Vlipids complexes would help improve glycemic regulation and may help in the prevention and management of insulin resistance and metabolic syndrome including diabetes and obesity [START_REF] Hasjim | Characterization of a novel resistant-starch and its effects on postprandial plasma-glucose and insulin responses[END_REF][START_REF] Lau | Effect of fat type in baked bread on amylose-lipid complex formation and glycaemic response[END_REF].

I.2.5.3. Effect on the functional properties of starch

The functional properties of starch such as pasting behavior, viscosity, retrogradation, water and oil adsorption and swelling power are modulated in the presence of lipids. The effects are attributed to the formation of amylose-lipids complexes, as reviewed by Putseys et al.

(2010), Obiro et al. (2012) and [START_REF] Panyoo | Amylose-lipid complex production and potential health benefits: a mini-review[END_REF].

The formation of amylose-lipids complexes decreases solubility, leaching of amylose, swelling capacity and granule disruption of starch [START_REF] Bhatnagar | Extrusion processing conditions for amylose-lipid complexing[END_REF][START_REF] Galloway | Properties and structure of amylose-glyceryl monostearate complexes formed in solution or on extrusion of wheat flour[END_REF][START_REF] Krog | Influence of food emulsifiers on pasting temperature and viscosity of various starches[END_REF][START_REF] Mira | On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy wheat starch in aqueous suspension. Part I. Pasting and calorimetric studies[END_REF][START_REF] Navarro | Modelling of rheological behaviour in starchlipid systems[END_REF][START_REF] Raphaelides | Effect of fatty acids on the rheological behaviour of pea starch dispersions during heating[END_REF].

This results in a decrease or absence in the first pasting peak viscosity (D 'Silva et al., 2011;[START_REF] Singh | Effect of fatty acids on the rheological properties of corn and potato starch[END_REF]. On the other hand, after a prolonged pasting time (30-120 min) beyond the initial peak viscosity, the second pasting peak viscosity increases again due to the formation of complexes of leached amylose with the added or native lipids (D 'Silva et al., 2011;[START_REF] Nelles | Maize starch biphasic pasting curves[END_REF][START_REF] Nelles | Granule integrity and starch solubility during slow, extended pasting of maize starch -Tthe second viscosity peak[END_REF]. It was shown that during this second increase in paste viscosity, the starch granules are completely disintegrated [START_REF] Nelles | Granule integrity and starch solubility during slow, extended pasting of maize starch -Tthe second viscosity peak[END_REF] and no micron-scale structures could be observed (Obiro et al., 2012).

The formation of amylose complexes also competes and reduces the retrogradation of starch. As a consequence, at high starch concentrations, the addition of ligands results in a weaker starch gels or a no or reduced gel formation (D 'Silva et al., 2011;[START_REF] Raphaelides | Rheological studies of starch-fatty acid gels[END_REF][START_REF] Richardson | Wheat starch gelatinization-the effects of sucrose, emulsifier and the physical state of the emulsifier[END_REF]. This behavior can provide possible means for improving the mouth feel of starch-containing foods (D 'Silva et al., 2011). In addition, the formation of amylose complexes has an anti-stalling effect and increases the shelf-life of gluten-free bread formulations due to the unavailability of amylose for retrogradation [START_REF] Nunes | Impact of emulsifiers on the quality and rheological properties of gluten-free breads and batters[END_REF][START_REF] Purhagen | The anti-staling effect of pre-gelatinized flour and emulsifier in gluten-free bread[END_REF]. On the other hand, at low starch concentration, the complexation of amylose with flavor compounds induces the gelation [START_REF] Conde-Petit | Gelation of low concentration starch systems induced by starch emulsifier complexation[END_REF]Conde-Petit & Escher, 1995;[START_REF] Nuessli | Influence of starch flavour interactions on rheological properties of low concentration starch systems[END_REF]. This is attributed to the intergranular networks composed of the inclusion complexes which provide the physical cross-links between granules.

Pastes of V-amylose complexes are very spreadable at high concentration. Their flow properties are very similar to commercial shortening [START_REF] Byars | Rheological properties of dispersions of spherulites from jet-cooked high-amylose corn starch and fatty acids[END_REF]. In addition, the gelation by amylose complexes with sodium palmitate is pH-dependent and thermally reversible [START_REF] Byars | Influence of pH and temperature on the rheological properties of aqueous dispersions of starch-sodium palmitate complexes[END_REF]. The gelling properties of these materials suggest practical applications as thickeners and as dispersants for lipids in foods, lotions and water-based lubricants [START_REF] Byars | Influence of pH and temperature on the rheological properties of aqueous dispersions of starch-sodium palmitate complexes[END_REF].

I.2.5.4. Encapsulation of flavors and bioactive molecules

The capacity of formation inclusion complexes of amylose has been exploited for the encapsulation of a wide range of compounds that serve as flavor components, nutraceutical, pharmaceutical, or bioactive substances [START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF]Obiro et al., 2012;[START_REF] Panyoo | Amylose-lipid complex production and potential health benefits: a mini-review[END_REF][START_REF] Putseys | Amylose-inclusion complexes: formation, identity and physico-chemical properties[END_REF]. Among these are fatty acids [START_REF] Bhosale | Preparation of spherulites from amylose-palmitic acid complexes[END_REF][START_REF] Lesmes | Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes[END_REF][START_REF] Yang | Delivery of bioactive conjugated linoleic acid with selfassembled amylose-CLA complex[END_REF], esters of vitamins [START_REF] Kong | Molecular encapsulation of ascorbyl palmitate in preformed V-type starch and amylose[END_REF][START_REF] Ma | Formation of inclusion complexes of starch with fatty acid esters of bioactive compounds[END_REF], ibuprofen [START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF][START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF], genistein [START_REF] Cohen | Structural and functional properties of amylose complexes with genistein[END_REF][START_REF] Cohen | Improving bioavailability and stability of genistein by complexation with high-amylose corn starch[END_REF], salicylic acid [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF], and many flavors such as linalool, citronellol, limonene, β-pinene, geraniol, menthol and menthone, camphor, thymol, etc. [START_REF] Ades | Complexation with starch for encapsulation and controlled release of menthone and menthol[END_REF][START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF][START_REF] Heinemann | Influence of amylose-flavor complexation on build-up and breakdown of starch structures in aqueous food model systems[END_REF][START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF]Nuessli et al., 2003;[START_REF] Tapanapunnitikul | Water solubility of flavor compounds influences formation of flavor inclusion complexes from dispersed high-amylose maize starch[END_REF][START_REF] Wulff | Molecular encapsulation of flavours as helical inclusion complexes of amylose[END_REF][START_REF] Yeo | Inclusion complexation of flavour compounds by dispersed high-amylose maize starch (HAMS) in an aqueous model system[END_REF]) [START_REF] Itthisoponkul | Inclusion complexes of tapioca starch with flavour compounds[END_REF][START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. The inclusion complexes with some drugs such as rifampicin [START_REF] Ribeiro | Synthesis and characterization of acetylated amylose and development of inclusion complexes with rifampicin[END_REF], nimesulide and praziquantel [START_REF] Carbinatto | Preparation and characterization of amylose inclusion complexes for drug delivery applications[END_REF] were also reported, although the size of the complexing agents seems to be incompatible with the regular helical cavity.

The use of V-amylose as an encapsulation and delivery system presents many advantages.

First, amylose is readily available at relatively low cost. The polymer is considered nontoxic and biodegradable (Obiro et al., 2012). Second, the formation of V-amylose complexes has been demonstrated to increase thermal and oxidative stability of the ligands [START_REF] Cohen | Structural and functional properties of amylose complexes with genistein[END_REF][START_REF] Cohen | Improving bioavailability and stability of genistein by complexation with high-amylose corn starch[END_REF][START_REF] Lalush | Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid[END_REF][START_REF] Szejtli | Inclusion complexes of unsaturated fatty acids with amylose and cyclodextrin[END_REF][START_REF] Yang | Delivery of bioactive conjugated linoleic acid with selfassembled amylose-CLA complex[END_REF]. The increased stability would be useful for protecting the thermal-or oxygen-sensitive molecules such as unsaturated fatty acid, vitamins, etc. during storage and processing. Third, the formation of complexes increases the retention time of volatile ligands, i.e. flavors [START_REF] Arvisenet | Influence of physicochemical interactions between amylose and aroma compounds on the retention of aroma in foodlike matrices[END_REF][START_REF] Jouquand | Formation of amylose complexes with C6aroma compounds in starch dispersions and its impact on retention[END_REF][START_REF] Wulff | Molecular encapsulation of flavours as helical inclusion complexes of amylose[END_REF]. Fourth, the complexes are stable in gastric conditions and slowly release the ligand in the intestine. It can thus be used as intestinally targeted and controlled release delivery system [START_REF] Cohen | Structural and functional properties of amylose complexes with genistein[END_REF][START_REF] Cohen | Improving bioavailability and stability of genistein by complexation with high-amylose corn starch[END_REF][START_REF] Lalush | Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic acid[END_REF][START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF][START_REF] Yang | Delivery of bioactive conjugated linoleic acid with selfassembled amylose-CLA complex[END_REF][START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF]. Finally, [START_REF] Cohen | Improving bioavailability and stability of genistein by complexation with high-amylose corn starch[END_REF] reported that amylose-genistein complexes increased the bioavailability of the ligand and suggested that starch can affect the bioavailability of additional food components.

I.2.5.5. Other potential applications

V-amylose have been shown to have potential applications to produce aerogels [START_REF] Kenar | Starch aerogel beads obtained from inclusion complexes prepared from high amylose starch and sodium palmitate[END_REF], water-resistant paper [START_REF] Fanta | Increased water resistance of paper treated with amylose-fatty ammonium salt inclusion complexes[END_REF], films with increased surface hydrophobicity and high elongation [START_REF] Fanta | Films prepared from poly (vinyl alcohol) and amylose-fatty acid salt inclusion complexes with increased surface hydrophobicity and high elongation[END_REF] and fat replacers [START_REF] Singh | Starch-lipid composites in plain set yogurt[END_REF][START_REF] Singh | Amylose-potassium oleate inclusion complex in plain set-style yogurt[END_REF][START_REF] Singh | Yogurt fermentation in the presence of starch-lipid composite[END_REF]. In addition, amylose-lipids complexes were shown to potentially inhibit azoxymethane-induced preneoplastic lesions (precursors of colon cancer) [START_REF] Zhao | Inhibition of azoxymethane-induced preneoplastic lesions in the rat colon by a stearic acid complexed high-amylose cornstarch using different cooking methods and assessing potential gene targets[END_REF][START_REF] Zhao | Inhibition of azoxymethane-induced preneoplastic lesions in the rat colon by a cooked stearic acid complexed high-amylose cornstarch[END_REF].

I.3. Objectives of the thesis

One of the main objectives of this thesis was to systematically study the crystallization of amylose in solution in the presence of a variety of organic molecules in different conditions, in order to understand the effect of the nature of complexing agents and several crystallization parameters (crystallization temperature, concentration of amylose and complexing agent, DP of amylose, presence of DMSO) on the formation of amylose complexes and their crystal structure.

Another important objective was to collect the crystallographic data on the different allomorphic families, especially those that we have identified for the first time during this works, in order to propose a conformation and a packing arrangement of amylose helices for each allomorph. In particular, our study have focused on the determination of the molecular and crystal structure of V1-butanol whose model was still hypothetical and of historical importance since it is one of the first molecules that have been used to recrystallize amylose on a large scale.

We have also investigated the encapsulation and release properties of a bioactive molecule, namely ibuprofen, by its V-amylose complex, as an example to evaluate the potential of V-amylose as a delivery system for drugs. A crystallographic study was conducted to understand the effect of the crystal structure on the release properties.

Chapter II

Materials and methods

II.1. Materials

II.1.1. Native amylose

Potato amylose was purchased from Sigma-Aldrich and further purified as previously reported [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Briefly, 30 g of amylose was dissolved in 1 L of dimethyl sulfoxide (DMSO) by stirring for 12 h at room temperature. The solution was centrifuged (2500 g, 20 min) to remove residual aggregates. Amylose was precipitated by addition of an equal volume of ethanol, kept at 4 °C for 12 h, filtered (glass filter G5) and extensively rinsed with ethanol, acetone and diethyl ether, successively, using a Büchner device hooked to a water suction pump. Amylose was then dried for one week in an exhaust hood at room temperature. II.1) so this fraction will be referred to as DP6500 in the following. The Mw/Mn ratio of native amylose was 2.58, indicative of a high polydispersity. (enzyme activity of 0.5 U.mL -1 ). Amylose was then precipitated from the reaction medium with 1/4 volume of 2-propanol at 4 °C for 12 h, and collected by centrifugation (2500 g, 20 min).

The resulting polymer was purified using the protocol previously described for native amylose.

The DP distribution was determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after dissolution in 1 N NaOH [START_REF] Potocki-Veronese | Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties[END_REF]. The DPw of the total fraction was 86 (Figure II.2, Table II.1) so it will be referred to as DP86 in the following. The synthesized amylose has a lower polydispersity compared to the native amylose, indicated a lower Mw/Mn of 1.25. Part of the total DP86 fraction was further fractionated by preparative gel filtration in a P6DG column (Biorad). In particular,

we have used three fractions with DPw of 60, 80 and 130, which will be referred to as DP60, DP80 and DP130, respectively (Table II.1 and Figure II.2). The Mw/Mn of all synthesized amylose fractions was close to 1, indicating a narrow size distribution.

II.1.2.2. Amylose synthesized in vitro by phosphorylase

Amylose fractions synthesized in vitro by phosphorylase [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF] with DPw = 28, 192 and 601, referred to as DP28, DP192, and DP600, respectively, were purchased from Shoko Co Ltd (Japan) and used without further purification. The characteristics of all fractions used in this study are summarized in Table II.1.

II.1.3. Complexing agents

121 organic molecules were tested for their complexing ability, as listed in Table II.2.

They have different functional groups such as carboxylic acids, alcohols, amines, ketones, aldehydes, amides, esters, ethers, etc. They can be saturated or unsaturated with open chain (straight or branched) or ring structure. In addition, molecules with very similar structures such as isomers or those belonging to homologous series were also tested. A number of complexing agents are flavors and bioactive molecules such as: ibuprofen, ketoprofen, salicylic acid, azelaic acid, menthol, linalool, citral, carvacrol, etc.

Table II.1. Molecular characteristics of the fractions used in this study. DPw is the weightaverage degree of polymerization. Mw and Mn are the weight-and number-average molecular weights, respectively.

Fraction DP50 a,c DP80 a,c DP86 Therefore, they were used at 5-80 vol%. Water-immiscible pentanoic acid was used at 1-5 vol%.

The crystallization mixtures were incubated at different temperatures (25, 40, 50, 60, 75, 90, 100 and 115 °C). After 1-2 weeks, the precipitates were recovered by centrifugation and washed with water containing the same amount of complexing agent present in the reaction medium.

Recrystallization. The complexation mixture containing crystals was heated up to about 90 °C to redissolve crystals, then cooled back to the crystallization temperature to allow the complexes to recrystallize. The procedure could be repeated several times. The lamellar crystals obtained were generally more individualized.

II.2.2. Treatments of the complexes

II.2.2.1. Hydrated / wet samples

V-amylose crystals are sensitive to dehydration. Therefore, it is important to characterize the crystals in a hydrated state to maintain the crystal structure. Suspensions of lamellar crystals were centrifuged and the supernatant was removed. The crystals were then deposited on absorbent paper for a few minutes to remove the solvent in excess.

II.2.2.2. Dry samples

Wet samples were dried using two methods: drying in primary vacuum or lyophilization.

II.2.2.3. Treatment with solvents

Wet crystals were dispersed in methanol, 2-propanol or tert-butanol. Some crystals were kept in suspension, while others were recovered by centrifugation and dried in vacuum.

II.3. Characterization techniques

II.3.1. Differential interference contrast (DIC) optical microscopy

DIC optical microscopy, also known as Nomarski microscopy, is used to enhance the contrast of unstained and transparent samples. A light polarized at 45° is divided into two rays polarized at 0 and 90° that are then focused to pass through two adjacent points in the sample (around 0.2 µm apart). If the travel regions of the two rays differ in refractive index or thickness, they will experience a different optical path length and thus have different phases. After traveling through the sample, they are recombined into one ray polarized at 135°. This generates an interference, brightening or darkening the image depending on the optical path difference.

A drop of crystal suspension was deposited on a glass slide and covered with a glass slip. The specimens were observed in DIC mode using a Zeiss Axiophot II microscope equipped with polarized light. The images were recorded with an Olympus SC50 digital camera operated by the Olympus Stream software.

II.3.2. Atomic force microscopy

Atomic force microscopy (AFM) is a type of scanning probe microscopy with a high resolution on the order of a few angstroms. AFM uses a cantilever with a sharp tip (probe) at For a good statistical distribution, at least 20 measurements were performed for each sample.

The surface roughness was calculated as

𝑅𝑞 = √ 1 𝑁 ∑ (𝑍 𝑖 -𝑍 ̅ ) 2 𝑁 1 2
where 𝑍 ̅ is the average height.

II.3.3. X-ray diffraction (XRD)

II.3.3.1. Diffraction by crystals

A crystal consists of a periodic spatial arrangement of atoms. Mathematically, a crystal can be considered as a three-dimensional arrangement of points called a point lattice. The lattice can be reproduced by repeating a small unit, referred to as unit cell which is defined by three

II.3.3.2. Experimental procedure

After decantation, the crystals were further centrifuged and the pellets was allowed to settle onto a piece of bolting cloth inside a closed chamber with a 95% relative humidity controlled humidity (95% relative humidity). After a few days of equilibration, strips of the resulting crystal mats were introduced into glass capillaries which were then flame-sealed immediately and X-rayed in a Warhus vacuum chamber using a Philips PW3830 generator operating at 30 kV and 20 mA (Ni-filtered CuKα radiation, λ = 0.1542 nm). Two-dimensional diffraction patterns were recorded on Fujifilm imaging plates, read off-line using a Fujifilm BAS 1800-II bioimaging analyzer. The resulting ring patterns were radially averaged to get diffraction profiles. The diffraction data were calibrated using a calcite powder standard and the unit cell parameters refined using the CelRef module of the LMGP package (Laugier & Bochu).

II.3.4. Transmission electron microscopy (TEM)

II.3.4.1. Principle

A beam of electrons is transmitted through thin (< 500 nm for a polymer) specimens to form an image of their volume with a resolution of a few angstroms. A beam of electrons is generated by an electron gun and accelerated by an anode (Figure II.4). The illumination system consists of a number of electromagnetic lenses that modify the trajectory of the incident electrons. The condenser lenses modify the size, position and intensity of the beam. The condenser aperture controls the parallelism and coherency of electron beam that irradiates the specimen. The objective lens forms a first magnified image using the electrons that have passed through the specimen. This image is further magnified by intermediate and projective lenses and visualized on a fluorescent screen, or recorded using a digital camera.

The TEM can be operated in imaging or diffraction mode (Figure II.5a and II.5b). In imaging mode, the intermediate lens is adjusted so that its object plane is the image plane of the objective lens. The objective aperture is inserted at the back focal plane of the objective lens to increase the contrast by blocking all the electrons scattered at large angles. A so-called bright-field image is formed by the electrons transmitted through the specimen. In diffraction mode, the objective aperture is removed. The selected area aperture is used to select a circular region of the specimen from which the diffraction pattern is recorded. Its diameter is generally 500 nm or 1 µm. The intermediate lens is readjusted so that its object plane is the back focal plane of the objective lens, allowing projecting the diffraction pattern of the specimen onscreen. By comparison with XRD that were used to record powder (ring) patterns on a large number of crystals, selected area electron diffraction (SAED) allowed probing individual crystals and thus record spot patterns. However, polymer crystals are extremely sensitive to radiation damage from the electron beam and are decrystallized in a matter a seconds at room temperature. Therefore, the samples must be observed under low dose conditions. The crystal structure can also be preserved if the specimen is kept at low temperature during the observation.

II.3.4.2. Experimental procedure

Drops of dilute crystal suspensions were deposited onto carbon-coated grids previously glow-discharged in a Pelco easiGlow station, and allowed to dry. The specimens were observed under low dose illumination with a FEI-Philips CM200 'Cryo' microscope operating at 200 kV.

For electron diffraction, the TEM grids were mounted on a Gatan 626 specimen holder and fast-frozen into liquid nitrogen just after air-drying and prior to being introduced in the microscope. The holder was then cooled down with liquid nitrogen and the observation was made at -176 °C. Base-plane ED patterns were recorded at a voltage of 200 kV, from selected areas of about 1 µm 2 . Images and diffraction patterns were recorded on a TVIPS TemCam F216 camera. Some ED patterns were also recorded on Fujifilm imaging plates. The diffraction patterns were calibrated using a gold-coated carbon film as standard.

In the specific case of the V1-butanol complex (see Chapter III), the intensity of the reflections in the base-plane ED diagram was measured semi-automatically using a tailor-made program, as described by [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. All isotopes that contain an odd number of protons and/or neutrons (e.g. 1 H and 13 C) have a nonzero nuclear spin. A spinning charge generates a magnetic field that results in a magnetic moment proportional to the spin. In the presence of an external magnetic field Bo, two spin states exist: one up and one down, where one aligns with the magnetic field and the other opposes it. The sample is submitted to an oscillation frequency, usually referred as a radiofrequency pulse, whose energy exactly corresponds to the difference in energy of the two spin states (∆E), inducing the excitation of this set of nuclei from the low to the high energy state.

Once the radio-frequency pulse is switched off, the nuclei magnetization returns to the initial equilibrium state by precessing about the main field Bo which is called free induction decay. It is the precession of the magnetization that is detected as the signal in the nuclear magnetic resonance (NMR) spectroscopy.

The ∆E or the resonance frequency is proportional to the spin magnetic moment.

However, not all nucleus of an isotope with the same magnetic moment yield a resonant signal at the same frequency values. The difference arises from the differing electronic environment of the nucleus of interest, which depends on the local geometry (binding partners, bond length, bond angle, etc.). The variation of the resonance frequencies of the same kind of nucleus, due to the variation in the electron distribution, is called a chemical shift. The extent of the chemical shift is given with respect to a reference frequency or reference sample. The chemical shift  is usually expressed in parts per million (ppm) by frequency, calculated from the expression

𝛿 = 𝜈 𝑠𝑎𝑚𝑝𝑙𝑒 -𝜈 𝑟𝑒𝑓 𝜈 𝑟𝑒𝑓
where 𝜈 𝑠𝑎𝑚𝑝𝑙𝑒 is the absolute resonance frequency of the sample and 𝜈 𝑟𝑒𝑓 is the absolute resonance frequency of a standard reference compound, measured in the same magnetic field Bo.

The electronic environment around a nucleus is generally anisotropic, so its chemical shift is also anisotropic and depends on the orientation of the molecules with respect to the magnetic field. On the one hand, in liquid-state NMR, such anisotropic information, chemical shift anisotropy (CSA) is averaged out due to a fast and random molecular tumbling and only a single isotropic chemical shift value is observed with a narrow peak width. On the other hand, in solid-state NMR, molecules are immobile and often oriented in random directions with respect to the magnetic field, which gives rise to a broad lineshape for each nucleus in a static condition. Such a lineshape is characteristic to the solid-state structure and dynamics of the molecule, and thus provides important information for structural analysis. However, the peak broadness results in peak overlap and makes interpretation of NMR spectra very challenging.

To overcome this problem, the anisotropic NMR interactions are suppressed by macroscopically rotating the sample at the 54.44° magic angle with respect to the magnetic field [START_REF] Andrew | Nuclear magnetic resonance spectra from a crystal rotated at high speed[END_REF], typically at a frequency of a few to tens of kHz. This technique, namely magic angle spinning (MAS), which is essentially mimicking molecular motion in solution state, makes the lineshapes narrower. Therefore, the resolution improves and a more precise structural analysis of molecular solids is possible.

Cross polarization (CP) is another important technique in solid-state NMR, which allows to efficiently detect a dilute spin such as 13 C. This exploits the polarization of abundant spins, such as 1 H, which is dipolar coupled with the rare spin. The natural polarization of abundant spins is transferred to the dilute spin during a so-called contact time, by using a low power pulse on both channels for dilute and abundant spins. The CP provides an enhancement of signals from dilute spins as well as faster repetition rate of the measurement.

The combination of CP and MAS techniques is a powerful analytical tool for structural analysis of solid organic materials [START_REF] Snape | Quantitative reliability of aromaticity and related measurements on coals by 13C nmr A debate[END_REF]. However, the quantitativeness of CP/MAS NMR is not always valid in specific cases [START_REF] Smernik | The use of spin counting for determining quantitation in solid state 13C NMR spectra of natural organic matter 2. HF-treated soil fractions[END_REF][START_REF] Smernik | Effects of added paramagnetic ions on the 13C CP/MAS NMR spectrum of a de-ashed soil[END_REF][START_REF] Coates | Interpretation of infrared spectra, a practical approach[END_REF]. In particular, this is a problem for a solid system where molecules with different dynamics co-exist. The direct excitation of carbon, or "single-pulse" (SP) NMR was suggested as a valid alternative for quantitative analysis for such systems, although the direct excitation of dilute spins requires much longer acquisition times compared to the case of CP.

II.3.5.2. Experimental procedure

Solid-state 13 C NMR analyses were performed by Yu Ogawa (CERMAV) on a Bruker

Avance III 400 MHz spectrometer ( 13 C frequency of 100.6 MHz) using CP/MAS. The spinning speed was set at 12 kHz, with a sweep width of 29761 Hz, and a recycle delay at 2 s. Each spectrum was averaged over about 6000 scans. The 13 C chemical shifts were calibrated with respect to that of the glycine carboxyl group (176.03 ppm). SP/MAS solid-state NMR experiments were carried out as well using a 13 C 90° pulse of 4 µs and a delay time of 64 s.

II.3.6. Fourier-transform infrared (FT-IR) spectroscopy

II.3.6.1. Principle

Infrared (IR) spectroscopy is a type of vibrational spectroscopy that studies the interaction of infrared radiation with matter. It covers a range of techniques, mostly based on absorption spectroscopy. The IR portion of the electromagnetic spectrum is usually divided into three regions: near-IR (14000-4000 cm -1 ), mid-IR (4000-400 cm -1 ) and far-IR (400-4 cm -1 ).

The mid-IR spectroscopy provides characteristic fundamental vibrations that are employed for the elucidation of molecular structure and was used in this study. The infrared spectrum is a plot of measured infrared light absorbance (or transmittance) versus frequency or wavelength of the light. The typical frequency units used in IR spectroscopy are reciprocal centimeters, also called wavenumbers, with the symbol cm -1 . The frequency (𝑣, sec -1 ), wavelength (𝜆, cm), and wavenumber (𝑊) are related to each other via the following equation:

𝜈 = 1 2𝜋 ( 𝑘 𝜇 ) 1 2 ⁄
where 𝑘 is the force constant of the bond and 𝜇 is reduced mass which refers to (M1M2)/(M1+M2) where M1 and M2 are the masses of the two atoms, respectively. Therefore, different molecules vibrate at different frequencies because their structures are different and thus can be distinguished using infrared spectroscopy.

The attenuated total reflection (ATR) mode allows analyzing samples (powders, pastes, liquids) directly without further preparation. The specimen is deposited onto a diamond crystal through which IR light is passing. The multiple reflections of the beam creates an evanescent wave that penetrates into the specimen and the signal is collected by a detector.

II.3.6.2. Experimental procedure

FT-IR spectra of hydrated complexes (desorbed in a chamber with 95% relative humidity) were recorded with a Perkin-Elmer Spectrum 2 FT-IR spectrophotometer equipped with an ATR accessory. Measurements were done in the 4000-400 cm -1 range with a resolution of 2 cm -1 and eight repeat scans were averaged for each spectrum.

II.3.7. Density measurement

The measurement was only carried out for Vibuprofen and V1-butanol crystals. Fragments of mats of crystals equilibrated at 95% RH were floated in 1,2-dichloroethane (d = 1250 g.L -1 ) to which 1,1,2-tricloro-1,2,2,-trifluoroethane (d = 1560 g.L -1 ) was slowly added. When the film remained in equilibrium in the mixture of liquids, its density was equal to that of the liquid which was measured using a pycnometer. Regular left-and right-handed amylose helices having 6, 7, or 8 glucosyl units per turn and the hydroxymethyl groups in gauche-gauche (gg) conformation [START_REF] Horii | Solid-state 13 C-NMR study of conformations of oligosaccharides and cellulose[END_REF] were generated by propagating a α-D-glucosyl residue in 4 C1 chair conformation using the parameters n (number of residues per turn) and h (rise per residue) [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF].

II

The glucosyl residues were taken from the previously reported rigid helices [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF][START_REF] Putaux | Morphology and structure of A-amylose single crystals[END_REF]. The energy of the resulting helices was not further optimized.

II.3.8.1.2. Packing energy calculation and geometry optimization

The non-bonded interaction was considered using Buckingham's potential [𝐸 = 𝐴𝑒𝑥𝑝(-𝐵 × 𝑟) -𝐶/𝑟 6 ], where the A, B, C parameters are different for each type of atom pair [START_REF] Dauchez | Vibrational molecular force field of model compounds with biologic interest. II. Harmonic dynamics of both anomers of glucose in the crystalline state[END_REF]. This function was applied with a cutoff of 1 nm. The molecule geometry was optimized using the Universal Force Field [START_REF] Rappé | UFF, a full periodic table force field for molecular mechanics and molecular dynamics simulations[END_REF] in the Forcite module of Material Studio (Forcite).

II.3.8.1.3. Model refinement

The structure refinement against experimental electron diffraction data was conducted using the SHELXL program [START_REF] Sheldrick | Crystal structure refinement with SHELXL[END_REF]). An intensity average was calculated for groups of symmetry-related reflections. The program used the atomic scattering factors for electrons from the International Tables for Crystallography [START_REF] Cowley | Parameterizations of electron atomic scattering factors[END_REF]. The models were refined using the conjugate-gradient least-square (CGLS) regression method with an isotropic approach of the thermal parameters. The so-called "1-2" and "1-3" distances are defined as the distances between an atom and its first and second neighbor, respectively. The isotropic U-values of equivalent atoms were restrained to be equal, and 1-2 and 1-3 distances were restrained using the DFIX and DANG instructions. Anti-bumping restraints were applied if the two atoms were closer to each other than the target distance. At the beginning of the refinement, the standard deviation sd for 1-2 and 1-3 distances was set 10 times lower than the default value (0.02 in the first DEFS parameter) to restrain the conformation of amylose. The resolution limit was first set to 0.35 nm, then decreased to 0.3 nm and finally to 0.24 nm. In the final refinement, sd was set to the default value (0.02) to allow the helix to further relax. The reliability factor R1 was calculated as:

𝑅1 = ∑||𝐹 𝑜𝑏𝑠 | -|𝐹 𝑐𝑎𝑙 || ∑|𝐹 𝑜𝑏𝑠 |
where 𝐹 𝑜𝑏𝑠 and 𝐹 𝑐𝑎𝑙 are the observed and calculated structure factors, respectively.

II.3.8.2. Molecular dynamics of Vibuprofen

Atomistic molecular dynamics simulations were performed using the Gromacs 5.1 package [START_REF] Hess | GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation[END_REF] and the Gromos 56A6CARBO_R force field [START_REF] Plazinski | Revision of the GROMOS 56A6CARBO force field: improving the description of ring-conformational equilibria in hexopyranose-based carbohydrates chains[END_REF] with a modified Lennard-Jones repulsive parameter for the CH1 atom type. In the simulations, the motion equations were solved by a standard leapfrog algorithm with integration step of 2 fs.

The length of covalent bonds was constrained by using the LINCS algorithm. All the equilibration and production runs were achieved in the NPT ensemble (constant number of particles, pressure, and temperature). The velocity-rescaling algorithm was used for temperature control with a coupling time of 0.1 ps. The pressure was regulated to 1 bar using a Berendsen pressure coupling algorithm with a pressure coupling constant of 2.0 ps [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF].

The pressure was anisotropically regulated. The long-range interactions were calculated by using the particle-mesh Ewald summation method with a cut-off distance of 0.9 nm and the long-range dispersion force was corrected for both energy and pressure. All analyses was performed using GROMACS tools. Trajectories were visualized with the VMD software [START_REF] Humphrey | VMD: visual molecular dynamics[END_REF] and final models were displayed using PyMOL [START_REF] Delano | PyMOL. DeLano Scientific[END_REF].

In another procedure, the molecule geometry was optimized by the Forcite module in Materials Studio (Forcite) using the Universal Force Field [START_REF] Rappé | UFF, a full periodic table force field for molecular mechanics and molecular dynamics simulations[END_REF]. The non-bonded electrostatic and van der Waals forces were controlled by the Ewald summation method. Water was packed into the cell using the Amorphous Cell module with the Universal Force Field. The module builds molecules in a cell using a Monte Carlo protocol and minimizes close contacts between atoms, while ensuring a realistic distribution of torsion angle [START_REF] Akkermans | Monte Carlo methods in materials studio[END_REF][START_REF] Chen | Molecular modeling of temperature dependence of solubility parameters for amorphous polymers[END_REF].

II

II.3.9.2. Determination of ibuprofen content in the complex

The ibuprofen content was evaluated by two methods. In the first one, 15 mg of the freeze-dried complex was introduced in a 5 mL volumetric flask and completed with ethanol 96%. The mixture was sonicated for 30 min and then filtered (PVDF, 0.22 µm). The ibuprofen concentration in the filtrate was determined spectrophotometrically at 263.9 nm. In the second method, 20 mg of freeze-dried complex was mixed with 10 mL of a 0.1 M NaOH aqueous solution, agitated (500 rpm) for 2 h at 37 °C, filtered (PVDF, 0.22 µm), and assayed at a wavelength of 264.3 nm. In both methods, the ibuprofen weight percentage was calculated according to the following equation:

%𝑖𝑏𝑢 = 100 × 𝐶 𝑖𝑏𝑢 × 𝑉 1000 × 𝑚 𝑐𝑜𝑚𝑝𝑙𝑒𝑥
in which V is the volume of solvent in mL (ethanol or 0.1 M NaOH solution), and mcomplex is the weight of the freeze-dried complex in mg.

II.3.9.3. Determination of the solubility of ibuprofen in the dissolution media

Excess ibuprofen was introduced in 15 mL of each medium in a 25 mL Erlenmeyer flask equilibrated at 37 °C in a water bath. The mixture was stirred at 500 rpm for 2 h to reach equilibrium. Then, 2 mL of the solution was filtered (PVDF, 0.22 µm) and the pH was adjusted to 12.69 by adding a known volume of a 5 M NaOH solution. Ibuprofen concentrations were determined by measuring the absorbance at a wavelength of 264.3 nm after appropriate dilution.

The whole test was made in triplicate.

II.3.9.4. In vitro dissolution studies

The experiments were performed using a basket apparatus (Sotax AT7, Sotax Switzerland) and the rotating speed of the baskets was set at 50 rpm. Briefly, 190 mL of a dissolution medium (0.1 M HCl or phosphate buffers pH 5.5, 6.8 and 7.2) was placed into the vessel and equilibrated at 37 ± 0.5 °C. A sample of the freeze-dried complex containing 10 mg ibuprofen was placed in the basket and the position of the latter was adjusted so that the sample was fully immersed. At appropriate intervals during 6 h, a 2 mL sample was taken from the release medium, filtered (0.22 µm PVDF filter), adjusted to pH 12.69 with a 5 M NaOH solution and spectrophotometrically assayed at 264.3 nm for cumulated drug release. A similar experiment was performed in the presence of amylose as reference to control the absence of detectable UV signal due to the presence of the polymer in the considered wavelength region.

Also, a dissolution study of pure ibuprofen in each medium was conducted at 37 °C by incubating 10 mg of drug instead of the complex into 190 mL acidic or buffer solutions. It should be noted that all the drug release experiments were performed under conditions that ensure that the maximum concentration corresponding to 100% of ibuprofen release in the vessel did not exceed 70% of the saturating ibuprofen concentration.

In addition, a two-stage dissolution test was carried out with the complex. The procedure was as follows: a first step in acidic medium (pH 1.2) was performed at 37 ± 0.5 °C after introducing 120 mg of the complex in the basket, rotating at 50 rpm, in 170 mL of 0.1 M HCl.

Samples were collected at predetermined time intervals and spectrophotometrically assayed as previously described. Then, 20 mL of a solution equilibrated to 37 ± 0.5 °C, containing either 1.293 g (KH2PO4.2H2O) and 0.832 g NaOH or 2.493 g KH2PO4.2H2O, 0.245 g Na2HPO4.12H2O and 0.680 g NaOH, was added to the acidic medium, in order to adjust the pH to 6.8 and 5.5, respectively. The release study at this stage was performed during 6 h. All the tests were made in triplicate.

Chapter III

Crystal and molecular structure of V 1-butanol

III.1. Introduction

As presented in Chapter I, five allomorphic families of V-amylose lamellar crystals containing compact 6-, 7-or 8-fold helices have been reported in the literature, including V6I, V6II, V6III, V7 and V8. The detailed crystal structure of V6I, V6III, V7 and V8 has been resolved based on ED or fiber XRD data, in combination with molecular modeling [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Cardoso | From rice starch to amylose crystals: alkaline extraction of rice starch, solution properties of amylose and crystal structure of V-amylose inclusion complexes[END_REF][START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF]Winter & Sarko, 1974). On the other hand, the crystal structure of V6II is still speculative.

V6II complexes were first prepared with n-butanol [START_REF] Rundle | The configuration of starch in the starch-iodine complex. IV. An X-ray diffraction investigation of butanol-precipitated amylose[END_REF][START_REF] Schoch | Fractionation of starch by selective precipitation with butanol[END_REF]. Several other complexing agents such as ketones [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF], fatty acids [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF], alcohols [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], esters [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF] were then shown to induce the formation of this crystalline structure.

In the V6II family, V1-butanol has been the most studied complex. Although crystalline fibers [START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF]) and single crystals [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Manley | Chain folding in amylose crystals[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes[END_REF] of V1-butanol have been prepared and characterized by ED and XRD, no definitive three-dimensional model is available yet. Hypotheses for the helical conformation and the arrangement of amylose helices for V6II have been proposed [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes[END_REF]. From diffraction data, it was found that the helical repeat was about 0.8 nm, i.e. the same as that of 6-fold V6I complexes. In addition, the reversible conversion into the V6I structure without a change in morphology supports the occurrence of the 6-fold helix for these crystals. [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF] and III.1c). Interestingly, as previously mentioned in other studies [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Manley | Chain folding in amylose crystals[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes[END_REF], the crystals frequently showed cracks along the longer dimension of the rectangular lamellae. These cracks, which are likely caused by anisotropic shrinkage during the drying of the crystal in air or in the vacuum of the TEM, are absent when the crystals are observed under frozen-solvated conditions. The ED diffraction pattern is identical to those reported by [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF] and [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. It can be indexed according to an orthorhombic unit cell (space group P212121) with lattice constants a= 2.65 ± 0.01 nm, b = 2.74 ± 0.01 nm, in agreement with those calculated from the XRD pattern (a =2.655 ± 0.001 nm, b = 2.708 ± 0.001 nm, c = 0.798 ± 0.001 nm) (Figure III.2b). These results confirm the cell parameters given earlier [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. A list of independent intensities for reflections up to a 0.24 nm resolution, averaged from several ED patterns, is presented in Annex 1, Table S.III.1. of V1-butanol. Only one signal was detected for each carbon site suggesting that the helix was rather symmetrical, in agreement with a previous study (Le [START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF]. The resolved resonances at 102.4, 81.8, 74.7, 71.7 and 61.0 ppm can be assigned to C1, C4, C3, and C2-C5 and C6 carbon atoms of the glucosyl units, respectively. The broad peak centered at 61.0 ppm is an overlap of the resonance of C6 carbon of the glucosyl units and that of C(OH) carbon of 1-butanol. Besides, the sharp peaks at 35.0, 19.4 and 14.3 ppm correspond to the resonances of other carbon atoms of 1-butanol, suggesting that the molecule is not mobile and would be located in crystallographic positions. In addition a shoulder at 100.4 ppm was also observed, indicating the presence of some B-amylose [START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF]Veregin et al., 1987a).

As noted in previous studies, the chemical shifts of the Cl and C4 atoms depend on the values explored by the torsion angles about the glucosidic linkages and thus reflect the global helical conformation of the chain [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF]Veregin et al., 1987a). [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF] showed that the 13 C chemical shifts were indistinguishable between 6-and 7-fold helices but the C1 and C4 signals were shifted upfield by about 1 ppm with respect to the V8 structure. [START_REF] Kawada | Solid-state NMR and X-ray studies on amylose complexes with small organic molecules[END_REF] also found that the complexes prepared with lauric (dodecanoic) acid which would contain 6-fold helices, presented the same C1 chemical shift at 103 ppm to that of Vthymol which is a 7-fold complex [START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. In contrast, several studies indicated that there are significant differences in the C1 resonance between the 6-, 7-, and 8-fold complexes. In particular, the C1 signal is located at 102.2-102.7, 103.3-103.4, and 105 ppm for 6, 7-, and 8-fold complexes, respectively [START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF][START_REF] Rondeau-Mouro | Structural investigation of amylose complexes with small ligands: inter-or intra-helical associations[END_REF]. However, all the above studies are in agreement regarding the upfield shifts of the resonance in theses complexes as opposed to V8

complexes. In the present study, the C1 chemical shift in V1-butanol was found at 102.4 ppm, which means the complexes would have 6-or 7-fold helices.

Previous works reported that the C6 resonance is correlated with the conformation of the hydroxymethyl group [START_REF] Horii | Solid-state 13 C-NMR study of conformations of oligosaccharides and cellulose[END_REF][START_REF] Veregin | Correlation of 13 C chemical shifts with torsional angles from high-resolution, 13 C-CP-MAS NMR studies of crystalline cyclomalto-oligosaccharide complexes, and their relation to the structures of the starch polymorphs[END_REF]. For cyclodextrins, the resonances in the range 59.6-61.7 ppm and 62.7-65.9 ppm are related to gauche-gauche (gg) and gauchetrans (gt) conformations, respectively [START_REF] Veregin | Correlation of 13 C chemical shifts with torsional angles from high-resolution, 13 C-CP-MAS NMR studies of crystalline cyclomalto-oligosaccharide complexes, and their relation to the structures of the starch polymorphs[END_REF]. Similar correlations have been found for cellulose, showing 60-62.6 ppm for gg, 62.5-64.5 ppm for gt, and 65.5-66.5 ppm for trans-gauche (tg) [START_REF] Horii | Solid-state 13 C-NMR study of conformations of oligosaccharides and cellulose[END_REF]). In the case of V1-butanol, because of the overlap between resonances of C6 carbons of amylose with that of carbon atoms of 1-butanol (Figure III.3), it is impossible to unambiguously determine the conformation of the hydroxymethyl group. However, the chemical shift of C6 in V1-butanol is rather broad, in agreement with previous studies (Le [START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF]Veregin et al., 1987a). This suggests a broad distribution of hydroxymethyl conformation, and all gg, gt, and tg conformations would be present in V1-butanol. The few reflections observed in ED or powder XRD patterns are not sufficient for a crystal structure determination by conventional direct refinement methods. Therefore, the dataset has been complemented by additional data of various origins. Since V6II complexes with similar diffraction patterns can be obtained with many guest molecules having a molecular geometry different from that of 1-butanol, it seems reasonable to assume that amylose is in an arrangement of minimum packing energy, which might be further stabilized by the complexing agent and the water molecules. As a consequence, a packing analysis would allow selecting the possible amylose helical conformations and helix packing arrangements that can be used as a starting models for the structure refinement. The approach used the present study is similar to that described by [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF] with some modifications.

III.2.3.2. Selection of helical conformation and exhaustive search of helix position

Six regular left-and right-handed amylose helices, having 6, 7, or 8 glucosyl residues per turn and a pitch of 0.8 nm were built as described previously [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. These helices will be referred to as L6, L7, L8, R6, R7, and R8, respectively. As shown by the 13 C NMR data, all gg, gt, and tg conformations would be present in V1-butanol. Therefore, to simplify the procedure of packing analysis, the hydroxyl group on the C6 atom was initially removed This is not surprising since the theoretical helix diameter (1.37 nm) calculated from the unit cell parameters is equal to the outer diameter of 6-fold helices. On the other hand, 7-and 8-fold helices which outer helix diameters are 1.50 nm and 1.62 nm respectively, would be too large for the unit cell size, leading to the observed collision.

In the 6-fold family, L6 and R6 helices have similar packing energy profiles (Figure

III.5).

Each of them presents three local energy minima with the helix centered at the fractional coordinates (0.00,0.00), (0.08,0.17) and (0.08,0.33) in the (a,b) plane. All the structures corresponding to these local minima were retained for further analysis. It is important to note that the energy calculation only took into account Buckingham's non-bonded interactions of amylose chains, while the contribution of the electrostatic interactions and the interactions with solvent/complexing molecules are not involved in the calculation. For this reason, the minima identified here might not be the minima of the whole system but should be very close to them.

By comparing the experimental XRD profile with those calculated from the models, it was found that the models with a L6 or R6 helix centered at the (0.00,0.06) fractional coordinates showed the best agreement. Strictly, these models did not correspond to an energy minimum but their packing energy was very close (Table III.2). Therefore, these models were also selected as starting models for structure refinement. For an easy identification of each model, a name was given based on the helix type and position. In total, eight models were selected, shown in

III.2.3.3. Addition of the C6 hydroxyl group, 1-butanol and water

Since the C6 hydroxyl group would adopt a wide range of orientations in the V6II structure, it was added with random conformation at each glucosyl residue. For 1-butanol and water, an initial rough number of these molecules inserted into the structure was estimated from the crystal density. The experimental density was 1.38 ± 0.02 g.cm -3 , in agreement with the value reported by [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. Considering the total volume of the unit cell

(5.8088×10 -21 cm 3 ) and the total mass of the four amylose chains (6.4588×10 -21 g), the total mass of water and 1-butanol would consequently be 1.5574×10 -21 g. Previously, [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF] estimated, based on the decrease in cell volume after drying, that each unit cell of V1-butanol would contain four 1-butanol molecules, along with some water molecules, located in the inter-helical space. If there is one 1-butanol located within each helical cavity, then the remaining mass corresponds to about 16 interstitial water molecules per unit cell.

After the addition of the C6 hydroxyl group, 1-butanol and water into the selected models, the molecular geometry was optimized using the Universal Force Field in two steps. First, the atoms of the rigid amylose helix were constrained and only 1-butanol and water molecules were allowed to move. In the second step, all the atoms were free to move. Figure III.8 shows the example of the L6_0817 and L_0006 packing models of rigid helices (a,d) and the corresponding geometry-optimized ones after addition of C6 hydroxyl group, 1-butanol and water (b,e). It is noted that after the geometry optimization, the helix is more relaxed, losing the regular hexagonal symmetry while the hydroxymethyl conformation can be gg or between gg and tg. The geometry-optimized structures were input to the refinement against ED data using SHELX (see Chapter II, § II.3.8.1.3). In general, the refinement procedure allowed to find the atomic arrangements that increased the matching between observed and calculated intensities, indicated by a decrease in the R1-factor. However, the refined structures and the corresponding R1-factor depended on the starting models. The R1-factor measures the agreement between the experimental and calculated structure factors after minimization (see Chapter II, § II.3.8.1.3). R1 is close to 1 when the helical conformation is completely lost. Therefore, in the first several refinement steps, the sd was fixed 10 times lower than the default value in order to preserve the starting molecular conformations, i.e. helical conformation. At the same time, the resolution was first limited to 0.35 nm, then to 0.30 nm and finally to 0.24 nm. In the final refinement step, the default value of sd was used to allow the helix to relax while the resolution remained limited to 0.24 nm.

The refinement results are summarized in However, there is no evidence for H-bonds between O2 and O3 of each pair of contiguous glucosyl residues which were reported in previous studies for the V6I structure [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF]. Besides, several intermolecular H-bonds between neighboring helices were also found for V6II while they are absent in V6I (Table III.5) [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF]. In addition, water and 1-butanol molecules in the interstitial space form an H-bonding network between themselves and with the adjacent single helices. These H-bonds together with the interhelical ones would ensure the stability of the orthorhombic packing in V6II structure. The removal of water would break down the H-bonding network leading to the disruption of the crystal structure and a conversion to hexagonal V6a.

III.3. Conclusions

The present structure determination yielded a satisfactory model for V1-butanol regarding the diffraction data in combination with 13 C CP/MAS NMR, density value and packing energy analysis. Since the refinement is based on base-plane ED, the atomic positions along c-axis are less certain than those in the (a,b) plane. In order to ascertain the 3D information on the structure, ED patterns must be recorded on crystals rotated about selected axes of the reciprocal space, e.g. a* and b* to observed the hkl (l≠0) reflections. Our final model confirms the hypothesis made by [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF].

V6II crystals could be obtained with other complexing agents (see Chapters IV and V) but some variation in the diffracted intensities was observed. Different guest molecules may result in some variation in atomic positions of amylose chains but the helical conformation and the helix position are expected to remain closely similar. The procedure described in this chapter can be applied to determine the structure of these isomorphous crystals, which can help to know the correlation between the nature of guest and the variation in diffraction data.

Chapter IV

Morphology and crystal structure of different forms of V-amylose

IV.1. Introduction

The polymorphism of V-amylose has been the subject of many studies [START_REF] Bear | Complex formation between starch and organic molecules[END_REF][START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Bluhm | Detailed structure of the Vh-amylose-iodine complex: a linear polyiodine chain[END_REF][START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF][START_REF] Rutschmann | Formation of inclusion complexes of starch with different organic compounds. IV, ligand binding and variability in helical conformations of V amylose complexes[END_REF][START_REF] Sarko | Crystal structure of the KOH-amylose complex[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Uchino | Inclusion compound formation of amylose by sealed-heating with salicylic acid analogues[END_REF]Winter & Sarko, 1974;[START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF][START_REF] Zobel | X-Ray diffraction of oriented amylose fibers. II. Structure of V amyloses[END_REF]. As reviewed in Chapter I, only five allomorphic families containing compact 6-, 7-and 8-fold single helices have been identified: V6I (Vh), V6II (Vn-butanol), V6III (Vglycerol), V7 (V2-propanol) and V8 (V1-naphthol). On the other hand, previous modeling works suggested that V-amylose can exist in other forms. For instance, it was proposed that a structure with as many as 10 residues per turn could form if appropriate larger guests were used [START_REF] French | Allowed and preferred shapes of amylose[END_REF][START_REF] French | Accessible conformations of amylose[END_REF]. So far, knowing the nature of a complexing agent is not enough to predict the structure of the resulting complex, although there are some indications of the dependence of the helical conformation on the size of the guest molecule. However, the crystallization of amylose into new structures likely requires testing new complexing agents and explore specific crystallization conditions. Some evidence showed that the structure of complexes may vary with the concentration of complexing agent and DMSO [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], but in previous investigations, the different complexes were usually prepared in similar conditions.

This chapter describes the preparation of different families of lamellar V-amylose crystals and the collection of their crystallographic data in order to propose a conformation and packing arrangement of amylose helices in each allomorph. We have tested the crystallization of amylose in the presence of 121 organic molecules (see Chapter II, Table II.2) in different crystallization conditions. Their morphology was characterized using TEM and, for a few of them, AFM. The unit cell parameters and symmetry were determined in hydrated/solvated and dry states from ED and XRD data. The complexes were also studied by FT-IR and the amylose conformation was determined by 13 C solid-state NMR. Finally, tentative molecular models built with symmetrical rigid amylose helices were proposed based on the collected crystallographic data, showing probable arrangements of helices in the unit cells. The conditions of formation of the complexes and the crystallization parameters affecting the complex structure will be discussed in Chapter V.

IV.2. V6I complexes

IV.2.1. Complexing agents

This allomorph was obtained with complexing agents that contain linear carbon chains with different functional groups such as carboxylic acids (-COOH), amides (-CONH2), alcohols (-OH), amines (-NH2) or aldehydes (-CHO). No tested alkanes and esters yielded V6I despite containing linear carbon chains. This suggested that the functional groups play a role in the formation of the complexes. Quinoline, (-)-borneol and trans-decalin did not yield V6I crystals although they were previously reported to induce this allomorph [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. In addition, no branched or cyclic molecules produced V6I. This result was expected as the size of these molecules is not compatible with the cavity of the 6-fold helices or the inter-helical spaces in V6I. with previous works [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. with the previous studies that, upon drying, V6I transforms into V6a (Va), which exhibits a smaller unit cell [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF][START_REF] Shogren | X-ray diffraction study of crystal transformations in spherulitic amylose/lipid complexes from jet-cooked starch[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. Indeed, the diffraction profile of the dry complex shown in nm. The slight difference in unit cell parameters between V6I and V6a would result from the loss of water molecules from V6I which reduces the inter-helix space but does not alter the helical conformation [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF]. Besides, the transition from V6I to V6a would not depend on the release of the complexing agent since some guests such as long-chain fatty acids and alcohols would not be removed from the complexes by drying. After rewetting with water, the complexes yielded an XRD profile identical to the initial hydrated complexes (Figure

IV.2.2. Morphology and crystal structure

IV.3c

), supporting the fact that water is the key factor that controls the structural transition.

Figure I

.4 shows the model proposed for V6I [START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF] and corresponding V6a [START_REF] Murphy | The structure of V amylose dehydrate: a combined X-ray and stereochemical approach[END_REF] projected on the (a,b) plane of the orthorhombic unit cells. V6a is more compact than V6I. In both structures (space group P212121), each unit cell contains two anti-parallel 6-fold left-handed helices packed onto a hexagonal lattice with guest molecules located inside the helix cavity. If the hexagonal symmetry is used, the crystal would have a statistical arrangement of 'up-and-down' amylose helices [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF].

IV.3. V6II complexes

IV.3.1. Complexing agents

Similar to those of V6I, V6II complexes were obtained with linear compounds like fatty acids, alcohols, amines, aldehydes and stearamide. Indeed, most of these compounds yielded both V6I and V6II. This result is unexpected as only a few complexing agents with such ability have been reported, including n-propanol, n-butanol and n-pentanol [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], adipic, suberic and sebacic acids [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], and hexanoic acid [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. In addition, the long-chain fatty acids and alcohols (8-18 carbons) which were previously reported to yield only V6I [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], were shown to induce V6II in this study. V6II was also obtained with some esters, which did not induce V6I. Interestingly, isopropyl myristate yielded V6II although the molecule contains a branched-chain moiety (isopropyl). V6II was maybe formed because the molecule contains a long straight-chain aliphatic part (myristyl) that predominantly interacts with the helical cavity and controls the helicity. The remaining part with a larger size would be located outside the helix. In fact, other branched molecules including isopentyl acetate and cyclic compounds did not yield the V6 complex. This result agrees with previous conclusions that the 6-fold helix complexes are obtained with linear complexing agents whose diameter in cross-section is about 0.3 nm [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. -f). This morphology is similar to that previously described for V6II complexes of n-butanol and n-pentanol [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Manley | Chain folding in amylose crystals[END_REF][START_REF] Yamashita | An electron microscopie study on crystals of amylose V complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes[END_REF]. Besides, cracks were observed along the longer dimension of the lamellae. These cracks are associated with the stress due to the shrinkage which occurs when the crystals are dehydrated in vacuum [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Manley | Chain folding in amylose crystals[END_REF]. The cracks were observed by TEM on dry crystals prepared in pure water with most complexing agents yielding the V6II structure. However, if the crystals were prepared in the presence of 1-2% of DMSO as a co-solvent, they became more stable and the cracks were observed after several hours of exposure in vacuum.

IV.3.2. Morphology and crystal structure

In contrast, the V6II crystals prepared with diols (Figure IV.5b-c) and diethyl malonate were as stable as those obtained with other complexing agents prepared in the presence of DMSO. This suggests that the complexing agents and co-solvent DMSO play an important role to stabilize the crystal structure. The drying behavior of V6II crystals will be further discussed in the next section. diffraction pattern is also identical to that previously reported for V6II prepared with n-butanol and n-pentanol [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF], and thus can be described with an orthorhombic unit cell (space group P212121). However, for consistency with the other V-amylose allomorphs described in this work, and following the convention proposed by [START_REF] Donnay | Rules for the conventional orientation of crystals[END_REF], i.e. a < b, we have switched the a and b values with respect to those given in the previous studies. Consequently, the average unit cell parameters are a = 2.65 ± 0.04 nm and b = 2.69 ± 0.03 nm, in good agreement with those calculated from the XRD pattern shown in

IV.3.3. Effect of drying on the crystal structure

As noted above, there are two different drying behaviors observed for V6II complexes: i) crystals show the cracks just after being exposed to vacuum, and ii) crystals are more stable and the cracks are observed after keeping the crystals for long time in vacuum. In both cases, the cracks occur along the longer dimension of the lamellae and the crystals yield diffraction These patterns were almost identical to those reported for V6III prepared with glycerol, DMSO or ethylenediamine [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF][START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF]Winter & Sarko, 1974) [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. b) Partial dry uncracked crystal having a pseudo-tetragonal V6III structure [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF]Winter & Sarko, 1974). c) Thoroughly dried crystal, exhibiting cracks running parallel to the long dimension of the crystals, having a pseudo-hexagonal structure V6a.

The above results reveal that the pseudo-tetragonal V6III is much more stable compared to the orthorhombic V6II and thus the transition into V6a takes a longer time. Interestingly, V6III exhibits a larger inter-helix space but requires a lower water content to stabilize the structure.

Previous studies on the transition of V6II into V6a during drying did not report the presence of V6III as an intermediate structure [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF]). In fact, our results

show that the transition from V6II to V6III only occurs for complexes with specific guests such as diols and diethyl malonate or complexes prepared in the presence of 1-2% of DMSO as a co-solvent. The role of complexing agents and DMSO as well as the driving forces that allow for the formation of V6III are still unclear.

According to [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF], the transition from V6II to V6a observed for complexes with n-butanol or n-pentanol would be promoted by the removal of the volatile complexing agents located in the inter-helix spaces from the crystals. However, such release may not be observed for non-volatile complexing agents such as long-chain mono-or dicarboxylic acids. This suggests that the departure of water would be the major cause of the structural transition rather than that of the complexing agent. Indeed, after rewetting in water,

IV.4. V6III complexes with glycerol

IV.4.1. Formation of complexes

In the present study, V6III lamellar crystals were directly obtained only with glycerol.

Analogous molecules such as 1,3-propanediol, 1,2-propanediol, 2-propanol did not yield V6III in the similar crystallization conditions. So far, V6III have been obtained with three complexing agents: DMSO (French & Zobel, 1967;[START_REF] Winter | The crystal structure of amylose-DMSO complex[END_REF] and ethylenediamine [START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF] in the form of crystalline films, and glycerol in the form of lamellar crystals. A common preparation condition of V6III with these complexing agents is that water is nearly absent. For example, [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF] prepared V6III lamellar crystals in glycerol at 100 °C in the presence of less than 0.2 wt% water. In the present study, V6III could be prepared with glycerol in the presence of up to 10 wt% water but at a lower temperature (40 °C). With a higher concentration of water, V6I was formed instead. In addition, V6III crystals were also prepared by dissolving amylose in DMSO at 100 °C, and addition of 9 or 999 volumes of glycerol at 90-100 °C. This method is simple and does not require a removal of water.

IV.4.2. Morphology and crystal structure

Figure IV.11a shows a typical V6III crystal prepared with glycerol. This crystal has a more or less square contour, consisting of a stack of square-shaped layers that may have grown by a dislocation-induced spiral growth. This morphology is similar to that previously reported for Vglycerol [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF].

The Vglycerol crystals had a good stability in vacuum, i.e., they did not exhibit any cracks et al. (1996) who reported that a ≠ b (a = 1.93 nm, b = 1.8 nm). Our results also confirm that Vglycerol is isomorphous to VDMSO (French & Zobel, 1967;Winter & Sarko, 1974) and Vethylenediamine [START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF]. VDMSO has similar unit cell parameters (a = b = 1.92 nm) [START_REF] Winter | The crystal structure of amylose-DMSO complex[END_REF] while those of Velthylenediamine are slightly smaller (a = b = 1.89 nm) [START_REF] Simpson | X-Ray diffraction of ethylenediamine-amylose complex[END_REF]. In the present study, both base-plane ED and XRD data did not allow identifying the c-parameter of the unit cell. [START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF] reported c = 0.83 nm for Vglycerol (corresponding to one helical pitch), which is about three times lower than that of VDMSO (c = 2.24 nm, corresponding to 3 helical pitches) [START_REF] Winter | The crystal structure of amylose-DMSO complex[END_REF].

The detailed molecular structure of Vglycerol is still unknown. However, the packing of amylose helices in Vglycerol should be similar to that of VDMSO described by Winter and Sarko (1974). Within the unit cell, there are two antiparallel left-handed 6-fold helices close-packed on a tetragonal lattice, as shown Figure IV.12. The complexing agent can be located both inside and between the helices. to 20 carbons, linear ketones (2-pentanone), linear alcohols with the hydroxyl group located at endo-positions such as 2-propanol and 1,3-butanediol, and many branched and cyclic compounds. In general, this result is in agreement with the previous investigations [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]Nuessli et al., 2003a;[START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]. However, V7 lamellar crystals were also successfully prepared with some of molecules which were previously reported as unable to form a complex with amylose, such as citral, 2-naphthol and quinoxaline [START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF]). In addition, V7 was also obtained with fatty acids having 6-20 carbons and quinoline which have been reported to form 6-and 8-fold complexes, respectively [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF].

IV.5.2. Morphology and crystal structure

V7 lamellar crystals prepared with different complexing agents had an almost identical morphology (Figure IV.13). Typical crystals were long rectangular lamellae, usually grown from the same nucleation site and forming flower-like aggregates. Individual lamellae were rarely seen. However, the crystals were more individual after successive recrystallizations. Stacks twinned at about 90° were sometimes observed. The lamellar thickness determined by AFM and averaged from 2 different samples is 7.9 ± 0.9 nm (Annex 2, Table S They all resemble those previously reported for complexes prepared with 2-propanol and acetone [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF], fenchone, menthone and geraniol (Nuessli et al., 2003a), thymol, linalool and terpineol [START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. They can be accounted for a two-dimensional orthorhombic unit cell, with average parameters a = 2.81 ± 0.03 nm and b = 2.95 ± 0.05 nm.

The powder XRD patterns of different complexes are similar as well ( This arrangement can be described by alternating motifs of 4 hexagonally-packed helices along with other 4 helices with a larger interhelical space. The complexing agent would be located both inside and between the helices, together with some water molecules.

IV.5.3. Effect of drying on the crystal structure

All V7 complexes were sensitive to drying. The dry crystals yielded a XRD pattern different from that of the initial hydrated state, suggesting a structural transition. However, depending on the complexing agents, two drying behaviors were observed for the V7 complexes.

Upon thorough drying, the complexes of all complexing agents, except 1,3-butanediol, exhibited similar XRD profiles, as shown for decahydro-2-naphthol in Figure IV.16b. These diagrams are nearly identical to those previously reported for the V7a obtained by drying the V7 complexes with branched alcohols, ketones, fatty acids and ring compounds (Nuessli et al., 2003b;[START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF]. They can be indexed based on a hexagonal unit cell with a = b = 1.480 ± 0.014 nm and c = 0.794 ± 0.008 nm or a pseudo-hexagonal orthorhombic unit cell with a = 1.480 ± 0.014 nm, b = 2.563 ± 0.024 nm and c = 0.794 ± 0.008 nm. However, since the 7-fold helix cannot be crystallographically located onto the 6-fold or 2-fold screw axes of P212121, only the pseudohexagonal orthorhombic unit cell with the space group P1 can be considered. Alternatively, V7a can adopt the space group P212121 by doubling the unit cell (a = 2.563 ± 0.024 nm, b = 2.960 ± 0.028 nm and c = 0.794 ± 0.008 nm). In both cases, the 7-fold amylose helices would be organized in a hexagonal net, similar to that of the V6a structure, as shown in Figure IV.17. behavior of V7 crystals with 1,3-butanediol would be due the ability of the complexing agent to adapt with both 6-and 7-fold helical conformation. Indeed, 1,3-butanediol was shown to induce the formation of both 6-and 7-fold helices.

The above structural transition behavior of V7 complexes would be the result of the departure of water rather than complexing agent. Indeed, the solid complexing agents such as cis-decahydro-1-naphthol or 2-naphthol are unlikely to be removed by drying. In addition, the V7 structure is completely recovered after rewetting with water (Figure IV.16), suggesting that the guest molecules remained inside the complexes. However, the above results raise the question of the location of complexing agents in the V7 crystals. If the complexing agent is located between the helices in V7 crystals and is not removed after drying, it is expected to disturb the hexagonal arrangement of amylose helices. The limited inter-helix space in V7a

would not allow the accommodation of the complexing agent.

IV.6. V8 complexes

IV.6.1. Complexing agents and formation of complexes

V8 lamellar crystals were prepared by crystallization of amylose in the presence of 1-naphthol, quinoline and salicylic acid. To our knowledge, salicylic acid is the third complexing agent that yields this V-amylose allomorph. In addition, although [START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF] reported that salicylic acid did not form crystalline complexes from amylose solutions, we have successfully prepared Vsalicylic acid lamellar crystals for the first time. respectively. They all resemble regarding the tetragonal symmetry and the positions of the corresponding reflections, and are similar to those previously reported for V1-naphthol and Vquinoline [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF]). These patterns are accounted for a tetragonal unit cell of space group P43212 with average parameters a = b= 2.30 ± 0.01 nm. The XRD patterns are similar as well, as shown in Figure IV.19g-i.

IV.6.2. Morphology and crystal structure

The average unit cell parameters calculated from XRD data are a = b= 2.313 ± 0.006 nm and c = 0.790 ± 0.001 (Annex 2, Table S.IV.3), in good agreement with the ED data.

However, differences in relative intensities of the ED spots could be noted between the different complexes. Some reflections in the ED pattern of V1-naphthol and Vsalicylic acid are absent in the pattern of Vquinoline. This would be due to the double diffraction that occurs when the crystals are thick, as previously noted for V1-naphthol crystals [START_REF] Putaux | Morphology and structure of A-amylose single crystals[END_REF]. A slight difference in reflection intensity was also observed in XRD patterns. Especially, the 1 1 0 reflection at d-spacing of 1.628 nm (2θ = 5.43°) is observed for Vsalicylic acid but is absent for the other complexes. These differences would be accounted for the contribution of the complexing agent. Since their structures are isomorphous, the three complexes would have similar packings of amylose helices such as that proposed for V1-naphthol [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF] After rewetting with water, the complex yielded a diffraction pattern that was almost identical to that of the initial wet state (Figure

IV.21a,c).

The result implied that the complexing agent was not removed during drying and that water is essential for the crystallinity and stability of the complexes. Similar behaviors were observed for Vsalicylic acid and Vquinoline.

IV.7. A new V-amylose allomorph obtained with 4-hydroxybenzoic acid

The interaction between amylose and 4-hydroxybenzoic acid (HBA) has been the subject of several studies [START_REF] Goudah | Complex interaction of starches with certain drug pharmaceuticals[END_REF][START_REF] Zhu | Effect of phenolic compounds on the pasting and textural properties of wheat starch[END_REF]. However, no study have reported the existence of crystalline complexes. Besides, the investigation conducted by [START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF] showed that HBA was unable to form the complexes with amylose. In the present study, VHBA was successfully prepared in the form of lamellar crystals. Moreover, it appears to be a new V-amylose allomorph, exhibiting unique morphological features, and distinct XRD and ED patterns.

IV.7.1. Formation of complexes

The preparation of HBA complexes conducted in pure water and in DMSO/water mixture gave similar results. In the presence of 0.1-0.5 wt% of amylose and a saturation of HBA, the complexes crystallized out slowly at a relatively low temperature ≤ 50 °C. The crystals appeared after about one week of incubation and the crystallization finished after two weeks. In comparison to other complexes, nucleation and growth rates of VHBA seem to be slower. [START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF] may not have waited for enough time to observe the formation of crystals. S.IV.1). In addition, the h00, 0k0 reflections are systematically absent when h, k were odd, suggesting the P21212 or P212121 space group.

IV.7.2. Morphology and crystal structure

The orthorhombic unit cell would contain 2 amylose helices. Assuming a regular closepacked array of helices, the helix packing diameter would be 1.55 nm, smaller than the diameter of an 8-fold helix (1.62 nm) [START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF]) but larger than that of a 7-fold helix

(1.50 nm) [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]) and a 6-fold helix (1.37 nm) [START_REF] Mikus | The complexes of fatty acids with amylose[END_REF].

Therefore, only 7-fold or 6-fold helices can be packed in the unit cell. If the crystal contains 7-fold helices, the space group would be P1 rather than P212121 since 7-fold helices cannot be located on 2-fold screw axes. In contrast, if it contains 6-fold helices, the P212121 space group is possible but the inter-helical space will be larger since the helices have a smaller diameter.

A tentative model of VHBA with P212121 space group containing left-handed 6-fold helices is shown in Figure IV.24. The complexing agent would be located only in the inter-helix space with some water molecules. The quantification of HBA in the complex showed that there is about 1 HBA molecule for 6 glucose units, corresponding to 2 HBA molecule per unit cell. Our diffraction data is insufficient to determine the helical conformation of amylose. Further consideration of this point will be made using the solid-state NMR data presented in § IV.13.

IV.8. A new V-amylose allomorph obtained with (-)-borneol, R-(+)-camphor, cis-decahydro-1-naphthol, decahydro-2-naphthol, and 1,3-butanediol

A new allomorphic family of V-amylose lamellar crystals was obtained by crystallization of amylose in the presence of (-)-borneol, R-(+)-camphor, cis-decahydro-1naphthol, decahydro-2-naphthol and 1,3-butanediol. These complexes are isomorphous, having characteristic morphological features and ED and XRD patterns. This pattern is almost identical to that of V7a obtained by drying V7 complexes, and can be indexed using a pseudo-hexagonal orthorhombic unit cell with a = 1.505 ± 0.005 nm, b = a√3 = 2.607 ± 0.009 nm and c = 0.795 ± 0.006 nm. In contrast, the XRD pattern (Figure

IV.31b)
recorded on the dry complexes with 1,3-butanediol contains characteristic reflections of both V6a and V7a, which is similar to that obtained by drying the V7 complexes of 1,3-butanediol.

After rewetting, the initial structures were recovered (Figure IV.30c).

The above results strongly support that the present crystals contain 7-fold helices like in previously known V7 structures. Therefore, they would have orthorhombic unit cells rather than hexagonal ones, and the space group would be P1. The arrangement of amylose helices would be essentially similar to that of V7a but with more inter-helix space, as shown in model in Figure IV.32. The complexing agent would be located inside the helical cavity while the inter-helix space would accommodate some water molecules. S.IV.2). The c-parameter is slightly larger than those of other allomorphs that are between 0.79 and 0.83 nm. In addition, only h00, 0k0, and 00l reflections with even indices were observed, suggesting that the space group is either P212121 or a lower symmetry P1. The unit cell would contain 2 amylose helices.

Assuming a close packing of helices, the helix packing diameter is about 1.50 nm which is identical to that found for the 7-fold helix of V7 complexes. Therefore, the present complex probably contains 7-fold helices in a compact arrangement or 6-fold helices with a looser packing. 1-Naphthol can be located inside the helices. The stoichiometry of 1-naphthol determined by UV spectroscopy is 1 molecule of 1-naphthol per 7.3 glucosyl units. From this geometrical analysis, it is not possible to conclude whether the complexes contain 6-fold or 7-fold helices.

The helical conformation will be further studied by solid-state NMR, as presented in § IV.13. V7 complexes or V(-)-borneol) and isomorphous complexes. This supports the hypothesis that the complexes are composed of 7-fold rather than 6-fold helices. After rewetting in water, the initial structure was recovered, giving a diffraction diagram identical to that of the initial hydrated state. The results suggests that the complexing agent is not removed during the drying process and the structural transition is due to the departure of water located in the inter-helical space. In addition, it is interesting to note that the present Vquinoline, V7 complexes and V(-)-borneol and isomorphous complexes are commonly transformed into V7a, but after rewetting, the V7a is reverted into the corresponding initial structure and not other allomorphs. The mechanism for this selective transition is still unknown, but the presence of water is essential for the process.

IV.11. A new V-amylose allomorph obtained with salicylic acid

In addition to the previously described V8 allomorph, the crystallization of amylose in the presence of salicylic acid allowed obtaining another type of crystals, with a structure differing from other known allomorphs. Upon thorough drying, the diffraction peaks become broader but the peak positions remain the same, suggesting that the structure is rather stable upon drying. The slight decrease of crystallinity would be because the amylose helix would be less ordered due to the loss of water. After rewetting, the peaks sharpen like in the initial hydrated state. It is interesting to note that the diffraction pattern of the dry complexed is similar to that previously reported for complexes of salicylic acid and some analogues prepared by sealed-heating method [START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Uchino | Inclusion compound formation of amylose by sealed-heating with salicylic acid analogues[END_REF]. As noted, the XRD diffraction data obtained from the polycrystalline aggregates prepared by sealed-heating was not resolved enough for the authors to determine the crystal structure. However, they proposed the formation of 8-fold helices showing some similarities with γ-cyclodextrin complexes with salicylic acid.

IV

IV.12. Study the complexes by FT-IR spectroscopy

We have studied the IR spectra of different allomorphs of V-amylose. In addition, we also compared the IR spectra of V-amylose complexes to those of A-and B-amylose and DP6500 amylose which contains mainly amorphous amylose together with small fraction of B-amylose.

The IR spectra of untreated DP6500 amylose, A-and B-amylose and different allomorphs of V-amylose complexes in the hydrated state are shown in Figures IV. 45 and IV.46. The observed bands attributed to amylose in these spectra are listed in Table IV.1. In addition, the band near 1645 cm -1 is attributed to the adsorbed water. In V-amylose complexes, additional bands correspond to contributions from the complexing agents. Figure IV.45 shows that the spectra of different allomorphs of amylose and V-amylose are very similar. However, some differences between V-amylose complexes and the uncomplexed amylose forms were noted in the fingerprint region from 1500 to 400 cm -1 . As observed in Figure IV.46, lines near 1408, 1370, 1295, 1104, 1022, 946 cm -1 observed for V-amylose complexes are nearly absent or have a lower intensity by comparison to those of DP6500 amylose, A-and B-amylose. The variation in the vibrational frequencies would be related to the structure of different forms of amylose. As being reviewed in Chapter I, one of the main differences between V-amylose and other forms lies in the conformation of amylose. In the V-amylose complexes, amylose exists as compact single helices which are stabilized by intra-molecular hydrogen bonds, i.e. O6…O2 ' and O2…O3 ' . On the other hand, A-and B-amylose have a more extended helical conformation which does not allow intra-molecular H-bonds. DP6500 amylose is almost amorphous and thus exists in a random coil state, in which H-bonds occur in an irregular manner. The difference in conformation and hydrogen bonds results in differences in length and strength of the related bonds, especially C1-O-C4' bridge and those related to O2, O3 and O6, leading to differences in vibrational frequencies of these bonds. In previous works, the frequencies around 1408, 1370, 1295, 1104 and 1022 cm -1 have been assigned for the C-H, CH2, C-C and C-O-H related modes while that at 946 cm -1 is due to the α(14) linkage [START_REF] Cael | Infrared and Raman spectroscopy of carbohydrates. Part VI. Normal coordinate analysis of V-amylose[END_REF][START_REF] Cael | Infrared and Raman spectroscopy of carbohydrates. Part III: Raman spectra of the polymorphic forms of amylose[END_REF][START_REF] Santha | Raman and infrared spectra of starch samples of sweet potato and cassava[END_REF]. [START_REF] Cael | Infrared and Raman spectroscopy of carbohydrates. Part III: Raman spectra of the polymorphic forms of amylose[END_REF] and [START_REF] Cael | Infrared and Raman spectroscopy of carbohydrates. Part VI. Normal coordinate analysis of V-amylose[END_REF]. -: not observed; ND: not determined because the bands are present as shoulders or superimposed to those of complexing agents.

Among the vibrational lines, the one near 1022 cm -1 which is assigned to the C-O-H deformation shows the most remarkable difference between V-amylose and other amylose forms and thus can be used as a fingerprint for V-amylose. However, the relative intensity and the position of this line vary with the hydration state of V-amylose. As shown for V7 of IV.2. The assignment was made based on the corresponding liquid-state spectra [START_REF] Gidley | Molecular organization in starches: A carbon-13 CP/MAS NMR study[END_REF][START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF]. A shoulder or a weak peak at 100.4 ppm was also observed in spectra of several complexes corresponding to C1 resonance of B-type amylose [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF]Veregin et al., 1987a). In addition, resonances assigned to complexing agents were observed in the spectrum of V6I (1,6-hexanediol) and V6II (1,6-hexanediol) at 62.1 ppm, V6III (glycerol) at 60.1 ppm and 72.8 ppm, and VR-(+)-camphor at 57.9 ppm. and ψ (O1-C1-C4-C5) about the glucosidic linkages and thus reflect the helical conformation [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF]Veregin et al., 1987a). Our results show that the C1 resonance is shifted downfield with increasing helicity of amylose chain ( 3-81.9 ppm, thus at higher field than that of V6III (80.2-84.6 ppm) and V8 (82.9-83.4 ppm).

Our results partially agrees with the report of [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF] that mentions that the C1 and C4 chemical shifts are not sensitive to expansion of the amylose helix from a 6-to a 7fold repeat, but are shifted downfield by ~1 ppm in the V8 complex obtained with 1-naphthol.

The conformation of V-amylose in the new allomorphs was determined based on the position of the C1 resonance. V(-)-borneol, the isomorphous V(R)-(+)-camphor and Vquinoline have C1 resonances centered at a position similar to that of V7 complexes, and thus would contain 7-fold helix, in agreement with the fact that these complexes are commonly transformed into V7a upon drying. The new allomorph Vsalicylic acid has the same C1 resonance with V8 complexes, suggesting an 8-fold helical conformation. Difficulties arise for V4-hydroxybenzoic acid and V1-naphthol which C1 resonance is a multiplet rather than only one signal observed in other allomorphs. The splitting of the resonance reflects the presence of inequivalent environments within the material [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Horii | Structural study of amylose polymorphs by cross-polarization-magic-angle spinning, 13 C-NMR spectroscopy[END_REF]Veregin et al., 1987a). Therefore, we can assume that the single helices in V4-hydroxybenzoic acid and V1-naphthol are less symmetrical than those in other allomorphs. However, it must be noted that the C1 resonance is rather broad in most spectra, suggesting that the residues are in a number of environments described by a range of conformations averaging about the highest peak. Considering the highest signal of C1, V4hydroxybenzoic acid and V1-naphthol would contain 6-and 7-fold amylose helix, respectively. In general, the helical conformation determined from the C1 resonance in solid-state NMR is in good agreement with that predicted on the basis of helix packing diameter. Further works, such as a structural refinement using diffraction data, should be conducted to confirm the helicity of amylose in new allomorphs.

In previous works, the C6 chemical shift was found to be correlated with the exocyclic angle  (C4-C5-C6-O6) of glucosyl residues and thus reflected the hydroxymethyl group conformation (gg, gt and tg) determined from single-crystal XRD data. For cellulose, three C6 chemical shifts are observed at 60-62.6 ppm, 62.5-64.5 ppm, and 65.5-66.5 ppm, which are related to the gg, gt and tg conformations, respectively [START_REF] Horii | Solid-state 13 C-NMR study of conformations of oligosaccharides and cellulose[END_REF]. These values are in agreement with those proposed for cyclodextrins: 59.6-61.7 ppm for gg, and 62.7-65.9 for gt [START_REF] Veregin | Correlation of 13 C chemical shifts with torsional angles from high-resolution, 13 C-CP-MAS NMR studies of crystalline cyclomalto-oligosaccharide complexes, and their relation to the structures of the starch polymorphs[END_REF]. In V-amylose, all allomorphs have the C6 resonance (singlet or multiplet) that covers a wide range (57-66 ppm), and centers at 60.2-61.8 ppm. Therefore, it seems that all gg, gt and tg conformations are present in V-amylose but gg is the major conformation. Finally, V6I and V6II complexes exhibit very similar spectra, with a slightly upfield shift observed for the C1 resonance of V6II (102.4 ppm) with respect to that of V6I (102.6-102.7 ppm), in agreement with the conclusions of Le [START_REF] Bail | Structural investigation of amylose complexes with small ligands: helical conformation, crystalline structure and thermostability[END_REF]. The complexing agents appear to have no significant effect on the chemical shifts of the glucosyl carbons in these allomorphs.

On the other hand, the V6III spectrum exhibits by a triplet C4 resonance at lower field than that sensitive not only to the crystal structure but also to the nature of the complexing agents.

IV.14. Classification of complexesproposition of a nomenclature

In the present study, 5 new V-amylose allomorphs have been identified, increasing the number of known allomorphic families to 10. In order to facilitate the identification of different allomorphs, we have established a nomenclature system on the basis of that proposed by [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], in which the complexes are classified as a function of their helical conformation, interstitial space in the unit cell and complexing agent. Each allomorph is named "Vxy", where "V" represents V-amylose, "x" is an Arabic number (6, 7, 8) representing the helicity of amylose, subscript "y" is Roman (I, II, III, etc.) referring to an ascending order of interstitial space of "unit cell" containing the same number of helices.

Table IV.3 summarizes the helical conformation and the unit cell parameters and the name assigned for each allomorph. According to the present nomenclature, the name of V6I, V6II, V6III remains the same while V7 and V8 will be called V7II and V8II, respectively. The new allomorphs V4-hydrobenzoic acid, V(-)-borneol, V1-naphthol, Vquinoline and Vsalicylic acid are named V6IV, V7I, V7III, V7IV and V8I, respectively. Since one allomorph can be obtained with different complexing agents, it is necessary to add the name of the complexing agent in order to clearly define a complex [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. For example, V6I obtained with ethanol will be named V6I (ethanol). It is important to note that the present crystallographic data did not allow an unambiguous determination of the helical conformation V6IV (4-hydroxybenzoic acid) and V7III (1-naphthol). The nomenclature may thus be temporary and depend on future results.

Figure IV

.49 shows the models proposed for V6I [START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF][START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF], V6II [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF], V6III (Winter & Sarko, 1974), V7II [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF], V8II [START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF], as well as the new allomorphs. In all constructed V-amylose models, left-handed helices are arranged as antiparallel pairs, consistent with the concept of chain-folding. For V6I, V7I, V7III, V7IV and V8I, the proposed arrangements of amylose are rather compact, and the complexing agents would only be located inside the helix.

In contrast, there is more interstitial space in V6II, V6III, V6IV, V7II and V8II, to accommodate guest molecules. Besides, since V6IV was obtained with 4-hydroxybenzoic acid whose size appears to be incompatible with the cavity of a 6-fold helix, the cyclic complexing agent should be located in interstitial space. For other complexes, the complexing agents can possibly be located inside the helix. c interhelical space per helix = [unit cell volume -N.(π.d 2 /4)]/N, where N is the number of helix per unit cell; d is the external helix diameter and is 1.37, 1.50 and 1.62 nm for V6, V7, and V8 helix, respectively.

It is important to note that the proposed models describe possible arrangements of amylose helices in the allomorphs rather than their precise atomic positions. For example, as suggested from 13 C solid-state NMR data, the amylose helices are likely less symmetrical than the rigid ones used in the models, especially for V6IV and V7III. However, these models can be used as starting hypotheses for other studies by molecular dynamics or structure refinement.

IV.15. Conclusions

The investigation of the complexation of amylose with about 120 small organic molecules allowed identifying 10 different allomorphic families of lamellar V-amylose crystals.

Interestingly, five of them are new allomorphs. A new nomenclature was proposed for these allomorphs based on the helical conformation and relative interhelical space of unit cells.

Consequently, the 10 allomorphs of were named: V6I, V6II, V6III, V6IV, V7I, V7II, V7III, V7IV, V8I and V8II. Each allomorph can be obtained with different complexing agents. However, V6I, V6II and V7II appear to be more prevalent. Our results also confirmed that the helical conformation is related to the size of the complexing agent. V6 complexes were obtained with molecules with straight carbon chains, while branched-chain and cyclic molecules tended to yield 7-fold complexes. V8 complexes were obtained with 1-naphthol, quinoline and salicylic acid. However, it is still difficult to predict the crystal structure based on the nature of the guest since many complexing agents were shown to induce different crystal structures, helical conformation or helix packing.

The model lamellar crystals corresponding to V6II, V6IV, V7II, and V7III exhibited distinct morphologies. On the other hand, those with the V6I, V7I, V7IV and V8I structures showed very similar shapes. The morphology of V6III and V8II were similar as well. However, the different allomorphs could be distinguished without ambiguity by their base-plane ED or powder XRD patterns.

The FT-IR spectroscopy data showed an increase in intensity of vibrational bands near 1408, 1370, 1295, 1104, 1022 and 946 cm -1 for V-amylose compared to amorphous or A-and Bamylose. These differences can be accounted for different amylose helical conformations in these forms. In addition, the band near 1022 cm -1 can be used as a fingerprint for V-amylose.

The 13 C solid-state NMR study showed a significant correlation between C1 resonances and the helicity of amylose. The C1 resonance shifted downfield with increasing number of glucosyl units per turn from 6 to 8. The result thus allowed determining the helical conformation of the new allomorphs. In addition, the C1 resonance appears as a muliplet in V6IV and V7III

while it is a singlet in other allomorphs suggesting that V6IV and V7III would contain helices that are less symmetrical than those in other allomorphs.

Based on the above crystallographic data, tentative models were proposed for each allomorph. Water was shown to play an important role on the stability and the crystallinity of the complexes. Upon drying, V6I, V6II, V6IV, V7I, V7II, V7IV were transformed into compact hexagonal structures. For V7III, V8I and V8II, a significant loss in crystallinity was observed, but the crystal structure remained the same. V6III is the only structure that was stable upon drying.

Chapter V

Factors affecting the crystallization and crystal structure of V-amylose

V.1. Introduction

The crystal structure of V-amylose depends not only on the nature of the complexing agent but also on the conditions of crystallization [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. Evidence supports that the helical conformation of amylose is related to the size of the complexing molecules [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Rutschmann | Formation of inclusion complexes of starch with different organic compounds. IV, ligand binding and variability in helical conformations of V amylose complexes[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Yamashita | An electron microscopie study on crystals of amylose V complexes[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF]. Indeed, isomorphous complexes can be obtained with molecules with a similar size. The amylose helices are thought to be in a minimum-energy conformation stabilized by complexing agents and water [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

On the other hand, with a given molecule, V-amylose may occur in more than one crystal structure with different helical conformations or packing arrangements [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. This phenomenon is called polymorphism and has been observed for many other organic and inorganic systems, as reviewed by [START_REF] Bernstein | Polymorphism in molecular crystals[END_REF]. The formation of different allomorphs requires applying a variety of crystallization conditions [START_REF] Bernstein | Polymorphism in molecular crystals[END_REF][START_REF] Bernstein | Polymorphism-a perspective[END_REF]. The conventional crystallization of V-amylose in solution generally allows varying the following parameters: concentration of complexing agent and amylose, solvent, solvent mixture, temperature, cooling rate and DP of amylose. However, only concentration of complexing agent and solvent (with or without DMSO) have been reported to affect the resulting crystal structure [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Oguchi | Structural change and complexation of strictly linear amylose induced by sealed-heating with salicylic acid[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], whereas other factors control the yield, morphology and crystallinity of the complexes [START_REF] Bhosale | Preparation of spherulites from amylose-palmitic acid complexes[END_REF]Biliaderis & Galloway, 1989;[START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF][START_REF] Cardoso | From rice starch to amylose crystals: alkaline extraction of rice starch, solution properties of amylose and crystal structure of V-amylose inclusion complexes[END_REF][START_REF] Dvonch | Behavior of low molecular weight amylose with complexing agents[END_REF][START_REF] Gelders | Amylose-lipid complexation: a new fractionation method[END_REF]Godet et al., 1995b;[START_REF] Karkalas | Some factors determining the thermal properties of amylose inclusion complexes with fatty acids[END_REF]Nuessli et al., 2003;[START_REF] Whittam | Aqueous dissolution of crystalline and amorphous amylose-alcohol complexes[END_REF]. It must also be noted that there is still a lack of systematic studies of V-amylose polymorphism. In most of the previous investigations, the different complexes were usually prepared in similar conditions [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF]).

In the present study, the complexes were prepared in different conditions of solvent (with or without DMSO), concentration of complexing agents and amylose, temperature and DP of amylose. This method allowed identifying for the first time five new allomorphs of V-amylose (see Chapter IV) and complexes with molecules that had been reported to be unable to form V-amylose from solution (ethyl butyrate, citral, 4-hydroxybenzoic acid, salicylic acid, 2-naphthol and quinoxaline). Furthermore, by comparison to what was reported in the literature, polymorphism was observed for a large number of complexes. These results provide unique data on the factors that govern the formation of crystals and their structure.

V.2. Complexes with straight-chain n-alcohols, n-diols, n-amines, dicarboxylic acids, octanal, stearamide

The complexation of amylose with homologous series of straight-chain saturated n-alcohols (2-16 carbons), n-diols (2-6 carbons), n-amines (4-12 carbons), dicarboxylic acids

(2-12 carbons), octanal and stearamide were conducted in 0.1 wt% aqueous amylose solutions at different temperatures and concentrations of complexing agent. As shown in Table V.1, all complexing agents, except n-amines having 2-4 carbons and dicarboxylic acids having 2-6 carbons, formed crystalline complexes with amylose. It is still unclear why the short-chain dicarboxylic acids do not complex amylose while diols and monocarboxylic acids with the same number of carbons do. For the water-miscible short-chain n-amines, it is possible that the amylose complexes may be not stable in basic solutions of these complexing agents due to the increased solubility of amylose. Longer n-amines are more hydrophobic and thus favor a localization in the hydrophobic cavity of amylose helices. Therefore, their inclusion complexes may be more stable and able to crystallize.

All molecules having complexing ability induced V6-type complexes. The result agrees with the previous conclusion of [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF] that linear molecules, whose crosssectional diameter is about 0.30 nm, form 6-fold helices. Interestingly, most of them can form both V6I and V6II crystals, some examples of which are shown in Figure V.1. This rather general complexing behavior is unexpected as it had only been previously observed for some dicarboxylic acids, n-alcohols having 3-5 carbons and hexanoic acid while other n-alcohols and diols were reported to form only V6I [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF].

In these polymorphic systems, the formations of V6I and V6II depend on the crystallization temperature. A general rule is that V6II is preferred at relatively lower temperature (25-50 °C) while an incubation of the same complexation mixture at higher temperature (60-75 °C) favors the formation of V6I (Table V.1 and Annex 3, Figure S.V.1).

The result suggests that the compact structure V6I is more thermally stable than V6II.

The crystal structure of complexes with 1-propanol, 1,4-butanediol and 1,6-hexanediol also varies with the concentration of complexing agent: V6I is favored with a higher concentration of complexing agent compared to V6II (Table V.1 and Annex 3, Figure S.V.2). [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] reported a similar result for 1-propanol. However, for 1,4-butanediol, the author only obtained V6I for all tested concentrations of complexing agent. In fact, in the study of [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF], the complexes were prepared at 50 °C while the present study showed that 1,4-butanediol induces the formation of V6II at ≤ 40 °C. The variation of crystal structure with the concentration of complexing agent may be related to a dehydration effect [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. A

sufficiently high concentration of complexing agents may capture water molecules, leading to a dehydration of amylose. This effect favors the formation of the compact structure V6I over the looser V6II [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Indeed, previous studies showed that V6II was transformed into V6I upon a release of water molecules [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF][START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF][START_REF] Hinkle | X-ray diffraction of oriented amylose fibers. III. The structure of amylose-n-butanol complexes[END_REF]. The hypothesis can also be accounted for the fact that ethanol or 1,2-ethanediol, which have to be used at a high concentration (≥ 40 vol%) for the complexation, only yield V6I.

Furthermore, the crystallization of homologous series of n-alcohols and n-diols showed that the critical concentration required for the complex formation decreased with increasing chain length. This would be due to the increased hydrophobicity of the complexing agent which favors its association with amylose helices instead of water. In addition, the increase of complexing agent concentration allowed the crystallization to occur at higher temperature. This would be due to a higher dehydration effect which would decrease the solubility of amylose.

Moreover, the maximum crystallization temperature also increases with the chain length of complexing agents. For example, Vn-butanol crystallized at ≤ 50 °C, while Vn-pentanol crystallized at ≤ 60 °C and the complexes with longer n-alcohols could crystallize at up to 75 °C. A similar effect was also observed for n-amines. This result suggests that the thermal stability of the V-amylose crystals increases with the chain length of the complexing agents. The lack of stability of Vn-butanol at high temperature (60-75 °C) would be the reason why V6I crystals cannot be obtained with the complexing agent from the crystallization in aqueous solution. Indeed, [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] reported that to obtain V6I with n-butanol, DMSO must be added to increase the solubility of n-butanol and thus increases the concentration of the complexing agent. In that case, the formation of V6I would be a result of a dehydration effect on amylose.

V.3. Complexes with straight-chain saturated fatty acids

The crystallization of amylose from dilute solutions in the presence of a series of linear saturated fatty acids (C3 to C20) was investigated by varying the fatty acid concentration, crystallization temperature and solvent composition (DMSO:water in various ratios). Table V.2 summarizes the allomorphic types observed for complexes formed with each fatty acid. A general observation is that all complexing agents could induce more than one crystalline structure.

Specifically, propanoic acid (C3) formed V6I and V7II while all longer-chain fatty acids (C4-C20)

promoted the formation of V6I, V6II and V7II, such as those shown for Vbutanoic acid and Vdecanoic acid

(Figure V.2).
Table V.1. V-type complexes with n-alcohols, n-amines, dicarboxylic acids, octanal and stearamide as a function of crystallization temperature and concentration of complexing agent (C). The amylose solutions and complexing agents were mixed at 90 °C, then kept at a given crystallization temperature, or allowed to slowly cool down to room temperature in a Dewar container. For mixtures, the major and minor allomorphs are indicated. This result is unexpected as polymorphism was only previously reported for propanoic, butanoic and hexanoic acids (Table V.2). In addition, all the tested fatty acids induced the formation of both 6-and 7-fold helices whereas earlier works reported that the amylose conformation depended on the chain length of the complexing agent. In particular, the occurrence of V7II with long-chain fatty acids C6-C20 has never been reported.

Considering the cross-sectional diameter of the linear fatty acids (ca. 0.30 nm), V6 structures are expected rather than V7II. V7II is usually obtained with bulkier molecules such as branched alcohols or monoterpenes whose cross-sectional diameter is about 0.45-0.60 nm [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. In this context, [START_REF] Shogren | X-ray diffraction study of crystal transformations in spherulitic amylose/lipid complexes from jet-cooked starch[END_REF] proposed that the formation of 7-fold helices with fatty acids was due to the interaction of amylose with fatty acid "dimers", the association of which would be promoted by hydrophobic interactions between the alkyl moieties [START_REF] Mukerjee | Dimerization of anions of long-chain fatty acids in aqueous solutions and the hydrophobic properties of the acids[END_REF]. However, our results showed that V7II was favored by water-miscible fatty acids (C3 and C4) than by the poorly-soluble ones. Moreover, several complexes formed at high temperature (90 °C) at which the existence of fatty acid "dimers" in solution was unlikely.

Therefore, the 7-fold helical conformation is more likely controlled by a specific interaction between fatty acids and amylose rather than by the molecular dimension of the guest. As proposed by [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF], the inclusion of the polar head (i.e. the carboxyl group) of the fatty acid in the amylose helix could be responsible for its enlargement to the V7 conformation.

In contrast, straight-chain molecules bearing a different functional group such as aliphatic n-alcohols and aldehydes have been reported to only induce the formation of 6-fold helices [START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. Besides, previous numerical simulations and NMR data concluded that the linear alkyl moiety was included inside the 6-fold helix while the polar head remained outside (Godet et al., 1993;[START_REF] Kawada | Solid-state NMR and X-ray studies on amylose complexes with small organic molecules[END_REF]Lebail et al., 2000;[START_REF] Snape | Solid state 13 C NMR investigation of lipid ligands in V-amylose inclusion complexes[END_REF]. In addition, the hypothesis that the inclusion of the carboxyl group controls the helical conformation somewhat agrees with the fact that V7II is preferred with shorter fatty acids (C3-C5) while V6I is favored with C6-C20 fatty acids. It is possible that with a longer chain length, the influence of the linear aliphatic chain gets predominant over that of the carboxyl group in determining the size of helix and thus would induce mainly 6-fold helical complexes [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF]. Expanding from the modeling work of Godet et al. (1993), further simulations must be carried out to elucidate the role of the carboxyl group and its interaction with amylose in the determination of its helical conformation. been reported for many polymorphic systems as reviewed by [START_REF] Bernstein | Concomitant polymorphs[END_REF]. The phenomenon has been explained by the fact that so-called occurrence domains, defined by sets of conditions at which specific allomorphs crystallize, could be independent or overlap. When the occurrence domain is unique, only one form is obtained, which is usually the most thermodynamically stable. In contrast, in regions where the domains overlap, two or more forms would crystallize under identical conditions. These concomitant allomorphs are energetically close and their relative amount is mainly governed by kinetic stability [START_REF] Bernstein | Concomitant polymorphs[END_REF][START_REF] Bučar | Disappearing polymorphs revisited[END_REF].

Since we have only characterized the morphology and structure of the final products, the precise chronology of crystallization events was lost. The possibility that the amylose chains form nuclei and crystallize under conditions that depend on their length (so-called fractionated crystallization) or on the temperature (during cooling) cannot be ruled out. One allomorph would crystallize after the other and the allomorph formed first may even act as a seed for nucleation and epitaxial growth of the second one. As illustrated by Figures V.3a and V.3b, rectangular V6II and hexagonal V6I crystals are often associated with specific orientation

> V6I - - - - V7II > V6I " " 40 vol% V7II > V6I V6I > V7II V6I > V7II V6I > V7II - V7II > V6I " " 50 vol% V6I > V7II V6I > V7II V6I > V7II V6I > V7II - V6I > V7II C4 water-miscible liquid 5-10 vol% V7II > V6II > B - - - - V7II > V6II > B " " 20-40 vol% V7II > V6I V7II > V6I V7II > V6I V7II > V6I - V7II > V6I " " 50 vol% V6I > V7II V6I > V7II V6I > V7II V6I > V7II - V6I > V7II C5 water-immiscible liquid 2.5-5 vol% V6II V6I > V6II V6I > V6II - - V6II C6 water-immiscible liquid * V6II V6II > V6I V6I - - V6II > V6I C8 water-immiscible liquid * V6II V6II V6I V6I - V6II > V6I C10-C20 solid * V6II V6II V6I > V6II V6I V6I V6II > V6I
C3: propanoic acid, C4: butanoic acid, C5: pentanoic acid, C6: hexanoic acid, C8: octanoic acid, C10: decanoic acid, C12: dodecanoic acid, C14: tetradecanoic acid, C16: hexadecanoic acid, C18: octadecanoic acid, C20: icosanoic acid. *: the fatty acid was used at saturation -: no precipitation

> V7II V6I > V7II V6I > V7II V6I > V7II - V6I > V7II C4 5-10 vol% V7II > V6II > B - - - - V7II > V6II > B " 20-30 vol% V7II > V6I V7II > V6I V7II > V6I V7II > V6I - V7II > V6I " 40-50 vol% V6I > V7II V6I > V7II V6I > V7II V6I > V7II - V6I > V7II C5 2.5 vol% V7II > V6II V7II > V6I, V6II - - - n. t. " 5 vol% V6II > V7II V6II > V6I, V7II V6I > V7II - - V6II > V6I C6 * V6I > V6II V6I > V6II V6I > V7II V6I > V7II - V6I > V6II C8 * V6I > V6II V6I V6I V6I > V7II - V6I > V6II C10, C12 * V6I V6I V6I V6I > V7II V6I > V7II V6I > V7II C14-C20 * V6I V6I V6I V6I V6I > V7II V6I > V7II
C3: propanoic acid, C4: butanoic acid, C5: pentanoic acid, C6: hexanoic acid, C8: octanoic acid, C10: decanoic acid, C12: dodecanoic acid, C14: tetradecanoic acid, C16: hexadecanoic acid, C18: octadecanoic acid, C20: icosanoic acid. *: the fatty acid was used at saturation -: no precipitate was observed n. t.: not tested relationships that were not precisely analyzed in the present study. However, since the images are projections of the crystal assemblies, one cannot clearly identify the crystallization sequence of each allomorph. In the case of coexisting V6I and V7II crystals, the latter ones are mostly observed as outgrowths of the former (Figure V.3d), suggesting that they formed the last. V6II and V7II crystals did not appear to be associated but rather independent from one another (Figure V.3e,f).

In our study, the fatty acid concentration, temperature, and solvent composition were shown to play important roles to regulate the formation and crystal structure of Vfatty acids. In general, the V6I structure was favored at higher concentrations of fatty acid and DMSO (10 vol%), and at higher temperature (60-90 °C), whereas milder conditions of these parameters resulted in the formation of V6II and V7II (Tables V.3-V.5 and Annex 3, Figures S.V.3-S.V.6).

This result is in agreement with those previously obtained for other straight chain molecules (see § V.2): higher concentrations of complexing agent and higher temperatures favor more compact structures. Therefore, this support the hypotheses on the effect of dehydration and the relative thermal stability of allomorphs. In the case of butanoic acid (C4), we observed that V6II was more sensitive to dehydration than V7II, the former appearing only with a fatty acid concentration ≤ 10 vol% while the later was formed at higher concentrations. This also explains why V6II could not be obtained with propanoic acid for which at least 30 vol% was required to form the complexes. In addition, for C5-C8 fatty acids, V6II is formed at a lower temperature compared to V7II (Tables V.4 and V.5, and Annex 3, Figure S.V.5).

The impact of DMSO on the formation of specific allomorphs has been clearly

highlighted. This molecule is a good solvent of amylose but also of fatty acids which results in an increase of concentration of the weakly soluble fatty acids and therefore, their availability for complexation. [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] also observed different allomorphs for complexes prepared with n-butanol and n-pentanol that exhibited a V6II structure when crystallized in water and V6I

in DMSO:water mixtures. It must also be noted that DMSO can form crystalline complexes with amylose (French & Zobel, 1967;[START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF]. While [START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF] mentioned the occurrence of two allomorphs based on 6-and 7-fold amylose helices, Winter and Sarko (1974) described the VDMSO unit cell as pseudo-tetragonal and containing antiparallel 6-fold helices, with DMSO molecules located inside and between helices. However, these complexes were only formed under anhydrous conditions by casting amylose / DMSO solutions and letting them to dry in a vacuum oven. Nevertheless, this shows that in specific conditions, DMSO no longer acts as a solvent of amylose but rather like a complexing agent that specifically interacts with amylose chains to form helical inclusion compounds.

The most remarkable effect of DMSO was its crucial role in the formation of V7II. Our results showed that the complexes with C5-C20 fatty acids exhibited a V7II structure only if DMSO was present, preferably 1-2 vol%. A significant role of DMSO on the conformation of amylose has been suggested by other authors [START_REF] Cheetham | Amylose conformational transitions in binary DMSO/water mixtures[END_REF][START_REF] Germino | Amylose V complexes from dimethyl sulfoxide solutions[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF][START_REF] Jane | 13 C-NMR study of the conformation of helical complexes of amylodextrin and of amylose in solution[END_REF][START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF]. DMSO may induce specific conformations for both amylose and fatty acids, although, so far, the nature of the interactions remains unknown and no evidence were produced regarding the presence of DMSO in the crystal structure of Vfatty acids complexes. The formation of both 6-and 7-fold helices would be due to the fact the molecular size of the complexing agent is compatible with both helical conformations. As mentioned above, 6-fold helices are expected to be obtained with molecules with straight-chain moieties while branched-chain or cyclic molecules usually used 7-fold complexes [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF][START_REF] Hathaway | Method for obtaining amylose from cooked starch solutions[END_REF][START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF][START_REF] Zaslow | Characterization of a second helical amylose modification[END_REF]. In case of 1,3-butanediol, the complexing agent is composed of a straight chain of carbons, but the presence of a hydroxyl group at the endo-position enlarges its diameter in cross section. Therefore, the molecule has an intermediate size between those that favor 6-fold helix (straight-chain molecules) and 7-fold helix (branched-chain molecules). In this thesis, other molecules which have similar structural features such as 2-propanol and 1,2-propanediol also induced both 6-and 7-fold complexes. Similar to other polymorphic V-amylose presented previously, the structure of V1,3-butanediol is dependent on the concentration of the complexing agent and the crystallization temperature, as shown in Table V.6 and Annex 3, Figures S.V.7 and S.V.8. In general, the concentration of 1,3-butanediol necessary for the formation of different allomorphs can be arranged in the order: V7I ≥ V6I ≥ V7II ≥ V6II. The effect of concentration of complexing agent on the structure of V1,3-butanediol is thus similar to that observed for complexes prepared with n-propanol, 1,4-butanediol, 1,6-hexanediol, propanoic acid and butanoic acid, that the more compact allomorphs are formed at higher concentrations of complexing agent relative to the less dense ones. As discussed above, this effect can be explained by the dehydration effect of the complexing agent on amylose. Besides, the result also revealed that 6-fold complexes are preferred with lower concentrations of 1,3-butanediol than 7-fold ones for both orthorhombic (V6II < V7II) and compact hexagonal structures (V6I < V7I). The reason for this is still unclear.

On the other hand, the crystallization in the presence of the same concentration of 1,3-butanediol showed that the 7-fold complexes (V7I, V7II) are favored at higher temperature than 6-fold complexes (V6I, V6II). Previously, we also observed that V7II was preferred at higher temperature than V6II obtained with fatty acids. However, for the complexes of fatty acids such as propanoic acid or pentanoic acid, the occurrence domain of V7II was at relatively lower temperatures than V6I; this is different from the crystallization behavior of V1,3-butanediol. [START_REF] Biais | Structural and stoichiometric studies of complexes between aroma compounds and amylose. Polymorphic transitions and quantification in amorphous and crystalline areas[END_REF][START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF]). This can be because the concentration of amylose used in this study (0.1-0.5 wt%) is lower than in the previous works

(1.0%). Isopentyl acetate is the only ester that forms V7II. This result agrees with previous observations that linear carbon chains induce 6-fold helices, while branched chains enlarge the helix to 7-fold [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF]. Six-fold helices form with isopropyl myristate maybe because the linear chain (myristate), not the branched part, predominates the helix.

As shown in Table V.7 and Annex 3, Figure S.V.9, the formation of Vesters is significantly affected by the crystallization temperature and concentration of amylose. In general, the complexes formed very slowly (1-2 weeks) at relatively low temperatures (≤ 40°C), suggesting a weak interaction between amylose and the complexing agents. In addition, the precipitated product usually contained a mixture of V-and B-amylose, indicating a competition between complexation and retrogradation. In addition, the complexation of each ester is favored at a specific range of amylose concentration, usually between 0.1-0.5 wt%. Out of this range, B-amylose predominantly forms. Nuessli et al. (2003) reported a similar effect of amylose concentration on the formation of complexes with geraniol. When the concentration was > 0.5%, the retrogradation rate of amylose into B-type was much faster than the formation of complex.

V.6. Complexes with some bicyclic compounds

The complexes of amylose with several bicyclic compounds (trans-decalin, β-pinene, Our results disagree with those of [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] who observed the formation of hexagonal V6I

with trans-decalin and (-)-borneol. However, the retrieval of V7 lamellar crystals was consistent with the molecular dimension of the bicyclic compounds [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part III. X-ray studies on amylose and cyclodextrin complexes[END_REF].

-)-Borneol V7I V7I V7I V7I V7I - - R-(+)-Camphor V7I V7I V7I V7I V7I - - (
Along with 1,3-butanediol, the above bicyclic compounds (cis-decahydro-1-naphthol, decahydro-2-naphthol, (-)-borneol and R-(+)-camphor) are among a few complexing agents that can produce V7I. In contrast, V7II appears to be more prevalent as having been obtained with a variety of molecules such as linear fatty acids [START_REF] Takeo | Complexes of starch and its related materials with organic compounds. Part. X. X-ray diffraction of amylose-fatty acid complexes[END_REF] and ketones [START_REF] Takeo | Complexes of starchy materials with organic compounds: Part VI. X-ray diffraction of amylose-n-aliphatic ketone complexes[END_REF], 2-propanol and branched alcohols [START_REF] Yamashita | Single crystals of amylose V complexes. II. Crystals with 71 helical configuration[END_REF], monoterpenes and many cyclic compounds [START_REF] Biais | Étude structurale et mécanismes de formation de complexes amylose-ligand Influence sur la rétention de composés d'arôme dans une matrice amylacée[END_REF][START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF][START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]Nuessli et al., 2003;[START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. As noted, the main difference between the structure of V7I and V7II stays in the more or less compact packing arrangements of 7-fold amylose helix. In V7I structure, amylose helix would be closely packed on a hexagonal net with small inter-helix spaces which allow the location of some water molecules and not the bulky complexing bicyclic compounds. In contrast, the V7II exhibits more interstitial spaces which accommodate for an occupation of both complexing agents and water [START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. Some authors believed that the location of complexing agents between amylose helices would disturb the hexagonal arrangement of amylose helices leading to the formation of the orthorhombic structures [START_REF] Booy | Electron diffraction study of single crystals of amylose complexed with n-butanol[END_REF]Godet et al., 1995a;[START_REF] Simpson | A V7 conformation of dimethyl sulfoxide-amylose complex[END_REF]. Based on that hypothesis, the possibility of complexing agent to be located only inside the helix or both inside and between the helix is the decisive factor that controls the crystallization into V7I or V7II.

Since V7II have been obtained with numerous hydrophilic and hydrophobic complexing agents of different chemical structures, it may be impossible to figure out the specific interactions or structural features that allow their localization between the helix. However, the dimension of the complexing agents must be compatible with the inter-helix space they occupy [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]. As noted previously, (-)-borneol and R-(+)-camphor yielded V7I but not V7II. It is probable that these complexing agents are too bulky to enter the inter-helix void of V7II. A few molecules could induce both V7I and V7II such as cis-decahydro-1-naphthol, decahydro-2naphthol and 1,3-butanediol. In these cases, V7I is preferred with a higher crystallization temperature (100-115 °C for cis-decahydro-1-naphthol and decahydro-2-naphthol) or higher concentration of complexing agent (60 vol% for 1,3-butanediol). These drastic conditions would necessitate strong interactions between complexing agent and amylose to stabilize the structure at high temperature or to avoid the retrogradation of amylose caused by dehydration effect of high concentration of complexing agent. Most of complexing agents such as transdecalin and β-pinene would not meet this requirement, would thus induce V7II which is favored at milder conditions. In addition, it is interesting to note that cis-decahydro-1-naphthol and decahydro-2-naphthol are different from trans-decalin just by a hydroxyl group. Therefore, it is logical to assume that the functional group would have some specific interactions with amylose that increase the stability of the complexes.

V.7. Complexes with 1-naphthol

The complexes were prepared by addition of an excess amount of 1-naphthol into aqueous solutions of amylose having different DPs, equilibrated at a predetermined temperature varying from 60-90 °C. The complexing mixture was kept at the mixing temperature for 30-60 min, and then allowed cooling down in ambient air to induce the crystallization. Two different allomorphs were obtained V7III and V8II whose morphologies are presented in Figure V.9. The type of allomorph depends on the DP of amylose and mixing temperature (Table V.9 and Annex 3, Figure S.V.10). V8II is preferred with lower DP and higher mixing temperature compared to V7III. Amylose with an average DPw of 60-86 yielded only V8II while DP130 amylose produced a mixture of V8II and V7III in which V8II was the major form (> 95%)

whatever the mixing temperature. In the case of DP192 and DP6500 amylose, V8II and V7III also concomitantly crystallized but the major form depended on the mixing temperature. In particular, V8II was the major form if the mixing temperature was 90 °C while at lower temperatures, V7III was the main product. Previous studies revealed that the formation of crystalline V-amylose required a critical value of DP of amylose (about DP 20-30) [START_REF] Dvonch | Behavior of low molecular weight amylose with complexing agents[END_REF][START_REF] Gelders | Amylose-lipid complexation: a new fractionation method[END_REF]Godet et al., 1995b). However this is the first time that the variation of the V-amylose crystal structure (both helical conformation and molecular packing in the case of V1-naphthol) with the DP of amylose has been reported. The reason for this effect of DP of amylose is still unclear.

On the other hand, as previously suggested by [START_REF] Yamashita | Single crystals of amylose V complexes. III. Crystals with 81 helical configuration[END_REF], the dependence of the crystal structure on the mixing temperature rather than the crystallization temperature suggests that the association of the random-coil amylose chains with 1-naphthol into helical complexes occurs at high temperature. The helical conformation of amylose would be decided in this critical step and would not vary during the cooling down. The result also suggests that the 8-fold complex with 1-naphthol is more thermally-stable than the 7-fold one. V.10). Sometimes, V7II was also present as a trace and could only be identified by TEM.

Unlike for V1-naphthol, the crystal structure of Vquinoline does not depend on the DP of amylose. In fact, the parameter only affects the morphology of the crystals. In contrast, the solvent composition (DMSO concentration) and crystallization temperature appeared to be decisive factors that controlled the formation of different allomorphs. V8II was generally preferred at higher concentrations of DMSO and higher crystallization temperatures. As shown in Table V.10

and Annex 3, Figure S.V.11, the crystallization at 25 °C or in the presence of ≤ 10 vol% of DMSO mainly led to the formation of V7IV while V8II is the major form only in the presence of ≥ 15 vol% of DMSO at 40-50 °C. [START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF] previously prepared Vquinoline by slowly cooling down the complexation mixture from 90 °C to room temperature. The author noted that the presence of 15 vol% of DMSO leaded to the formation V8II, while V6I formed in the absence of DMSO. It is possible that the V7IV was incorrectly identified as V6I crystals in the previous study since the morphology of the two allomorphs is very similar. As discussed in previous sections, DMSO would increase the solubility of both amylose and the complexing agent, and thus increases the concentration of complexing agent in the solution. In addition, DMSO also affects the conformation of amylose in solution. These results would be accounted for by the effect of the solvent on the crystal structure of Vquinoline but the mechanism is still unknown.

V.9. Complexes with salicylic acid

The crystallization of amylose having a different DP was conducted in dilute solution in the presence of an excess amount of salicylic acid and in different conditions of crystallization temperatures and solvent compositions. Two crystalline allomorphs were identified, namely V8I and V8II whose morphology is shown in Figure V.11. [START_REF] Kuge | Complexes of starchy materials with organic compounds: Part II. Complex formation in aqueous solution and fractionation of starch by L-menthone[END_REF] formerly reported that salicylic acid did not form complexes with amylose in solution. Later, Oguchi et al.

(1998) prepared complexes with salicylic acid using the sealed-heating method and obtained two crystal types that, according to the authors, would consist of 7-and 8-fold amylose helices, respectively. The 7-fold complexes gave a powder XRD profile that is similar to that of V7a while the 8-fold complexes gave a similar diffraction pattern to V8I. Therefore, the result in the present study has been the first report of lamellar crystals of Vsalicylic acid prepared by crystallization in solution. In addition, Vsalicylic acid exhibited polymorphism but no 7-fold complex was identified. The crystallization conditions did probably not cover the occurrence domain of 7-fold complexes of Vsalicylic acid or the complexes cannot form in aqueous solution.

Table V.11 presents the allomorphic type of Vsalicylic acid corresponding to different crystallization conditions. It is observed that V8I is favored with higher DP of amylose, higher concentrations of DMSO and higher crystallization temperatures, relative to V8II (Annex 3, Figures S.V.12 and S.V.13). In terms of packing arrangement of amylose helices, V8I is more compact than V8II (see Chapter IV). Therefore, the crystallization behavior of Vsalicylic acid also supports that higher crystallization temperatures favor the more compact structure. The effects of DMSO and DP of amylose are not very clear, but these parameters may have an influence on the relative solubility between the two allomorphs. On the other hand, the crystallization behavior of Vsalicylic acid is similar to that of V1-naphthol regarding the preferred formation of V8II allomorph with the shorter amylose although both allomorphs of Vsalicylic acid consist of 8-fold helices while those of V1-naphthol have different helical conformations (7-and 8-fold). However, we did not observe any similar effect of DP of amylose on the formation of V8II in the case of Vquinoline. A similar remark can also be made regarding the effect of DMSO.

V.10. General conclusions

The results of the present study support the correlation between the dimensions of the complexing agent and the helical conformation: straight-chain molecules form 6-fold helices, while branched molecules and cyclic compounds induce 7-fold helical conformations, and some cyclic compounds form 8-fold helices (Table V.12). In addition, several molecules formed two different helical conformations. For example, straight-chain fatty acids and straightchain alcohols with the hydroxyl group located at endo-position induced both V6 and V7

complexes. Quinoline and 1-naphthol formed both V7 and V8 complexes. These molecules likely have an intermediate size that is compatible with both helical conformations. An exception is the case of 4-hydroxybenzoic acid which complex is supposed to contain 6-fold helices although the size of the complexing agent is not compatible with helical cavity. In this case, the complexing agent would be located between the helices.

Polymorphism was observed for crystals prepared with a large number of complexing agents and is thus more general than what was previously reported in the literature. The tendency of a complex to exhibit a polymorphic behavior is not only related to the nature of complexing agent but also to the accessibility of the various structures through crystallization.

Complexes of straight-chain molecules are more likely to exhibit polymorphism than the branched-chain or cyclic compounds. In addition, for straight-chain molecules, alcohols, fatty acids, aldehydes, amides, amines are likely more prone to form different allomorphs than esters and ketones. As noted, the occurrence domains usually varied between different allomorphs as well as between different complexing agents and are thus sometimes difficult to predict.

Therefore, a variety of crystallization conditions with the variation of different parameters (solvent composition, temperature of mixing and crystallization, concentration of complexing agent and amylose, DP of amylose, etc.) had to be explored in order to observe relevant structures. However, some general tendencies could be observed: i) more compact structures are favored at higher temperature;

ii) the crystal structure of complexes with highly soluble or water-miscible molecules usually depends on the concentration of the complexing agent. In that case, the more compact structures with a larger helix (containing a higher number of glucosyl units per turn) are favored at a higher concentration of the complexing agent. The mechanism of polymorphic crystallization is still unclear. Future works should be conducted to understand the effect of the guest and several crystallization parameters on the formation and crystal structure of V-amylose.

The result of the present study suggests that more new V-amylose structure remain to be discovered. This can be done by testing new complexing agents, but more importantly, by varying the conventional crystallization method in solution. The influence of some parameters such as solvents and additives should be further explored. As noted, in this study, only DMSO was tested as additive. In addition, using different preparation techniques is also recommended, e.g. insertion of complexing agent into a preformed V-amylose, sealed-heating, etc. The property of amylose to form inclusion complexes with a variety of small organic molecules has been exploited for the encapsulation of a wide range of compounds that serve as flavor components, nutraceutical, pharmaceutical, or bioactive substances [START_REF] Conde-Petit | Structural features of starch-flavor complexation in food model systems[END_REF]Obiro et al., 2012;[START_REF] Panyoo | Amylose-lipid complex production and potential health benefits: a mini-review[END_REF][START_REF] Putseys | Amylose-inclusion complexes: formation, identity and physico-chemical properties[END_REF]. In particular, complexes of amylose with ibuprofen (IBU), a widely used non-steroidal anti-inflammatory, analgesic and antipyretic agent, have been prepared by acidification of an alkali solution or in situ enzymatic polymerization [START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF][START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF]. The studies agree on the good in vitro stability of the complexes in a simulated gastric fluid and the gradual but sustained release of ibuprofen by enzymatic degradation of the amylose matrix in a simulated intestinal environment [START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF][START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF]. However, XRD profiles attested that the product prepared in previous studies contained a significant fraction of retrograded B-amylose along with semicrystalline V-type. Besides, the low crystallinity achieved with the preparation methods did not allow retreiving detailed information about the crystal structure.

Diesters

We have studied the crystallization behavior of amylose with IBU in solution in order to prepare highly crystalline complexes and determine their structure. We have proposed models based on crystallographic and spectroscopic data as well as molecular modeling. Finally, in order to validate the potential of V-amylose as a delivery system, we have studied the in vitro release profiles of the guest molecule as a function of pH and in relation with the crystal structure.

VI.2. Crystallization behavior and morphology of Vibuprofen complexes

Two batches of complexes were prepared. The first one was prepared from low amylose concentration solutions (0.1 wt%) with the aim of producing individualized lamellar single crystals for imaging (TEM and AFM) and ED crystallography analyses. Each crystallization was carried out using 10 mg amylose diluted in 10 mL of pure water. The second batch was prepared at a higher concentration (1 wt%) in order to produce a larger amount of complex (about 10 g). In that case, DMSO (10 vol%) was added as co-solvent to facilitate the dissolution of amylose. For spectroscopy characterization, the solid product was briefly washed to remove uncomplexed molecules and kept in suspension. However, for release studies, the complexes were extensively washed by centrifugation in water saturated with IBU prior to lyophilization.

VI.2.1. VIBU crystals from dilute solutions

We have investigated the formation of lamellar VIBU crystals as a function of the DPw of amylose (60 to 6500), concentration of ibuprofen (0.0025 to 0.04 wt%) and at different temperatures (25, 40, 60, 75 and 90 °C). The complexes prepared with longer amylose crystallized at higher temperature (Table V.1). This would be due to the decrease of solubility of amylose with increasing DP. In addition, Table VI.2 shows that the yield of complexation depended on the incubation temperature and concentration of IBU. When crystallized from a 0.1 wt% amylose solution and at a given temperature, the yield increased with IBU concentration and reached a maximum at saturation. However, the possibility that the initial amount of IBU has not been entirely complexed by amylose cannot be ruled out. Indeed, the crystallization at higher temperature required a higher amount of IBU. A similar behavior was observed for complexes with highly soluble fatty acids or alcohols. In the latter cases, the crystallization at high temperature was favored by the desolvation effect caused by the presence of high concentrations of complexing agent. In contrast, IBU being poorly water-soluble, the desolvation is negligible. The requirement of a higher concentration of IBU for complexation at higher temperature would likely be related to the increase in solubility of the complexing agent. As discussed in Chapter V, each complexing agent exhibits a critical concentration essential for the complexation which is correlated with its solubility in water. Indeed, as solubility increases, the association of complexing agent with water is favored. Therefore, a higher concentration of complexing agent is required to promote its association with amylose. This would be a result of a slower nucleation and growth rate.

Considering the very large number of crystals per aggregate, recrystallization steps were used, resulting in more individual single crystals. The series of images in Figure VI.3 shows that the length, width and "perfection" of the crystals vary with the DP of amylose. The crystals are longer for longer amylose chains while their rectangular shape is more clearly defined with the shorter chains. The morphology of these crystals appears to be characteristic of V7II complexes [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF][START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]. from which height profiles were determined. As seen in Table VI.3, the lamellar thickness slightly increased with increasing amylose DP, from 5.5 (±0.5) nm for DP60 to 7.0 (±0.4) nm for DP6500. The roughness (0.7 nm) was fairly similar for all samples. Assuming that crystals contain 7-fold helices with a pitch of 0.8 nm and that there is no chain-folding, the theoretical thickness would be proportional to the DP of amylose (Table VI.3 agreement with previously published results [START_REF] Buléon | Single crystals of V amylose complexed with isopropanol and acetone[END_REF][START_REF] Nishiyama | Molecular and crystal structure of 7-fold V-amylose complexed with 2-propanol[END_REF]Nuessli et al., 2003;[START_REF] Putaux | Single crystals of V-amylose inclusion complexes[END_REF]. The shape of the lamellar crystals and their base-plane ED and parameters are similar to those reported earlier for V7II crystals (see Chapter IV).

Consequently, we concluded that the unit cell was orthorhombic and that its three-dimensional space group was P212121. Experimental thickness (nm) 5.5 ± 0.5 5.7 ± 0.4 6.5 ± 0.3 7.0 ± 0.4 Roughness (nm) 0.7 0.7 0.5 0.7

Theoretical thickness (nm) 6.9 14.9 21.9 743.0 In addition, the reflections of the dry form are rather broad suggesting a slight loss in crystallinity. However, after rewetting in water or rehydrating in 95% RH chamber, the complexes produced a diffraction pattern that is almost identical to that of the wet sample. This result suggested that ibuprofen was retained in the crystal and the structural transition was due a loss of water molecules. Furthermore, water would not be bound very tightly since the structural transition occurred even when the crystal was exposed to the ambient air. The drying behavior of VIBU was generally observed for V7II crystals obtained with many other complexing agents (see Chapter IV).

As noted in Chapter IV, the transition to V7a from a V7II complex with a non-volatile guest which is not easily removed by drying raises an ambiguity about the location of the complexing agent. Indeed, the presence of the bulky molecules in the inter-helix space may perturb the hexagonal arrangement in V7a.

VI.2.2. VIBU crystals from concentrated solutions

A batch of VIBU crystals was prepared from a more concentrated aqueous solution of DP6500 amylose (1 wt%) in order to obtain a larger amount of material for the release experiments. DMSO (10 vol%) was used as co-solvent to facilitate the dissolution of amylose.

The crystallization occurred at 75 °C in the presence of a saturation of IBU. The solid precipitate was recovered by decantation followed by centrifugation (3600 g, 15 min), washed three times with water saturated with IBU and then freeze-dried. band of pure IBU observed at 1707 cm -1 is present in the spectra of the physical mixture and the complex but a new band appeared at 1730 cm -1 in VIBU's spectrum. By analogy with the results from several studies of complexes between IBU and β-cyclodextrins, the band at 1707 cm -1 would correspond to a very strong interaction between IBU through H-bonds between carbonyl groups while the band at 1730 cm -1 would be attributed to less structured H-bonded carbonyls which are formed as a result of complexation [START_REF] Braga | Solid state inclusion compound of S-ibuprofen in β-cyclodextrin: structure and characterisation[END_REF][START_REF] Bratu | FT-IR spectroscopy of inclusion complexes of beta-cyclodextrin with fenbufen and ibuprofen[END_REF][START_REF] Hussein | Comparative evaluation of ibuprofen/βcyclodextrin complexes obtained by supercritical carbon dioxide and other conventional methods[END_REF][START_REF] Mura | Interactions of ketoprofen and ibuprofen with β-cyclodextrins in solution and in the solid state[END_REF]Tozuka et al., 2006).

In addition, the band assigned to aromatic C-C stretching shifted from 1507 cm -1 in pure IBU or the physical mixture to 1512 cm -1 in the complexes. Several bands attributed to IBU, and that still appeared in the physical mixture, disappeared from the VIBU spectrum. This is the case for absorbances at 669 and 779 cm -1 , attributed to the deformation of the out-of-plane aromatic C-H bonds of IBU's phenyl group [START_REF] Ghorab | Enhancement of ibuprofen dissolution via wet granulation with β-cyclodextrin[END_REF] and bands that correspond to aromatic C-C stretching (1451 cm -1 ), O-H bending (1418 and 934 cm -1 ), C-O stretching (1268-1123 cm -1 ) and in-plane C-H bending (1091-1007 cm -1 ) [START_REF] Coates | Interpretation of infrared spectra, a practical approach[END_REF].

Furthermore, characteristic vibrational bands for V-amylose were observed at 1017, 1103, 1150, 1205, 1298, 1368 and 1411 cm -1 (see Chapter IV).

The 13 C CP/MAS NMR spectrum of never-dried VIBU crystals shown in Figure VI.10a is typical of V-amylose. In particular, the C1 resonance at 103.3 ppm is a singlet, while those of the A-and B-amylose would be a triplet and a doublet, respectively [START_REF] Gidley | Carbon-13 CP/MAS NMR studies of amylose inclusion complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between glycosidic linkage conformation and solid-state carbon-13 chemical shifts[END_REF][START_REF] Veregin | Investigation of the crystalline" V" amylose complexes by high-resolution 13 C CP/MAS NMR spectroscopy[END_REF]. Other clear peaks in the carbohydrate region can be assigned to specific carbons as summarized in Chapter IV, Table IV.2. Within the 50-110 ppm region, the spectrum is broadly similar to that shown by [START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF], although the resolution is higher in our case since, as can be judged from the XRD profiles, our specimen was significantly more crystalline. The contributions of guest IBU molecules appear at 20-40 ppm (CH/CH2/CH3) and 120-140 ppm (aromatic carbons). However, the peak corresponding to carbonyl groups (C=O), located in the spectrum of bulk ibuprofen at 181-183 ppm [START_REF] Braga | Solid state inclusion compound of S-ibuprofen in β-cyclodextrin: structure and characterisation[END_REF], is absent from the VIBU spectrum, suggesting a high mobility of the IBU molecules in the crystal lattice.

A SP/MAS spectrum was recorded to quantify the guest IBU molecules included in the complex, (Figure VI.9a). The stoichiometry of IBU and glucosyl residues, calculated as the ratio of the integral area of the signal from the C1 contribution of amylose (103.3 ppm) to those from aromatic carbons of ibuprofen (121-134 ppm), was found to be 1:14.6. Note that a sharp peak corresponding to residual DMSO is present at 39.6 ppm in the SP/MAS spectrum while it is nearly absent in the CP/MAS spectrum, which suggests that the molecule is highly mobile. As noted above, the transition of Vibuprofen into the hexagonal V7a without removal of ibuprofen raised the ambiguity about the location of IBU in the inter-helix space. Consequently, we have studied two types of models: in model 1, IBU is only located inside the helix while in model 2, IBU lies both inside and between the helices. The 13 C SP/MAS NMR analysis showed that the stoichiometry was about 1 IBU per 14.6 glucosyl units. As there would be a small amount of amorphous amylose and to simplify the calculation, the stoichiometry of the complexes was rounded to 1 ibuprofen per 14 glucosyl units. In addition, the amount of water added was based on the measured density (1.48 g.cm -3 ).

In model 1, built by Yu Ogawa (CERMAV), the unit cell structure of Vibuprofen was duplicated to build a P1 supercell having approximately 3 nm in each direction and containing 4 amylose chains. Each chain consisted of 28 glucose residues and was covalently bonded to its own periodic image to simulate an infinite chain. Periodic boundary conditions were applied in all three directions to model an infinite-size crystal. Two IBU molecules were placed inside each helix and water molecules were inserted both inside and between the helices. The supercell structure was first energy-minimized using the steepest-decent method, followed by the conjugate gradient method. The convergence criterion to stop minimization was a 10 kJ mol -1 nm -1 maximum force. The structure was then heated at a rate of 0.2 K ps -1 , and equilibrated at 300 K and 1 atm. The production run was performed for 50 ns at the same To construct model 2, a P1 supercell was generated by considering three helical turns.

Thus, it has a c-axis of 2.40 nm and the same a and b parameters as those of the Vibuprofen unit cell with space group P212121. One IBU molecule was inserted in each helical cavity and interstitial pocket. Thus, the proposed ratio of intra-helical to inter-helical ibuprofen was 2:1.

The stable positions of IBU were then searched by geometry minimization while constraining the amylose helices. Water were then placed into the structure and the structure was energyminimized using the same method. Finally, the system was optimized with all atoms free to move. The final structure was presented in Figures VI.11c,d.

In both models, the amylose helices have positions very similar to those in the model of V2-propanol (Annex 4, Figure S.VI.2). On the other hand, in both cases, the helices are much more deformed because the helices are allowed to freely relax in the P1 symmetry. The above results suggest that the interstitial IBU is not essential for the stability of the crystal structures.

Figure VI.12 compares the observed and calculated EDs for the two models. There are noticeable differences in reflection intensities between both patterns. Model 2 seems to give a slightly better fit. In addition, for model 1, some reflections of the calculated pattern are absent in the experimental one. The difference may arise from the location of IBU and water molecules that should be considered as one local solution among many others differing by the distribution of the guest molecules. As revealed by solid-state NMR results, IBU is quite mobile in the crystal structure and thus would not occupy a precise crystallographic position. In addition, it is important to note that these models were built on the basis of packing energy. A combination with a structural refinement against diffraction data, such as that previously used to solve the structure of V1-butanol may help increase the matching between the observed and calculated diffraction pattern. However, the difficulty is that the diffraction data with several reflections at low angles would not be sufficient to solve the structure of VIBU with a large number of parameters.

VI.4. In vitro release properties of ibuprofen from the complexes

VI.4.1. Ibuprofen content in the complexes

The amount of IBU in the complex was determined by UV spectroscopy using two different methods. Method 1 used ethanol to extract IBU from the complex without solubilizing amylose. In order to confirm the complete IBU extraction from the complex, a second method was implemented. The complex was dissolved in 0.1 M NaOH, resulting in the total release of IBU. The results obtained with both methods were in good agreement: IBU contents of 8.345 ± 0.089 wt% and 8.403 ± 0.132 wt% were determined by methods 1 and 2, respectively. The corresponding stoichiometry was calculated to be about 1 IBU molecule per 14 glucosyl units, in agreement with the value previously determined by 13 C SP/MAS NMR spectrometry ( § VI.2.2).

The result suggests that ibuprofen was totally extracted by ethanol. In addition, all the above quantification methods are reliable for the determination of ibuprofen content in VIBU.

VI.4.2. In vitro dissolution studies

The solubility of raw IBU powder at 37 °C increased with increasing pH (Table VI.4), in agreement with previous studies [START_REF] Levis | Effect of buffer media composition on the solubility and effective permeability coefficient of ibuprofen[END_REF][START_REF] Qiu | Studies on the drug release properties of polysaccharide multilayers encapsulated ibuprofen microparticles[END_REF][START_REF] Rivera-Leyva | Comparative studies on the dissolution profiles of oral ibuprofen suspension and commercial tablets using biopharmaceutical classification system criteria[END_REF][START_REF] Yiyun | Solubilization of non-steroidal anti-inflammatory drugs in the presence of tween series surfactants[END_REF]. The increase in solubility is due to the ionization of IBU. Indeed at pH higher than pKa (4.5) [START_REF] Newton | pKa values of medicinal compounds in pharmacy practice[END_REF][START_REF] Ràfols | A comparison between different approaches to estimate the aqueous pKa values of several non-steroidal anti-inflammatory drugs[END_REF], the ionized form of IBU is predominant. The lowest IBU solubility (75 mg.L -1 ) was observed in 0.1 M HCl corresponding to the acidic form of IBU. In order to ensure sink conditions, all dissolution tests were performed in a sufficient volume of medium with respect to the amount of IBU to be dissolved, taking into account that the maximum concentration corresponding to the release of 100% IBU should not exceed 52.6 mg.L -1 . In these conditions, the dissolution medium would not reach saturation. The dissolution rate clearly increased with increasing pH. This result is expected to correlate with the solubility in these media. One can note that in acidic medium (pH 1.2), the dissolved IBU molecular fraction was about 40% after 4 h while in higher pH environments, it reached 100% within the same period of time. Indeed, at pH 1.2, residual IBU particles remained stuck to the basket and at the air/liquid surface due to the poor powder wettability.

The release profiles of the VIBU complex were obviously different from those of pure IBU powder, as shown in Figures VI.14 and VI.15. The variation of pH affected the amount of released IBU rather than the dissolution rate. The maximum percentage of released drug was only about 12% in 0.1 M HCl, but increased to about 45% in the buffer solution pH 5.5 and 100% in buffer solutions with pH > 6.8. The dramatic difference in percentage of released guest would mainly be attributed to the different solubilities of IBU in these pH solutions. The release profiles also suggest that IBU could be located at different positions within the complex. Three different IBU fractions were selectively released by controlling the pH of the dissolution medium. The first fraction (about 12% of total IBU) was readily released in the 0.1 M HCl.

This fraction would be very loosely bound to V-amylose and may correspond to uncomplexed IBU entrapped inside amorphous regions of the sample. On the other hand, the fraction released at higher pH would correspond to complexed IBU that would comprise two distinct fractions:

one that is further released upon increasing the pH to 5.5 (about 30% of total IBU) and the other is the remaining fraction (about 60%) that is released at pH 6.8-7.2 (Figures VI.14 and VI.15).

The latter would correspond to the most tightly bound molecules. In addition, the total release of IBU at pH 6.8-7.2 resulted in the complete dissolution of the VIBU crystals. In contrast, the crystals appeared to be resistant in media pH 1.2 or pH 5.5. Since the amylose helical cavity is a relatively hydrophobic environment, hydrophobic ibuprofen should be more tightly bound when located inside the helix than in the interhelical spaces. On the basis of this hypothesis, the ratio between intra-and inter-helical ibuprofen is approximately 2:1, thus supporting model 2. The result also suggests that the interstitial ibuprofen is not essential for the stability of the crystal structure, in agreement with the modeling study. However, it must be noted that our experimental data do not allow an unambiguous confirmation of the location of IBU in the complex.

Furthermore, as the release of IBU mainly occurred at pH 5.5-7.2, the VIBU complexes can act as a delayed release system for intestinal targeting. The observation that VIBU was stable in gastric pH 1.2 but released in simulated intestinal fluid has recently been made in other studies [START_REF] Yang | Preparation, characterization, and properties of amylose-ibuprofen inclusion complexes[END_REF][START_REF] Zhang | Structural and release properties of amylose inclusion complexes with ibuprofen[END_REF]. However, the authors showed a sustained release profile in pH 6.8-7.2 buffers by comparison with the instantaneous dissolution revealed in the present work. Similar instantaneous and sustained releases have also been observed for complexes of ibuprofen with β-cyclodextrin [START_REF] Charoenchaitrakool | Utilization of supercritical carbon dioxide for complex formation of ibuprofen and methyl-β-cyclodextrin[END_REF][START_REF] Salústio | Release profile of ibuprofen in β--cyclodextrin complexes from two different solid dosage forms[END_REF]. For VIBU, the release rate is also most probably be related to the morphology of the complex. Indeed, the lamellar shape with a thickness lower than 10 nm, offers a high contact surface with the medium which should increase the release rate.

VI.5. Conclusions

Lamellar rectangular V-amylose single crystals were prepared by adding racemic IBU to hot dilute aqueous solutions of native and enzymatically-synthesized amylose (0.1 wt%) with an average DPw varying from 60 to 6500. In addition, a highly crystalline fraction of VIBU inclusion complexes was successfully prepared by crystallization in 1 wt% DP6500 amylose DMSO:water solution. The base-plane electron diffraction pattern, powder XRD profile, FT-IR and 13 C CP/MAS NMR spectra of the crystals concurred to show that the complex belongs to the V7II allomorphic family. Amylose is thus crystallized in an orthorhombic P212121 space group with unit cell parameters a = 2.824 (±0.001) nm, b = 2.966 (±0.001) nm and c = 0.800 (±0.001) nm, corresponding to four 7-fold left-handed amylose single helices per unit cell. IBU would be located both inside and between the helices along with some water molecules. Water was shown to be an essential part of the complexes, its elimination resulting in the collapse of the original orthorhombic V7II structure and a generation pseudo-hexagonal V7a structure. The transition was reversible by rewetting the dry complexes. A stoichiometry of 1 ibuprofen molecule per 14.6 glucosyl residues was determined by SP/MAS NMR spectroscopy, in good agreement with the value quantified by UV spectrometry from in vitro release experiments

(1 IBU per 14 glucosyl units). Two tentative models with or without interstitial IBU were built by molecular dynamics and geometry optimization using the previously published model of isomorphous for V2-propanol crystal as starting model. The organization of amylose helices were similar in both calculated structures, which suggests that the stability of the complex would not depend on the presence of IBU in the interstitial spaces.

In vitro dissolution studies showed that the amount of IBU released from the complexes increased with increasing pH. This correlated with possible locations of IBU in the complexes in relation with different interactions with amylose. For instance, the IBU molecules located inside the helix would be bound more tightly and have more important effect on the stability of the complexes than that located in the inter-helix space. The results also suggest that the inclusion complexes are potentially interesting for intestinal targeting which would thus be an advantage for improving the therapeutic effect of ibuprofen while avoiding stomach damage. cells: V6I, V6II, V6III, V6IV, V7I, V7II, V7III, V7IV, V8I and V8II. The lamellar crystals corresponding to V6I, V7I, V7IV and V8I structures exhibited very similar shapes. Those of V6III and V8II were similar as well while V6II, V6IV, V7II, and V7III exhibited distinct morphologies.

However, the allomorphs could be distinguished without ambiguity from their base-plane ED and powder XRD patterns.

Spectroscopy analyses were used to complement the crystallography data. FT-IR spectra of V-amylose showed an increase in intensity of specific vibrational bands compared to amorphous or A-and B-amylose. In addition, the band near 1022 cm -1 could be used as a fingerprint of V-amylose. These differences can be accounted for different amylose helical conformations in the various allomorphs. However, we consider these FT-IR data to be preliminary and the complex spectra should be analyzed and compared in more details.

Expanding from previous results from the literature, we observed that there is a significant correlation between the helicity of V-amylose and its C1 resonance in 13 C CP/MAS solid-state NMR spectra. The resonance is shifted downfield with increasing number of glucosyl units per turn. Three C1 chemical shifts were detected, likely corresponding to the 6-, 7-and 8-fold helices. In addition, the C1 resonance was a muliplet in V6IV and V7III while it was a singlet in other allomorphs, suggesting that V6IV and V7III would contain helices that are less symmetrical than those in other allomorphs. However, due to the multiplicity in the C1 chemical shift, the helical conformation of V6IV and V7III could not be unambiguously confirmed from the present data. Like for FT-IR spectroscopy, we consider these solid-state NMR results to be preliminary and they should be further investigated. In particular SP/MAS analyses are potentially interesting to evaluate the mobility of the guest molecules and determine the stoichiometry of the complexes.

Although we proposed tentative geometrical models for each allomorph, we have carried out a more detailed study of V1-butanol (Chapter III). On the one hand, this complex is of historical importance since 1-butanol was one of the first agents used in the 1940's to fractionate native starch by selective crystallization with amylose. On the other hand, among the 5 previously known crystal forms, it was the only one for which the arrangement of helices was still hypothetical, and 43 of our tested complexing agents yielded the V6II allomorph. Our structure determination approach was based on conformational and packing energy analyses, combined with classical crystalline polymer structure refinement. The resulting model with the lowest reliability factor was described by a P212121 orthorhombic lattice of antiparallel 6-fold left-handed single helices in which the hydroxyl groups exhibit some conformational disorder.

There are four 1-butanol and 16 water molecules distributed into four elongated interstitial pockets, and one 1-butanol molecule per unit cell located inside each helix. This result confirmed the model proposed by [START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. However, our structure was only refined against base-plane ED data. Therefore, the atomic positions along the c-axis are not precisely known. In order to ascertain the 3D structure, ED patterns must be recorded on crystals rotated about the main axes of the reciprocal space and the intensity of the diffraction spots quantitatively analyzed.

Due to time limitation and the large number of different guest molecules, we could not perform the same type of analysis for the 5 new allomorphs but we have proposed geometrical models based on the crystallographic and spectroscopic data. The helix packing in V6I, V7I, V7III, V7IV and V8I structures is rather compact, and the complexing agents would only be located inside the helices. In contrast, there is more interstitial space in V6II, V6III, V6IV, V7II and V8II

to accommodate guest molecules. In addition the complexing agents can possibly be located inside the helices, except for V6IV. In fact, since V6IV was obtained with 4-hydroxybenzoic acid whose size appears to be incompatible with the cavity of a 6-fold helix, the cyclic complexing agent should be located only in interstitial spaces. All the tentative geometrical models remain to be validated by a detailed structural analysis combining experiments and modeling.

Our results show that each allomorph can be obtained with different complexing agents.

However, V6I, V6II and V7II were more prevalent. In addition, the helical conformation was found to be related to the size of the complexing agent. V6-type complexes were obtained with molecules with straight carbon chains, while branched-chain and cyclic molecules tended to yield 7-fold complexes. V8-type complexes were obtained with 1-naphthol, quinoline and salicylic acid. However, it is still difficult to predict the crystal structure based on the nature of the guest since a complexing agent could induce different crystal structures.

Indeed, V-amylose polymorphism was observed with a large number of complexing agents and is thus a more general phenomenon than what was previously reported in the literature. The tendency of a complex to exhibit a polymorphic behavior is not only related to the nature of the complexing agent but also to the accessibility of the various structures through crystallization. Complexes of straight-chain molecules are more likely to exhibit polymorphism than the branched-chain or cyclic compounds. In addition, straight-chain molecules, alcohols, fatty acids, aldehydes, amides or amines are likely more prone to form different allomorphs than esters and ketones.

Different parameters including solvent composition, temperature of mixing and crystallization, concentration of complexing agent and amylose, DP of amylose were revealed to have important impact on the formation and crystal structure of V-amylose. A variety of crystallization conditions had to be explored in order to observe relevant structures. However, the mechanism of polymorphic crystallization is still unclear. Future works combining experimental data and simulations should be conducted to understand the effect of the chemical nature of the guest and several crystallization parameters on the formation and crystal structure of V-amylose.

Water was shown to play a crucial role on the stability and the crystallinity of the complexes. Upon drying, V6I, V6II, V6IV, V7I, V7II and V7IV were transformed into compact hexagonal structures. For V7III, V8I and V8II, a significant loss in crystallinity was observed, but the crystal structure remained the same. V6III is the only structure that was stable upon drying. Surprisingly, in many cases, after drying in vacuum resulting in a loss of crystallinity, the original structure was reversibly restored by rehydration in humid atmosphere. In many previous studies, the focus was on the entrapment and release of the complexing agent but in view of our results, the role of water must be further studied and clarified.

Our results also suggest that new allomorphs of V-amylose remain to be discovered, which can be done by testing new complexing agents together with varying the conventional crystallization method in solution. The influence of factors such as solvent composition and additives should be further explored. As noted, in this study, only DMSO was tested as additive.

In addition, using different preparation techniques is also recommended, e.g. insertion of complexing agent into a preformed V-amylose, sealed-heating, etc.

To evaluate the potential of V-amylose as a delivery system of bioactive agents, we have studied model amylose complexes prepared with racemic ibuprofen. Highly crystalline fractions of lamellar crystals were successfully prepared by crystallization in water or DMSO:water solution (0.1-1 wt% amylose). The base-plane electron diffraction pattern, powder XRD profile, FT-IR and 13 C CP/MAS NMR spectra of the crystals showed that the complex belonged to the V7II allomorphic family. Tentative models with or without interstitial ibuprofen were built by molecular dynamics and geometry optimization based on the arrangement that was previously published for the isomorphous V2-propanol structure. The result suggests that the stability of the structure does not depend on the presence of ibuprofen in the interstitial spaces. However, the fit between the experimental ED pattern and those calculated from the proposed models is still not good enough and further work should be done to improve the model using a complementary analysis of packing energy.

In vitro dissolution studies revealed that Vibuprofen crystals exhibited different release profiles. In particular, the variation of the pH of the dissolution medium induced the selective release of different fractions of ibuprofen from the complexes. This result suggests that ibuprofen would reside in different locations and have different types of interactions with amylose. For instance, the ibuprofen molecules located inside the helix would be bound more tightly and play a more important role on the stability of the complexes than those located in the inter-helix space. Since the release mainly occurs at higher pH, the inclusion complexes seem to be a potentially interesting delivery system of ibuprofen for intestinal targeting and would thus be an advantage for improving its therapeutic effect.

Finally, since all validated models of V-amylose only involve left-handed helices, the potential property of chiral selectivity should also be investigated in the future. Our preliminary tests with enantiomers of ibuprofen, carvone, and menthol (results not shown in this manuscript) that all formed V7II complexes did not reveal any chiral discrimination. However, future works should investigate the interaction of amylose with stereoisomers of other molecules having different sizes and carefully consider the kinetics of the complex formation. a data indexed on the basis of the orthorhombic unit cell with a = b/√3 = 1.53 ± 0.002 nm, c = 0.812 ± 0.002 nm. Root-mean-square error (RMSE) = √∑(2𝜃 𝑜𝑏𝑠 -2𝜃 𝑐𝑎𝑙 ) 2 𝑁 𝑟𝑒𝑓𝑙𝑒𝑐𝑡𝑖𝑜𝑛𝑠 ⁄ = 0.027. b data indexed on the basis of the orthorhombic unit cell: a = 2.790 ± 0.001 nm, b = 2.957 ± 0.001 nm, c = 0.798 ± 0.001 nm. RMSE = 0.009. c data indexed on the basis of the orthorhombic unit cell with a = 1.663 ± 0.001 nm, b = 2.518 ± 0.001 nm and c = 0.856 ± 0.001 nm. RMSE = 0.009. d data indexed on the basis of the orthorhombic unit cell with a = 2.702 ± 0.001 nm, b = 3.291 ± 0.002 nm. c = 0.786 ± 0.001 nm. RMSE = 0.019. a indexed based on an orthorhombic unit cell with a = 2.82(4) ± 0.00(1) nm, b = 2.96(6) ± 0.00(1) nm, c = 0.79(9) ± 0.00(1) nm.

b indexed based on a hexagonal unit cell with a = b = 1.48(2) ± 0.00(9) nm, c = 0.79(3) ± 0.00(5) nm.

  Figure I.1 whereas the proposed structure for the branched amylose is shown in Figure I.2. Native amylose would contain less than 1% branching points.

Figure I. 1 .

 1 Figure I.1. Chemical structure of linear amylose.

Figure I. 2 .

 2 Figure I.2. Proposed structure of the branched amylose. EL, extremely long; L, long; and S, short chains; Ø, reducing end[START_REF] Takeda | Structures of sub-fractions of corn amylose[END_REF]).

Figure I. 3 .

 3 Figure I.3. Conformational models proposed for amylose in solution: a) random coil having no helical character, b) interrupted helix with helical segments connected by short random coils,c) helix with the overall form of a random coil. Adapted fromBanks and Greenwood (1971a).

  . A random coil (Figure I.3a) has no helical character and the arrangement of successive units is random. In both interrupted helix (Figure I.3b) and helical models (Figure I.3c), the amylose chain organized in the helical portions are thought to be stabilized by intra-molecular hydrogen bonds. However, due to the flexibility of the amylose chain, any of the three structures can adopt the overall shape of a random coil model.

Figure I. 4 .

 4 Figure I.4. Conformation of amylose in dilute solution. Adapted from Banks & Greenwood (1971a).

Figure I. 5 .

 5 Figure I.5. X-ray diffraction profiles (a) and solid-state 13 C CP-MAS NMR spectra (b) of A and B starch recorded at 20% H2O. Adapted from Buléon et al. (2007).

Figure I. 6

 6 Figure I.6. a) TEM image of a single crystal of A-amylose; b) example of ED pattern properly oriented with respect to the crystal (Montesanti et al., 2010); c) scheme describing the orientation of double helices in the crystal (Putaux et al., 2011b); d) SEM image of A-amylose single crystals radially organized in a spherical aggregate[START_REF] Montesanti | A-type crystals from dilute solutions of short amylose chains[END_REF]; e) SEM image of A-amylose spherulites. The radial organization is well revealed in the fracture of one of them, as indicated by the arrow[START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

Figure

  Figure I.7. a) TEM image of B-amylose lamellar crystals prepared by in vitro crystallization[START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]; b) base-plane ED pattern recorded on a single crystal[START_REF] Buléon | Single crystals of amylose with a low degree of polymerization[END_REF]; c) SEM image of B-amylose spherocrystals[START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF].

Figure I. 8 .

 8 Figure I.8. Projection in the (a,b) plane of the molecular models of A-and B-amylose[START_REF] Popov | Crystal structure of A-amylose: a revisit from synchrotron microdiffraction analysis of single crystals[END_REF][START_REF] Takahashi | Crystal structure of B-amylose[END_REF].

Figure I. 9 .

 9 Figure I.9. Known V-amylose single helices. Adapted from[START_REF] French | Allowed and preferred shapes of amylose[END_REF].

Figure I. 10 .

 10 Figure I.10. V6I crystals: a) TEM image (bar: 1 µm) and b) corresponding base-plane ED pattern[START_REF] Helbert | Données sur la structure du grain d'amidon et des produits de recristallisation de l'amylose[END_REF]; c,d) projections on the (a,b) plane of the molecular models based on hexagonal[START_REF] Brisson | The crystal and molecular structure of VH amylose by electron diffraction analysis[END_REF] and orthorhombic unit cells[START_REF] Rappenecker | Detailed refinement of the crystal structure of Vhamylose[END_REF], respectively.

Figure I. 11 .

 11 Figure I.11. V6II crystals: a) TEM image (scale bar: 1 µm) of single crystals of V1-butanol and b) corresponding base-plane ED pattern; c) projection on (a,b) plane of the probable organization of amylose helices within the unit cell. The guest solvent molecules occupy the shaded areas. Adapted from Helbert & Chanzy (1994).

  and give sharp base-plane ED patterns that exhibit a square symmetry (Figure I.12a and I.12b).

Figure I. 12 .

 12 Figure I.12. V6III structure: a) TEM image (scale bar, 1 µm) of single crystals of Vglycerol and b) corresponding base-plane ED pattern[START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF]; c) projection in the (a,b) plane and d) 3D view of the VDMSO structure(Winter & Sarko, 1974a).

Figure I. 13 .

 13 Figure I.13. V7 structure: (a) TEM image of single crystals of V2-propanol and (b) corresponding ED pattern recorded at low temperature (Putaux et al., 2008); (c) projection on the (a,b) plane of the molecular structure (Nishiyama et al., 2010).

Figure I. 14 .

 14 Figure I.14. V8 structure: (a) TEM image of single crystals of V1-naphthol; (b) corresponding base-plane ED pattern recorded at low temperature; (c) projection of the (a,b) plane of the molecular model; (d) averaged high-resolution lattice image recorded along the helical axis with the inset of molecular model[START_REF] Cardoso | Single crystals of V-amylose complexed with α-naphthol[END_REF].

Figure I. 15

 15 Figure I.15. a) Molecular structure of VKOH; potassium ions are designated by ⊕ (Sarko & Biloski, 1980). b) Molecular structure of VKBr; bromide and potassium ions are designated by large and small open circle, respectively[START_REF] Senti | X-ray diffraction studies of addition compounds of amylose with inorganic salts[END_REF].

  The chain length distribution was determined by Noriyuki Isobe (Université de Tokyo) by size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) detection after dissolution of amylose in LiCl/1,3-dimethyl-2-imidazolidinone. The weight-average degree of polymerization DPw was 6474 (Figure II.1 and Table

Figure II. 1 .

 1 Figure II.1. SEC-MALLS Chromatogram of purified native potato amylose and dissolution in LiCl/DMAc.

Figure II. 2 .

 2 Figure II.2. HPAEC chromatograms of the total amylose fraction (DP86) synthesized in vitro by amylosucrase and 3 fractions (DP60, DP80 and DP130) obtained by fractionation of DP86.

  a synthesized in vitro by amylosucrase; b total fraction; c fractionated from the total fraction. d synthesized in vitro by phosphorylase e native amylose from potato (Sigma Aldrich) f values given by the manufacturer TableII.2. List of complexing agents lamellar crystals were prepared in aqueous solutions of amylose in different conditions (concentration of amylose, complexing agent and DMSO, DP of amylose and crystallization temperature). Amylose solutions (0.1-0.5 wt%) were prepared by two methods: i) dispersing amylose in water, bubbling with nitrogen for 20 min and then autoclaving at 160 °C for 30 min, and ii) dissolving amylose in DMSO at 90 °C for 1 h, then diluting in water (90 °C); the DMSO concentration varied from 2, 5, 10, 15, 20 and 30 vol%.Most complexing agents were added into amylose solution after the dissolution at 75-90 °C. Fatty acids having 10-20 carbons, dodecanedioic acid, n-aliphatic alcohols having 10-16 carbons, (-)-borneol, R-(+)-camphor, decahydro-2-naphthol, cis-decahydro-1-naphthol and trans-decalin were added to amylose suspension before autoclaving since these complexing agents are thermally stable. Most molecules are poorly soluble and were used at saturation for complexation. However, for water-miscible agents such as short-chain fatty acids (propanoic and butanoic acids) and alcohols (ethanol, n-propanol and 2-propanol) and diols having 2-6 carbons, the complexation results depended on the concentration of complexing agents.

  its end to scan the specimen surface (Figure II.3). When the tip is brought in proximity of the sample surface, the local forces between the tip and the sample result in a deflection of the cantilever and thus allows local imaging of the topography of the sample surface by recording the height of the probe that corresponds to a constant probe-sample interaction.

Figure II. 3 .

 3 Figure II.3. Typical configuration of an AFM.

  vectors a, b, and c and the interaxial angles between them, α, β, and γ. A plane which passes through 3 lattices points which are not aligned is called a lattice plane. In real space, a set of parallel lattice planes is described using (h k l) Miller indices and the (x, y, z) coordinates of any point in the lattice planes satisfies the equation: hx + ky + lz = n, where n is an integer number. When a radiation with a λ wavelength illuminates a crystalline specimen, part of the beam is deflected in specific directions due to Bragg diffraction (Figure II.4). The angle of the incidence of the parallel planes appearing at intervals d (d-spacing) is θ. The waves reflected by the two planes have an optical path difference of 2dsinθ. They interfere with each other to enhance the intensities when the optical path difference equals an integer multiple (n) of the incident wavelength λ. Therefore, the condition of diffraction for mutual intensity enhancement is given by the equation 2𝑑𝑠𝑖𝑛𝜃 = 𝑛𝜆, which is called Bragg's law. A diffraction pattern from a single crystal usually contains many distinct peaks, each corresponding to a different d-spacing and a different set of planes (h k l) in real space.

Figure II. 4 .

 4 Figure II.4. Geometry for interference of a wave scattered from two crystal planes separated by a spacing d.

  First, the 100 and 010 vectors of the base-plane diagram were determined by measuring the position of the 600 and 060 diffraction spots. 16 × 16 pixels 2 boxes were extracted around each spot at each point defined by these base vectors. The intensity was calculated by fitting the data with a Gaussian peak function and a linear background. The saturated peaks were interpolated by fitting the non-saturated base of the reflection with a Gaussian function. The intensities of the reflections were averaged according to the symmetry of the pattern and normalized using the strongest value as reference.

Figure II. 5 .

 5 Figure II.5. Schemes illustrating the imaging (a) and diffraction (b) modes to operate a TEM. In each case, the intermediate lens selects either the image plane (a) or the back focal plane (b) of the objective lens as its object.

  Figure II.6. Vibration modes of a CH2 portion in a CH2X2 where X can be any other atom.

  .3.9. Dissolution tests of V-ibuprofen complexes II.3.9.1. Ibuprofen quantification assaysThe ibuprofen fraction in the complexes, the ibuprofen solubility values and the in vitro dissolution characteristics of the complexed drug were determined using a Shimadzu UV-1603 ultraviolet spectrophotometer. In ethanol, the ibuprofen concentration (Cibu, mg.L -1 ) was calculated from the equation Cibu = 712.56*A -3.0633 (R 2 = 0.9998) by measuring the absorbance (A) at 263.9 nm. At pH 12.69 in a 0.1 M NaOH solution, the ibuprofen concentration was spectrophotometrically assayed at 264.3 nm and calculated from the equation Cibu = 517.44*A -4.4734 (R 2 = 0.9998).

  Figure III.1 shows electron micrographs of typical crystals of V1-butanol prepared from dilute aqueous amylose solutions. They generally consist of stacks of thin rectangular-shaped lamellae. Each lamella is about 2-5 µm wide and 8-12 µm long. Spiral dislocation growth was not observed but the crystals thickened by epitaxial growth oriented on the neighboring lamellae. In addition, cross-shaped or rosette-shaped twinned crystals consisting of two or three platelets with angles of intersection close to 60 or 90° were usually observed (Figure III.1b

Figure

  Figure III.2a shows a part of a typical base-plane ED pattern of V1-butanol recorded on a 1 µm 2 surface of the lamellar crystal in frozen-hydrated state at low temperature. The diffraction spots extend to a resolution of 0.24 nm and distributed symmetrically in a lattice defined by the two orthogonal axes a* and b* aligned parallel to the shorter and longer dimensions of the lamella, respectively.

Figure

  Figure III.1. TEM images of V1-butanol crystals: a) rectangular crystal favoring an epitaxial growth; b,c) twinned configurations of crystals at 60 and 90°, respectively. Scale bars: 2 µm.

Figure

  Figure III.2. a) ED pattern recorded on a frozen-hydrated V1-butanol crystal at low temperature; b) XRD powder pattern recorded on a hydrated film of V1-butanol crystals.

Figure

  Figure III.3. 13 C CP/MAS NMR spectrum recorded on hydrated crystals of V1-butanol.

  Figure III.4. Axial and longitudinal view of 6-, 7-and 8-fold left-and right-handed helices with C6 hydroxyl groups removed. Hydrogens were omitted for clarity.

  Figure III.5. Color packing energy map for left-handed (a) and right-handed (b) 6-fold helices when they are rotated and translated along the a-and b-axis in 1/8 th of the unit cell.

Figure III. 6 .

 6 Figure III.6. Comparison of the projection on the (a,b) plane of the unit cell with the lowest packing energy models for 6-(a), 7-(b) and 8-fold (c) left-handed amylose helices with C6 hydroxyl groups removed. Hydrogens were omitted for clarity.

Figure III. 7 .

 7 Figure III.7. Selected starting models for structural refinement: a) L6_0000; b) L6_0006; c) L6_0817; d) L6_0833; e) R6_0000; f) R6_0006; g) R6_0817; h) R6_0833.

The 1 -

 1 butanol and water molecules were directly inserted in the models and their positions were refined independently of the biopolymer molecules themselves. Using the average ED intensities to a resolution of 0.24 nm (72 diffraction spot) and the default value of the effective standard deviation (sd = 0.02 defined in the DEFS command) for restrained parameters, no model converged to the experimental diffraction intensities.

Figure III. 8 .

 8 Figure III.8. Projection on the (a,b) plane of L6_0817 (a-c) and L6_0006 (d-f) models: a,d) initial packing of rigid helices with C6 hydroxyl groups removed, b,e) geometry-optimized model after addition of C6 hydroxyl groups, 1-butanol and water molecules; c,f) final structure refined with SHELX.

Figure

  Figure III.9. Observed ED pattern of V1-butanol (upper half) and a calculated pattern (lower half) of the final model after SHELX refinement: a) L6_0817; b) L6_0006.

Figure

  Figure III.10 shows the observed XRD pattern recorded on a film of crystals (a) and those simulated from L6_0006 final structure with/without including the c-axis preferred orientation (band c). The observed and calculated patterns fit better when a preferred orientation was considered. This result is consistent with the fact that the lamellar V-amylose crystals favor a flat-on orientation when settling in the form of a mat, which is demonstrated by a fiber-like XRD pattern when the X-ray beam is directed parallel to the film surface. Since the pattern shown in Figure III.10a was recorded when the X-ray beam was perpendicular to the film surface (parallel to the c-axis), hkl (l≠0) reflections were almost not observed and the pattern was thus consistent with that including a preferred orientation.

Figure III. 11

 11 Figure III.11 shows the projection of the final molecular structure on the (a,b) and (a,c) planes of the unit cell. The atomic coordinates are given in Annex 1, TableS.III.2 whereas a selection of conformational parameters defining the helix geometry is listed in TableIII.4.Consistent with the 13 C CP/MAS NMR data, the present results confirm that the V1-butanol structure consists of 6-fold left-handed helices in which the hydroxyl groups exhibit some conformational disorder. Inside the P212121 orthorhombic unit cell, the helices are organized in an antiparallel fashion and in rows parallel to the b axis. These results validate the tentative model previously proposed by[START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. In addition, there are four 1-butanol and 16 water molecules distributed into four elongated interstitial pockets, and one 1-butanol molecule located inside each helix.

Figure

  Figure III.10. a) XRD profile recorded on a hydrated film of V1-butanol crystals; b) calculated powder profile; c) calculated profile with an imposed c-axis preferred orientation.

Figure III. 11 .

 11 Figure III.11. Projection on the (a,b) (a) and (a,c) (b) planes of the final structure of V1-butanol. Hydrogen atoms were omitted. Amylose and water molecules are in blue and in pink respectively.

Figure III. 12 .

 12 Figure III.12. Hydrogen bonds in the final structure of V1-butanol projected on the (a, b) plane of the unit cell.

Figure IV. 1

 1 Figure IV.1 shows TEM micrographs of typical V6I crystals with a well-defined hexagonal shape (Figure IV.1a-c). They were monolamellar or, more frequently, multilamellar due to the spiral growth with a screw dislocation center. They usually resulted from a slow crystallization at relatively high temperatures or successive recrystallizations. In other cases, the crystals exhibited an overall round or less defined hexagonal shape. Sometimes elongated or undefined shapes were also observed (Figure IV.1d-f). The lamellar thickness determined by

Figure

  Figure IV.2. a) TEM image of a V6I crystal of native amylose complexed with tetradecanoic acid (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

The

  Figure IV.3 shows the XRD profiles of V6I crystals in their initial hydrated state (a), after drying (b) and after rehydration (c). After thorough drying, the peaks became broader and located at higher diffraction angles compared to those of the hydrated crystals, indicating a slight loss of crystallinity and a decrease in unit cell parameters. This result is in agreement

  Figure IV.3b is typical for V6a and was indexed on the basis of a hexagonal unit cell with a = b = 1.32 nm and c = 0.81 ± 0.01 nm, or an orthorhombic unit cell with a = 1.32 nm, b = a√3 = 2.29 nm and c = 0.81 ± 0.01

Figure IV. 3 .

 3 Figure IV.3. XRD profiles of a) hydrated V6I crystals prepared with octadecanoic acid, b) crystals as in (a) after thorough drying in vacuum, c) crystals as in (b) after rewetting.

Figure IV. 4 .

 4 Figure IV.4. Packing of amylose helices in V6I (a) (Rappenecker & Zugenmaier, 1981) and corresponding V6a (b) (Murphy et al., 1975), projected on the (a,b) plane of the orthorhombic unit cells. Hydrogen atoms were removed for clarity.

Figure IV. 5

 5 Figure IV.5 shows TEM images of typical rectangular V6II lamellar crystals. The lamellar thickness determined by AFM and averaged from 4 different samples is 9.4 ± 1.2 nm (Annex 2, Table S.IV.4 and Figure S.IV.1b). Individual lamellae were rarely seen. Indeed, the crystal were usually built as compact stacks of lamellae that may result from an epitaxial growth. The rectangular platelets were isolated (Figure IV.5a,b) or occurred as twinned structures at an angle of about 60 or 90° (Figure IV.5c-f). This morphology is similar to that

Figure IV. 5 .

 5 Figure IV.5. TEM images of V6II crystals obtained with dodecanoic acid (a), 1,6-hexanediol (b), 1,3-butanediol (c), pentanoic acid (d), 2-propanol (e), and octadecanoic acid (f). Bars: 1 µm.

Figure

  Figure IV.6. a) TEM image of V6II crystals obtained with 1,3-butanediol (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure

  Figure IV.6c (a = 2.646 ± 0.009 nm, b = 2.705 ± 0.013 nm, and c = 0.807 ± 0.009 nm). The Miller indices of the XRD reflections for V1,3-butanediol are given in Annex 2, Table S.IV.1. In Chapter III, we have described the molecular structure of V1-butanol crystals based on analyses of the conformational and packing energy, combined with classical polymer structure refinement against ED data. The antiparallel helices are organized in rows parallel to the b axis, with 1-butanol molecules located inside and between helices (Figure III.8).

Figure IV. 7 .

 7 Figure IV.7. XRD profiles of a) hydrated V6II crystals prepared with 1,4-butanediol, b) crystals as in (a) after partial drying in vacuum, c) crystals as in (b) after thorough drying.

Figure

  Figure IV.8. a) TEM image of a V6II crystal prepared with 1,3-butanediol, partially dried in vacuum (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) base-plane ED pattern recorded from a V6II crystal obtained with 1,4-butanediol after partial drying in vacuum.

  patterns almost identical to that of V6a obtained by drying the V6I crystals (Figure IV.7c). Following the drying process of the more stable crystals, it is noted that the crystals rapidly yielded XRD (Figure IV.7b) and ED diagrams (Figure IV.8) exhibiting a tetragonal symmetry.

  (see § IV.3). This structure was rather stable and slowly transformed into V6a (Figure IV.7c).

Figure IV. 9

 9 Figure IV.9 shows the schematic representation of the rectangular crystal in different states and the corresponding amylose packing arrangement. In the initial V6II, amylose helices are organized in rows parallel to the b-axis of the unit cell or to the longer-dimension of the rectangular crystal. Upon partial drying, amylose helices are aligned along both a-and b-axes, leading to a small enlargement of a-parameter from 2.65 nm to 2.72 nm, while the b-parameter remained unchanged (2.72 nm). The small change in a-parameter does not cause any cracks on the crystal. Upon thorough drying, amylose helices are reorganized into a close-packed hexagonal arrangement, forming V6a. There is a slight decrease of b (0.08 nm) while a significantly decreases (0.43 nm), which is consistent with the formation of cracks along the b-axis. In addition, the present models show that V6a and the isomorphous V6III obtained by drying V6II would have orthorhombic unit cells with space group P212121, containing four amylose chains per unit cell. These unit cells are larger than those proposed for the V6a prepared by drying V6I and the V6III of glycerol, DMSO or ethylenediamine, which contain only two helices.

Figure IV. 9 .

 9 Figure IV.9. Schematic representation of a rectangular V6II crystal and corresponding amylose helices packing (insert) during the drying process. a) Initial uncracked crystal having an orthorhombic V6II structure[START_REF] Helbert | Single crystals of V-amylose complexed with n-butanol or n-pentanol: structural features and properties[END_REF]. b) Partial dry uncracked crystal having a pseudo-tetragonal V6III structure[START_REF] Hulleman | Single crystals of V amylose complexed with glycerol[END_REF] Winter & Sarko, 1974). c) Thoroughly dried crystal, exhibiting cracks running parallel to the long dimension of the crystals, having a pseudo-hexagonal structure V6a.

  the crystals recovered the V6II structure, as shown in Figure IV.10.

Figure IV. 10 .

 10 Figure IV.10. XRD profiles of a) hydrated V6II crystals obtained with pentanoic acid, b) crystals as in (a) after thorough drying in vacuum, c) crystals as in (b) after rewetting.

  or important decomplexation phenomenon. The corresponding base-plane ED pattern recorded at room temperature is shown in Figure IV.11b. This diagram can be indexed for a two-dimensional pseudo-tetragonal orthorhombic unit cell of space group P212121 with a = b = 1.92 ± 0.01 nm, in good agreement with data calculated from the powder XRD pattern shown in Figure IV.1c (a = b = 1.91 ± 0.01 nm) (Figure IV.1c, Annex 2, Table S.IV.1). The unit cell parameters found in the present study are different from those determined by Hulleman

Figure

  Figure IV.11. a) TEM images of a V6III crystal prepared with glycerol (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure IV. 12 .

 12 Figure IV.12. Packing of amylose helices in V6III structure, projected on the (a,b) plane of the pseudo-tetragonal orthorhombic unit cell. Hydrogen atoms were removed for clarity.

  .IV.4 and Figure S.IV.1c). The corresponding ED pattern recorded from individual V7 crystals prepared with different complexing agents were similar, such as that of Vpentanoic acid shown in Figure IV.14b.

  Figure IV.14c) and can be indexed on the basis of an orthorhombic unit cell with a = 2.813 ± 0.013 nm, b = 2.973 ± 0.011 nm and c = 0.797 ± 0.011 nm (Annex 2, Table S.IV.2). In the model determined for V2-propanol (Nishiyama et al., 2010), each unit cell contains four left-handed 7-fold helices packed in the P212121 symmetry with an alternation of up and down chains, as shown in Figure IV.15.

Figure IV. 13 .

 13 Figure IV.13. TEM images of V7 crystals prepared with 1,3-butanediol (a), pentanoic acid (b), β-citronellol (c), (S)-perillaldehyde (d), cis-decahydro-1-naphthol (e), 2-naphthol (f). Bars: 1 µm.

Figure

  Figure IV.14. a) TEM image of V7 crystals prepared with pentanoic acid (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure IV. 15 .

 15 Figure IV.15. Packing of amylose helices in the V7 structure projected on the (a,b) plane of the orthorhombic unit cell. Hydrogen atoms were removed for clarity.

Figure IV. 16 .

 16 Figure IV.16. XRD profiles of a) hydrated V7 crystals prepared with decahydro-2-naphthol, b) crystals as in (a) after thorough drying in vacuum, c) crystals as in (b) after rewetting.

Figure IV. 17 .

 17 Figure IV.17. Possible packings of helices in the V7a structure projected on the (a,b) plane of an orthorhombic unit with space group P1 (a) or a larger orthorhombic unit cell with space group P212121 (b).

Figure IV. 18 .

 18 Figure IV.18. XRD profiles of a) hydrated V7 crystals prepared with 1,3-butanediol; b) crystals as in (a) after thorough drying in vacuum.

Figure

  Figure IV.19a-cshows TEM images of single crystals of V8 complexes prepared with 1-naphthol, salicylic acid, and quinoline, respectively. The crystals have a square shape and consist of a stack of many thin lamellae. They are usually very thick, even when prepared in highly dilute condition (0.025 wt% amylose). For Vquinoline, mono-and multilamellar crystals were observed. This morphology is very similar to that observed for V6III of glycerol.

Figure

  Figure IV.19. a-c) TEM images of V8 crystals prepared with 1-naphthol (a), salicylic acid (b) and quinoline (c) (scale bars: 1 µm). d-f) base-plane ED patterns of V8 crystals prepared with 1-naphthol (d), salicylic acid (e) and quinoline (f). g-i) powder XRD diagram recorded from hydrated V8 crystals prepared with 1-naphthol (g), salicylic acid (h) and quinoline (i).

  . The model in Figure IV.20 shows that one unit cell contains two antiparallel left-handed 8-fold helix packed along space group P43212. The complexing agents would be located both inside and between the helices. The complexing agent:glucosyl unit stoichiometry was determined by UV spectroscopy after extraction of complexing agent from the freeze-dried complexes using ethanol. The value was measured to be 1:6.2, 1:5.0, and 1:8.3 for V1-naphthol, Vsalicylic acid, and Vquinoline, respectively.

Figure IV. 20 .

 20 Figure IV.20. Packing of amylose helices in V8 structure projected on the (a,b) plane of the orthorhombic unit cell. Hydrogen atoms were removed for clarity.

Figure IV. 21 .

 21 Figure IV.21. XRD profiles of a) hydrated V8 crystals prepared with 1-naphthol; b) crystals as in (a) after thorough drying in vacuum; c) crystals as in (b) after rewetting.

Figure IV. 22

 22 Figure IV.22 shows lamellar VHBA crystals prepared at 40 °C from a 0.2 wt% native amylose solution. The crystals are needle-shaped lamellae that radiate from a common nucleation site forming flower-like aggregates. Each lamella is about 0.1-0.3 µm wide and 10-20 µm long. This morphology is quite similar to that previously reported for some V7 crystals, but the VHBA crystals do not have a well-defined rectangular shape.

Figure IV. 22 .

 22 Figure IV.22. TEM images of a new form of V-amylose complexed with 4-hydroxybenzoic acid. Scale bars: 1 µm.

Figure

  Figure IV.23. a) TEM image of V-amylose complexed with 4-hydroxybenzoic acid (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure

  Figure IV.23b shows the base-plane ED pattern recorded from frozen-hydrated VHBA crystals while Figure IV.23c shows the powder XRD diagram of hydrated crystals. These patterns clearly differ from those of V6I, V6II, V6III, V7 and V8, indicating that this is a new V-amylose allomorph. If the helices are perpendicular to the lamellae as reported for other allomorphs, then the ED pattern shown in Figure IV.23b corresponds to a two-dimensional lattice in the zero layer of the reciprocal lattice. It can be indexed on the basis of an orthorhombic unit cell with a = 1.54 ± 0.01 nm and b = 2.81 ± 0.01 nm. These cell parameters are in good agreement with those calculated from XRD data (a = 1.550 ± 0.001 nm, b = 2.836 ± 0.001 nm and c = 0.790 ± 0.001nm) (Annex 2, TableS.IV.1). In addition, the h00, 0k0 reflections are

Figure IV. 24 .

 24 Figure IV.24. Proposed model of V4-hydroxybenzoic acid, composed of left-handed 6-fold helices packed into the orthorhombic unit cell, space group P212121. The model is projected on the (a,b) plane of the unit cell. Hydrogen atoms are not shown.

Figure IV. 25 .

 25 Figure IV.25. XRD profiles of a) hydrated V-amylose complexes with 4-hydroxybenzoic acid; b) crystals as in (a) after thorough drying in vacuum; c) crystals in (a) after freeze-drying; d) crystals as in (c) after rewetting.

Figure IV. 26 .

 26 Figure IV.26. Proposed model of the dry form of V4-hydroxybenzoic acid, composed of left-handed 6-fold helices packed into a pseudo-hexagonal orthorhombic unit cell. The model is projected on the (a,b) plane of the unit cell. Hydrogen atoms are not shown.

  Figure IV.27 shows lamellar crystals of V-amylose prepared with (-)-borneol, R-(+)-camphor, cis-decahydro-1-naphthol, decahydro-2-naphthol and 1,3-butanediol. The crystals have a more or less hexagonal shape. They can be monolamellar or more frequently

Figure IV. 27 .

 27 Figure IV.27. TEM images of a new form of V-amylose obtained with (-)-borneol (a,b), R-(+)-camphor (c), cis-decahydro-1-naphthol (d), decahydro-2-naphthol (e), 1,3-butanediol (f). Scale bars: 1 µm.

Figure

  Figure IV.28. a) TEM image of V-amylose complexed with (-)-borneol (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure IV. 29 .

 29 Figure IV.29. Base-plane ED patterns of V-amylose crystals obtained with cis-decahydro-1naphthol (a); decahydro-2-naphthol (b); 1,3-butanediol (c).

Figure IV. 30 .

 30 Figure IV.30. XRD profiles of a) hydrated V-amylose complexes with (-)-borneol, b) crystals as in (a) after thorough drying in vacuum, c) crystals as in (b) after rewetting.

Figure IV. 31 .

 31 Figure IV.31. XRD profiles of a) hydrated V-amylose complexes with 1,3-butanediol, b) crystals as in (a) after thorough drying in vacuum.

Figure IV. 32 .

 32 Figure IV.32. Proposed model of V(-)-borneol and the isomorphous complexes projected on the (a,b) plane of the unit cell. Hydrogen atoms are not shown.

Figure IV. 35

 35 Figure IV.35 shows a possible packing of left-handed 7-fold helices in the unit cell with space group P1. Such arrangement allows for a statistical or regular alternation of up and down chains.

Figure IV. 33 .

 33 Figure IV.33. TEM images of a new form of V-amylose complexed with 1-naphthol. Bars: 1 µm.

Figure

  Figure IV.34. a) TEM image of V-amylose complexed with 1-naphthol (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect to the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure IV. 35 .

 35 Figure IV.35. Proposed model for a new allomorph of V1-naphthol based on 7-fold left-handed helices. The model is projected on the (a,b) plane of the unit cell of space group P1. Hydrogen atoms are not shown.

Figure IV. 36 .

 36 Figure IV.36. XRD profiles of a) hydrated V-amylose complexed with 1-naphthol; b) crystals as in (a) after thorough drying in vacuum; c) crystals as in (a) after freeze-drying; d) crystals as in (c) after rewetting.

Figure IV. 37 .

 37 Figure IV.37. TEM images of crystals of a new form of V-amylose complexed with quinoline prepared from DP80 (a) and D130 (b) amylose. Scale bars: 1 µm.

Figure

  Figure IV.38. a) TEM image of a crystal of DP80 amylose complexed with quinoline (scale bar: 1 µm); b) corresponding base-plane ED pattern correctly oriented with respect the crystal in (a); c) corresponding powder XRD diagram recorded from hydrated crystals.

Figure IV. 39 .

 39 Figure IV.39. Proposed model for a new allomorph of Vquinoline based on 7-fold left-handed helices. The model is projected on the (a,b) plane of the unit cell of space group P212121. Hydrogen atoms are not shown.

  Figure IV.41 shows TEM images of typical crystals of the new form of Vsalicylic acid. The crystals consist of stacks of many lamellae with both epitaxial and dislocation-centered spiral growths. The shape of individual lamellae cannot be defined with precision. In general, the crystals exhibit an overall shape that is close to those of V6I type, V(-)-borneol type and the new allomorph of Vquinoline.

Figure IV. 40 .

 40 Figure IV.40. XRD profiles of a) hydrated V-amylose complexed with quinoline; b) crystals as in (a) after thorough drying in vacuum; c) crystals as in (b) after rewetting.

Figure IV. 41 .

 41 Figure IV.41. TEM images of a new form of V-amylose complexed with salicylic acid. Bars: 1 µm.

Figure

  Figure IV.42b shows the typical base-plane ED pattern recorded from one frozenhydrated lamella of Vsalicylic acid. This pattern shows a centrosymmetry (𝐼 ℎ𝑘𝑙 = 𝐼 ℎ ̅ 𝑘 ̅ 𝑙 ̅ ) and is different from those of other V-amylose allomorphs. If the crystals are composed of helical amylose chains perpendicular to the lamellar surface, then the ED pattern can be indexed based on a monoclinic unit cell with a = 3.21 ± 0.01 nm, b = 3.23 ± 0.01 nm, and γ = 116.9 ± 0.3°. The unit cell parameters well agree with those calculated from powder XRD pattern (Figure IV.42c) (Annex 2, Table S.IV.3), given as a = 3.245 ± 0.006 nm, b = 3.246 ± 0.005 nm, c = 0.793 ± 0.001 and γ = 116.62 ± 0.13°. Since there is no systematic absence based on the h and k indices, the space group would be P2 or P21. Each unit cell would contained four amylose helices.Assuming a close packing, the center-center distance of helices is 1.62 nm which is equal to the helix diameter of 8-fold helices. Therefore, all 6-, 7-and 8-fold helices could be packed into the unit cell. A tentative packing model of left-handed 8-fold helices in space group P21 is shown in Figure IV.43. Salicylic acid may be located inside the helix cavity. The stoichiometry of the complexes determined by UV spectroscopy is 1 salicylic acid per 6.1 glucosyl units. Additional details on the helical conformation obtained by solid-state NMR will be given in § IV.13.

Figure IV. 43 .

 43 Figure IV.43. Proposed model for a new allomorph of Vsalicylic acid based on 8-fold left-handed helices. The model is projected on the (a,b) plane of the monoclinic unit cell of space group P21. Hydrogen atoms are not shown.

Figure IV. 44 .

 44 Figure IV.44. XRD profiles of a) hydrated V-amylose complexed with salicylic acid; b) crystals as in (a) after thorough drying in vacuum; c) crystals as in (b) after rewetting.

Figure

  Figure IV.45. FT-IR absorbance spectra of untreated DP6500 amylose (a), A-amylose (b), B-amylose (c), V6I of 1,6-hexanediol (d); V6II of 1,6-hexanediol (e); V6III of glycerol (f); V7 of 4-tert-butylbenzoic acid (g); V8 of salicylic acid (h); (i-m): new allomorphs of V4-hydroxybenzoic acid (i), V(-)-borneol (j), V1-naphthol (k), Vquinoline (l) and Vsalicylic acid (m).

Figure

  Figure IV.46. FT-IR absorbance spectra of untreated DP6500 amylose (a), A-amylose (b), B-amylose (c), V6I of 1,6-hexanediol (d); V6II of 1,6-hexanediol (e); V6III of glycerol (f); V7 of 4-tert-butylbenzoic acid (g); V8 of salicylic acid (h); new allomorphs of V4-hydroxybenzoic acid (i), V(-)-borneol (j), V1-naphthol (k), Vquinoline (l) and Vsalicylic acid (m).

  ibuprofen in Figure IV.47, there is a decrease in intensity and the band is slightly shifted to lower frequencies upon drying, suggesting a decrease in bond strength. As presented above, V7 is transformed into a more compact hexagonal structure V7a upon removal of water. As amylose helices get closer, intermolecular H-bonds between the hydroxyl groups would be formed, leading to a shift of the position of the C-O-H deformation bands.

Figure IV. 47 .

 47 Figure IV.47. Effect of drying on the FT-IR spectra of Vibuprofen.

Figure

  Figure IV.48. 13 C CP/MAS NMR spectra in two different regions of V6I of hexadecanoic acid (a), V6II of hexanoic acid (b), V6III of glycerol (c), new allomorph V4-hydroxybenzoic acid (d), new allomorph Vborneol (e), V7 of butanoic acid (f), new allomorph V1-naphthol (g), Vquinoline (h), new allomorph Vsalicylic acid (i), V8 of salicylic acid (j).

Figure

  In particular, V6I, V6II and V6III complexes have a C1 signal in closely equal range 102.3-102.7 ppm, regardless the nature of complexing agents, while the C1-site in V7 and V8 complexes gives rise to resonance at 103.3-103.6 ppm and 103.9-104.3 ppm, respectively. On the other hand, we found no significant correlation between C4 resonances and the helical conformations. V6I, V6II and V7 have C4 signals in the range of 81.

  Figure S.IV.2), which is consistent with slight differences in unit cell parameters and peaks intensity observed in ED and XRD. This suggests that the chemical shifts in V8 complexes are

Figure IV. 49 .

 49 Figure IV.49. Tentative geometrical molecular models projected on the (a,b) plane of the unit cell of V-amylose allomorphs. Symmetrical left-handed helices with the hydroxymethyl group in gg conformation were used. Hydrogen atoms were removed for clarity.

Figure V. 1 .

 1 Figure V.1. TEM images of V6I (a-d) and V6II crystals (e-h) obtained with 1-hexadecanol (a,e), dodecanedioic acid (b,f), octanal (c,g) and stearamide (d,h). Scale bars: 1 µm.

Figure V. 2 .

 2 Figure V.2. TEM images of V6I (a,d), V6II (b,e) and V7II (c,f) crystals obtained with butanoic acid (C4) (a-c) and decanoic acid (C10) (d-f). Scale bars: 1 µm.

Tables V. 3

 3 to V.5 summarize the results obtained with different solvent compositions, fatty acid concentrations and crystallization temperatures. For a given fatty acid, different allomorphs can crystallize alone or concomitantly, depending on the crystallization conditions. Selected examples of coexistences of the allomorphs are illustrated by TEM images (Figure V.3) and XRD diagrams (Figure V.4). The concomitant crystallization has previously

Figure V. 3 .

 3 Figure V.3. Examples of coexisting crystals with different morphologies and allomorphic types prepared with a given fatty acid: a,b) V6I and V6II prepared in water with the saturation of hexanoic acid at 50 °C and tetradecanoic acid at 60 °C, respectively; c,d) V6I and V7II prepared in the presence of 10 vol% DMSO with 50% of butanoic acid at 60 °C and octadecanoic acid at 90 °C, respectively; e,f) V6II and V7II prepared with 5 vol% butanoic acid in water and 3 vol% pentanoic acid at 40 °C in the presence of 2 vol% DMSO, respectively (TEM images).

Figure V. 4 .

 4 Figure V.4. XRD profiles of V-amylose crystallized from aqueous solutions in the presence of a series of linear fatty acids. Unique allomorphs: a) V6I prepared with tetradecanoic acid; b) V6II prepared with hexanoic acid; c) V7II prepared with pentanoic acid. Coexisting allomorphs: d) V6II and V7II prepared with 3 vol% pentanoic acid at 40 °C in the presence of 2 vol% DMSO; e) V6I and V7II prepared in the presence of 2 vol% DMSO with octadecanoic at 90 °C; f) V6I and V6II prepared in water with tetradecanoic acid at 60 °C. The crystals have been equilibrated at 95% RH. For mixtures (d-f), the major and minor allomorphs are indicated.

Figure V. 5 .

 5 Figure V.5. TEM images of V6I (a), V6II (b), V7I (c) and V7II (d) V-amylose crystals prepared with 1,3-butanediol. Scale bars: 1 µm.

  cis-decahydro-1-naphthol, decahydro-2-naphthol, (-)-borneol and R-(+)-camphor) were prepared at different temperature (25-115 °C). The result is presented in Table V.8. trans-Decalin and β-pinene formed V7II at relatively low temperature (≤ 40 °C). (-)-Borneol and R-(+)-camphor yielded V7I at ≤ 75 °C. On the other hand, cis-decahydro-1-naphthol and decahydro-2-naphthol produced both V7I and V7II (Figure V.7), and V7I was preferred at higher temperatures. In addition, the crystallinity of the complexes increased with the crystallization temperature (Figure V.8), which would be due to a slower nucleation and growth at higher temperatures.

Figure V. 6 .

 6 Figure V.6. TEM images of V-amylose complexed with ethyl butyrate (a), ethyl valerate (b), ethyl caproate (c), butyl acetate (d), pentyl acetate (e), diethyl malonate (f), isopropyl myristate (g) and isopentyl acetate (h). Scale bars: 1 µm.

Figure V. 7 .

 7 Figure V.7. TEM images of V7I (a,c) and V7II (b,d) crystals prepared with cis-decahydro-1naphthol (a,b), and decahydro-2-naphthol (c,d). Scale bars: 1 µm.

Figure V. 8 .

 8 Figure V.8. XRD profiles of hydrated V-amylose complexes prepared with decahydro-2naphthol (a) and (-)-borneol (b) at different temperatures.

Figure V. 9 .

 9 Figure V.9. TEM images of V1-naphthol crystals: a) V7III crystals prepared with DP6500 amylose; b) V8II crystals prepared with DP130 amylose.

Figure V. 10 .

 10 Figure V.10. TEM images of Vquinoline crystals: a) V7II crystals prepared with DP6500 amylose, b) V7IV crystals obtained with DP80 amylose, c) V8II crystals obtained with DP6500 amylose.

Figure V. 11 .

 11 Figure V.11. TEM images of Vsalicylic acid crystals: a) V8I prepared with DP6500 amylose, b) V8II crystals prepared with DP86 amylose. Scale bars: 1 µm.

Figure VI. 1

 1 Figure VI.1 shows the optical and low-magnification TEM images of typical VIBU crystals from DP6500 amylose prepared in the presence of saturated ibuprofen at 75 °C. These micrographs shows that the crystals radially grow from a common origin forming of flowerlike aggregates. Higher magnification TEM images of crystals prepared at different crystallization temperatures are presented in Figure VI.2. The crystals are lamellae with a more or less rectangular shape. The size of the lamellae increases with the crystallization temperature.

  Figure VI.1. a) Interference contrast optical micrograph (a) and low-magnification TEM image (b) of aggregates of VIBU crystalline complexes (0.1 wt% DP6500 amylose, 75 °C).

Figure VI. 2 .

 2 Figure VI.2. TEM images of lamellar single crystals of VIBU prepared using DP6500 amylose at 25 °C (a), 40 °C (b), 60 °C (c) and 75 °C (d). Scale bars: 1 µm.

Figure VI. 3 .

 3 Figure VI.3. TEM images of lamellar single crystals of V-amylose complexed with ibuprofen: a,b) DP6500; c,d) DP192; e,f) DP130; g,h) DP60 (0.05 wt%, 75 °C for DP6500 amylose and 60 °C for others). More individual crystals were obtained by repeated recrystallization.

  ). The difference between the measured and calculated thickness supports the model of chain folding of amylose helices in Vamylose crystals.Base-plane electron diffraction patterns were recorded at low temperature from individual frozen-solvated lamellar crystals. Despite some differences in terms of shape and size, the crystals prepared from all amylose fractions yielded similar patterns. A typical diagram recorded on a specimen area of about 1 µm 2 of a single crystal prepared from DP60 amylose is shown in Figure VI.5b. It contains about 150 diffraction spots, up to a resolution of 0.35 nm, distributed along a rectangular network defined by two orthogonal reciprocal base vectors a* and b* very close in length. The stronger spots are located on the 5 th and 6 th rows and the odd reflections on the a* and b* axes are systematically absent. The calibration of about 40 spots allowed calculating the unit cell parameters a = 2.83 ± 0.01 nm and b = 2.96 ± 0.01 nm, in good

Figure VI. 4 .

 4 Figure VI.4. AFM images of lamellar single crystals of DP192 (a) and DP60 (b) amylose complexed with ibuprofen.

Figure

  Figure VI.5. a) TEM image of a lamellar single crystal of DP60 amylose complexed with ibuprofen; b) corresponding base-plane electron diffraction pattern. The pattern is rotated by 90° with respect to the crystal in (a).

Figure VI. 6 .

 6 Figure VI.6. XRD profiles of hydrated VIBU crystals (D6500 amylose) (a), after thorough drying in vacuum at room temperature (b), and rewetting (c).

Figure VI. 7

 7 Figure VI.7 shows an optical micrograph and TEM images of the resulting crystals that exhibit a less defined rectangular shape and a smaller width compared to those prepared in a 0.1 wt% amylose aqueous solution at the same crystallization temperature (Figure VI.2d). This morphological difference can be due to a faster nucleation and growth since the crystallization was carried out in much higher amylose concentration (1 wt%) (Mersmann, 2001). The powder XRD profiles of hydrated VIBU crystals (Figure VI.8a) is well defined and almost identical to that shown in Figure VI.6a for the sample prepared from 0.1 wt% amylose. After freeze-drying, the complexes yielded a typical diffraction pattern of V7a (Figure VI.8b). This diffraction

Figure VI. 7 .

 7 Figure VI.7. Optical (a) and TEM (b,c) images of aggregates of VIBU crystalline complexes prepared in a 1 wt% amylose and 1:9 DMSO:water solution at 75 °C.

Figure VI. 8 .

 8 Figure VI.8. XRD profiles of: a) hydrated VIBU crystals prepared in 1 wt% amylose and 1:9 DMSO:water mixture at 75 °C; b) crystals as (a) after freeze-drying; c) crystals as in (b) after partial dissolution in 0.1 M HCl at pH 1.2; d) crystals in (b) after partial dissolution in phosphate buffer at pH 5.5.

Figure VI. 9 .

 9 Figure VI.9. FT-IR spectra of racemic ibuprofen (a), DP6500 amylose (b), physical mixture of amylose and 10 wt% ibuprofen (c) and VIBU crystalline complex (d). The series on the right focuses on the 1780-1580 cm -1 region corresponding the contribution of carbonyl groups.

Figure

  Figure VI.10. 13 C CP/MAS (a) and SP/MAS (b) NMR spectra of VIBU crystalline complexes prepared from 1 wt% DP6500 amylose in 1:9 DMSO:water solution.

  Figures VI.11a and VI.11b respectively.

Figure VI. 11 .

 11 Figure VI.11. Projection on the (a,b) plane (a-c) and (a,c) plane (b-d) of the two molecular models proposed for Vibuprofen. In model 1 (a-b), IBU is located only inside the helices. There are 2 IBUs per 4 turns of left-handed 7-fold helix. In model 2 (c-d), IBU is located both inside and between the helices. There is 1 intrahelical IBU per 3 turns of helix and another IBU located in the adjacent interstitial pocket.

Figure

  Figure VI.13 shows the dissolution profiles of the IBU raw powder in different media.

Figure

  Figure VI.15. Release of ibuprofen from VIBU complexes in a 2-stage dissolution test: acid stage (0-2 h): 0.1 M HCl; buffer stage (2-8 h): phosphate buffer at pH 5.5 (0.1 M) or pH 6.8 (0.05 M) at 37 °C.

Figure S .

 S Figure S.III.1. Definition of the dihedral bond angles Φ, Ψ and  for a maltose unit.

  the on basis of the hexagonal unit cell: a = b = 1.377 ± 0.002 nm, c = 0.813 ± 0.002 nm. Root-mean-square error (RMSE) = √∑(2𝜃 𝑜𝑏𝑠 -2𝜃 𝑐𝑎𝑙 ) 2 𝑁 𝑟𝑒𝑓𝑙𝑒𝑐𝑡𝑖𝑜𝑛𝑠 ⁄ = 0.012. b data indexed on the basis of the orthorhombic unit cell: a = 2.656 ± 0.002 nm, b = 2.724 ± 0.003 nm, c = 0.790 ± 0.004 nm. RMSE = 0.042. c data indexed on the basis of the orthorhombic unit cell with a = b = 1.906 ± 0.006 nm, c unknown. RMSE = 0.038. d data indexed on the basis of the orthorhombic unit cell with a = 1.550 ± 0.001 nm, b = 2.836 ± 0.001 nm and c = 0.790 ± 0.001 nm. RMSE = 0.014.

a

  data were indexed on basis of the monoclinic unit cell: a = 3.245 ± 0.006 nm, b = 3.246 ± 0.005 nm, c = 0.793 ± 0.001 nm, γ = 63.38 ± 0.14 o . Root-mean-square error (RMSE) = √∑(2𝜃 𝑜𝑏𝑠 -2𝜃 𝑐𝑎𝑙 ) 2 𝑁 𝑟𝑒𝑓𝑙𝑒𝑐𝑡𝑖𝑜𝑛𝑠 ⁄ = 0.014. b data were indexed on basis of the orthorhombic unit cell: a = b = 2.306 ± 0.002 nm, c = 0.789 ± 0.001 nm. RMSE = 0.028.

  Figure S.IV.1. AFM images (tapping mode) of V-amylose crystals: a) V6I of octadecanoic / stearic acid; b) V6II of octadecanoic / stearic acid; c) V7II of 1,3-butanediol; d) V7III of 1-naphthol.

Figure S .

 S Figure S.IV.2. 13 C CP/MAS NMR spectra of: V6I of hexadecanoic acid (a) and 1,6-hexanediol (b); V6II of 1-butanol (c), 1,6-hexanediol (d) and hexanoic acid (e); V6III of glycerol (g); V6IV of 4-hydroxybenzoic acid; V7I of (-)-borneol (h) and (R)-(+)-camphor (i); V7II of ibuprofen (j), 2-propanol (k) and butanoic acid (l); V7III of 1-naphthol (m); V7IV of quinoline (n); V8I of salicylic acid (o); V8II of salicylic acid (p) and quinoline (q).

Figure S.V. 1 .

 1 Figure S.V.1. XRD profiles of V-amylose prepared at different temperatures in the presence of saturation of complexing agent: 1-octanol (a), 1-heptylamine (b), nonanedioic acid (c), octanal (d) and 1,6-hexanediol (e).

Figure S.V. 2 .

 2 Figure S.V.2. XRD profiles of V-amylose prepared by slow cooling to room temperature in a Dewar bottle in the presence of different concentrations of 1,4-butanediol (a) and 1,6-hexanediol (b).

Figure S.V. 3 .

 3 Figure S.V.3. XRD profiles of Vpropanoic acid (C3) complexes prepared at different temperatures in the presence of 40 and 50 vol% of propanoic acid in pure water or in a 1:9 DMSO:water mixture.

Figure

  Figure S.V.4. XRD profiles of Vbutanoic acid (C4) complexes prepared with different concentrations of butanoic acid in pure water or in a 1:9 DMSO:water mixture, at 40 and 60 °C.

Figure S.V. 5 .

 5 Figure S.V.5. XRD profiles of Vpentanoic acid (C5) complexes prepared in pure water and in a 1:9 or 1:49 DMSO:water mixture at different temperatures and concentrations of pentanoic acid.

Figure

  Figure S.V.6. XRD profiles of Vhexanoic acid (C6), Voctanonic acid (C8) and Vicosanoic acid (C20) complexes prepared at different temperatures in pure water and in a 1:9 DMSO:water mixture.

Figure S.V. 7 .

 7 Figure S.V.7. XRD profiles of V1,3-butanediol prepared in the presence of different amounts of 1,3-butanediol at different temperatures.

Figure S.V. 8 .

 8 Figure S.V.8. XRD profiles of V1,3-butanediol prepared in the presence of different amounts of 1,3-butanediol by slow cooling to room temperature.

Figure S.V. 9 .

 9 Figure S.V.9. XRD profiles of V-amylose complexed with ethyl butyrate (EB), ethyl valerate (EV), butyl acetate (BAT), pentyl acetate (PAT), diethyl malonate (DEM) and isopentyl acetate (IPAT) prepared at in different amylose concentrations (wt%) at 25 or 45 °C or cooling down in a Dewar bottle.

Figure

  Figure S.V.10. XRD profiles of V1-naphthol as a function of mixing temperature at which an excess amount of 1-naphthol was added into DP6500 amylose solution. The crystals were formed as cooling down to room temperature.

Figure S.V. 11 .

 11 Figure S.V.11. XRD profiles of Vquinoline as a function of DMSO concentration. The crystallization occurred at 40 °C in 0.1 wt% DP6500 amylose solution with the presence an excess amount of quinoline.

Figure S.V. 12 .

 12 Figure S.V.12. XRD profiles of Vsalicylic acid prepared from different amylose fractions in aqueous solution at different temperatures.

Figure S.V. 13 .

 13 Figure S.V.13. XRD profiles of Vsalicylic acid prepared from different amylose fractions in aqueous solution in the presence of different amounts of DMSO at 25 °C.

Figure S .

 S Figure S.VI.1. XRD profiles of Vibuprofen (a) and V2-propanol (b) crystalline complexes (DP6500 amylose).

Figure S.VI. 2 .

 2 Figure S.VI.2. Axial view of the molecular model of the V2-propanol crystal drawn from the CIF data file provided as supplementary data by Nishiyama et al. (2010). The hydrogen atoms were omitted for clarity.

Table I

 I

	Enzyme	Substrate
	Starch (glycogen) synthase	ADP-glucose (UDP-glucose)
	Isoamylase	Starch
	CGTase	Cyclodextrin
	D-enzyme	Maltodextrin
	Amylosucrase	Sucrose
	Glucan phosphorylase	G-1-P
	Sucrose phosphorylase + glucan phosphorylase	Sucrose
	Cellobiose phosphorylase + glucan phosphorylase	Cellobiose
	I.1.3.

.1. Enzymes that synthesize amylose in vitro and corresponding substrates

[START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF]

.

Solvents and conformation of amylose in solution I.1.3.1. Solvents

  .,

	2006;	Random coil
		in
	water and neutral, aqueous potassium chloride
	Expanded coil	Helix
	in	in
	formamide,	neutral solution + complexing agent,
	DMSO,	alkaline solution + complexing agent,
	and aqueous alkali	and aqueous solution (pH 12) in the presence
		of 0.3 M potassium chloride

Table I

 I

		.2. Known V-amylose allomorphs.
	Allomorph	Crystal system Cell parameters (nm)	Helicity		References
			a	b	c	n p (nm) h (nm)
	V6I (Vh)	hexagonal	1.37 1.37	0.81	6 0.81	0.13	(1)
		orthorhombic 1.37 2.37	0.81	6 0.81	0.13	(2)
	V6II (V1-butanol) orthorhombic 2.65 2.74	0.80	6 0.80	0.13	(3)
	V6III (VDMSO) orthorhombic 1.92 1.92 2.44/0.81 6 0.81	0.14	(4,5)
	V7 (V2-propanol) orthorhombic 2.83 2.95	0.80	7 0.80	0.11	(6)
	V8 (V1-naphthol)	tetragonal	2.32 2.32	0.79	8 0.79	0.10	(7)
	VKBr	tetragonal	1.07 1.07	1.61	4 1.61	0.40	(8)
	VKOH	orthorhombic 0.88 1.23	2.24	6 2.24	0.37	(9)
	n: number of monomers per turn; p: pitch; h: raise per monomer; (1) Brisson et al. (1991);
	(2) Rappenecker and Zugenmaier (1981); (3) Helbert and Chanzy (1994); (4) French and Zobel
	(1967); (5) Winter and Sarko (1974a); (6) Nishiyama et al. (2010); (7) Cardoso et al. (2007);
	(8) Senti and Witnauer (1952); (9)

.14c,d)

	contains
	antiparallel 8-fold left-handed helices packed in a tetragonal unit cell (space group P43212) with
	a = b = 2.32 nm and c = 0.79 nm. 2.25 1-naphthol molecules are located inside the helix while
	two other molecules are located in-between. No water molecule was included in the model. The
	proposed model superimposed well onto high-resolution TEM lattice images, which is considered
	to be the strongest evidence for the 8-fold helical model (Figure I

  a,b DP130 a,c DP28 d DP192 d DP600 d DP6500 e

	DPw	60	80	86	130	28	192	601	6474
	Mw	9738	12816 13788	21078	4500 d 31200 d 97500 d 1048827
	M w M n	1.05	1.07	1.25	1.07	1.06 f	1.01 f	1.04 f	2.58

Table III . 1 .

 III1 Lowest packing energy for different models built with rigid amylose helices.

Table III .

 III 2. Helix position and packing energy of selected models.

	Model name	Helix center position (fractional coordinates) a b	Packing energy (kJ/glucosyl unit)
	L6_0000*	0.00	0.00	-196.4
	L6_0006	0.00	0.06	-177.3
	L6_0817*	0.08	0.17	-205.9
	L6_0833*	0.08	0.33	-199.0
	R6_0000*	0.00	0.00	-205.2
	R6_0006	0.00	0.06	-180.7
	R6_0817*	0.08	0.17	-203.0
	R6_0833*	0.08	0.33	-207.6
	* corresponding to the local minima
	III.2.3.4. Structure refinement

Table III

 III

	.3 and the examples of the L6_0817
	and L6_0006 refined models are presented in Figure III.8c,f. The L6_0006 refined structure

gave the best fit with the experimental data, with the lowest R1-factor of 0.13. Note that the L6_0817 model, which initially had the lowest packing energy in the grid-search (Table

III

.2) did not exhibit the lowest R1 after refinement (Table III.3 and Figure III.9).

Table III .

 III 3. R1-factor of selected models refined against ED data using SHELX. d is the resolution of used reflections; sd is the effective standard deviation for restraints of distances between bonded atoms and bond angles, defined in the DEFS instruction in SHELX. The model with the lowest R1 is in boldface.

			R1-factor
	Model	d > 0.35 nm	d > 0.30 nm	d > 0.24 nm	d > 0.24 nm
		sd = 0.002	sd = 0.002	sd = 0.002	sd = 0.02
	L6_0000	0.60	0.40	0.42	0.20
	L6_0006	0.23	0.28	0.33	0.13
	L6_0817	0.71	0.64	0.60	0.40
	L6_0833	0.68	0.60	0.54	0.43
	R6_0000	0.63	0.61	0.58	0.42
	R6_0006	0.47	0.45	0.42	0.34
	R6_0817	0.78	0.77	0.74	0.33
	R6_0833	0.88	0.80	0.77	0.57

Table III .

 III 5. Hydrogen bonding in the final structure of V1-butanol.

	Bond Atom1 Atom2	Length (Å)	Symmetry operation of atom 1	Symmetry operation of atom 2
			Intrahelical hydrogen bonds
	O61	O12	3.3	x,y,z	x,y,z
	O61	O52	2.7	x,y,z	x,y,z
	O61	O62	2.8	x,y,z	x,y,z
	O63	O14	3.0	x,y,z	x,y,z
	O63	O54	2.3	x,y,z	x,y,z
	O64	O15	3.0	x,y,z	x,y,z
	O64	O55	2.3	x,y,z	x,y,z
	O64	O65	2.3	x,y,z	x,y,z
	O66	O11	2.9	x,y,z	x,y,1+z
	O11	O36	2.9	x,y,z	x,y,-1+z
	O51	O66	2.4	x,y,z	x,y,-1+z
	O31	O62	2.6	x,y,z	x,y,-1+z
	O63	O23	3.3	x,y,z	x,y,1+z
	O64	O34	3.3	x,y,z	x,y,1+z
	O11	O51	2.2	x,y,1+z	x,y,1+z
	O11	O21	3.0	x,y,1+z	x,y,1+z
			Interhelical hydrogen bonds
	O56	O26	3.0	x,y,z	-1/2-x,-y,1/2+z
	O63	O33	3.3	x,y,z	1/2-x,-y,1/2+z
	O34	O31	3.3	x,y,z	-x,-1/2+y,-1/2-z
	O25	O22	3.3	x,y,z	-x,-1/2+y,-1/2-z
		Hydrogen bonds with 1-butanol and water
	O36 O_Bu2	3.2	x,y,z	-1/2+x,1/2-y,-z
	O32	OW1	2.8	x,y,z	x,y,z
	O25	OW1	3.2	x,y,z	-x,-1/2+y,-1/2-z
	O55	OW2	3.1	x,y,z	-x,-1/2+y,1/2-z
	O32	OW3	3.0	x,y,z	x,y,-1+z
	O23	OW3	2.5	x,y,z	x,y,-1+z
	O53	OW4	2.3	x,y,z	x,y,-1+z
	O24	OW4	3.1	x,y,z	1/2-x,-y,-1.5+z
	O_Bu2 OW1	2.3	x,y,z	x,y,z
	O_Bu2 OW2	2.3	x,y,z	x,y,z
	OW1	OW2	2.3	x,y,z	x,y,z
	OW3	OW4	2.5	x,y,z	x,y,-1+z

Table III .

 III 4. Selected geometrical parameters of the six glucosyl residues of the 6-fold helix in the final structure. The glucosidic torsion angles Φ and Ψ are defined by (O5-C1-O1-C4) and (C1-O1-C4-C5) respectively. The bond angle τ is defined by (C1-O1-C4). The torsion angle

	describing the hydroxymethyl conformation  is defined by (O5-C5-O6-C6) which is -60, 60
	and 180° for gg, gt and tg respectively (Annex 1, Figure S.III.1).
	Residue number	Φ (°)	Ψ (°)	τ (°)	 (°)	O1-O4 (nm)
	1	93(1)	-129(0)	117(4)	-154(1)	0.43
	2	91(2)	-113(9)	118(6)	40(8)	0.44
	3	96(3)	-114(3)	119(3)	-142(0)	0.44
	4	100(1)	-143(8)	117(0)	-179(8)	0.44
	5	76(0)	-112(8)	117(3)	26(2)	0.43
	6	103(3)	-103(2)	117(2)	-93(4)	0.44

Table IV .

 IV 1. FT-IR wavenumbers (cm -1 ) of untreated DP6500 amylose, A-and B-amylose and V-amylose complexes.

	Amylose a A-type B-type	V6I b	V6II b	V6III c	V7 d	V8 e	VHBA f	V(-)-borneol f V1-naphthol f Vquinoline f	Vsalicylic acid f Assignment
	3306.3	3306.8 3306.0 3307.0 3306.3 3306.5 3306.5 3305.8 3296.8 3306.5	3307.5	3306.0	3296.5	O-H stretching
	2923.5	2931.5 2929.8 2933.8 2932.3 2929.8 2930.3 2930.8 2927.5 2931.8	2919.5	2926.3	2931.8	C-H stretching
	1455.0	1456.0 1459.0 1455.5 1455.5 1455.0 1458.0 ND	ND	1455.5	1455.5	1452.0	1455.5	CH2 sym def.
	1408.0	1418.0 1428.0 1406.5 1407.0 1407.5 1413.3 -	1411.5 1408.5	1408.5	1407.5	1408.5	C-H def.
	1367.0	-	-	1370.0 1368.5 1368.0 1372.0 1373.0 1371.5 1371.0	1365.0	1372.5	1373.5	C-H def.
	-	-	1358.0 -	-	-	-	-	-	-	-	-	-
	1338.0	1337.5 1340.0 1335.5 1335	1334.5 1336.5 1342.0 1335.5 1343.0	1334.0	1335.0	1339.0	C3-O-H and C6-H def
	1302.5	-	-	1295.0 1294.5 1295.0 1298.3 1296.0 -	1298.8	1303.5	1299.5	1297.3	C-H def of ring hydrogens
	1240.3	1244.8 1246.8 1241.5 1241.5 1238.0 1242.8 ND	ND	1241.5	1242.0	1244.8	ND	C-O-H and C-H def of ring H
	1206.8	1207.3 1204.8 1207.0 1207.3 1206.5 1206.0 ND	ND	1206.0	1207.0	1207.0	ND
	1149.												C-O-H, CH2 def

Table IV .

 IV 2.13 C chemical shifts (ppm) of carbons of glucosyl residues of hydrated V-amylose allomorphs.

	Complex	C1	C4	C2,3,5	C6
	V6I of hexadecanoic acid	102.7	81.3	75.0, 71.8	60.2
	V6I of 1,6-hexanediol	102.6	81.8	74.7, 72.0	62.1
	V6II of 1-butanol	102.4	81.8	74.7, 71.7	61.0
	V6II of 1,6-hexanediol	102.4	81.9	74.8, 71.7	60.8
	V6II of hexanoic acid	102.4	81.5	75.1, 71.7	60.1
	V6III of glycerol	102.3	82.6 a,b 74.3, 72.1	61.8
	New allomorph of 4-hydroxybenzoic acid	102.4 a,c 81.2 a,d 73.8, 73.5, 72.0 61.0 a,e
	New allomorph of (-)-borneol	103.3	82.2 a,f 76.4, 74.1, 72.0 60.4
	New allomorph of (R)-(+)-camphor	103.3	82.2 a,f 74.4, 72.0	60.4
	V7 of ibuprofen	103.3	81.9	74.3, 71.8	60.1
	V7 of isopropanol	103.6	81.9	74.6-74.3, 71.8 60.3
	V7 of butanoic acid	103.4	81.9	74.9-74.1, 71.7 60.1
	New allomorph of 1-naphthol	103.4 a,g 82.8 a,h 76.1, 73.8, 72.2 60.8 a,i
	New allomorph of quinoline	103.4	82.8	72.4	60.7
	New allomorph of salicylic acid	103.9	83.1	73.7, 72.8, 72.0 60.8
	V8 of salicylic acid	103.9	83.4	73.8, 73.1, 72.2 60.5
	V8 of quinoline	104.3	82.9	75.1, 74.0, 72.1 60.3
	a weight-highest values; b individual values: 84.2, 82.6, 80.2 ppm ; c individual values: 102.4,
	101.4, 98.5 ppm, d individual values: 83.7, 81.2, 78.9 ppm; e individual values: 64.3, 61.0, 57.8
	ppm; f individual values: 83.9, 82.2, 79.8 ppm; g individual values: 105.9, 103.4, 98.5 ppm;
	h individual values: 84,4, 82.8, 79.8 ppm; i individual values: 63.4, 60.8, 59.8, 57.8 ppm.

Table IV .

 IV 3. Symmetry, unit cell parameters and interhelical space per helix of V-amylose allomorphs determined from XRD powder data.

	Allomorph	Name	Space group	Crystal system	a (nm)	Unit cell parameters b (nm)	c (nm)	Number of helices per unit cell	Interhelical space per helix (nm 3 ) c
	V6I	V6I	P212121 orthorhombic 1.372 ± 0.006 2.376 ± 0.006 0.809 ± 0.008 P6522 hexagonal 1.372 ± 0.006 1.372 ± 0.006 0.809 ± 0.008	2 1	0.126
	V6II	V6II	P212121 orthorhombic 2.646 ± 0.009 2.705 ± 0.013 0.807 ± 0.009	4	0.254
	V6III	V6III P212121 orthorhombic 1.913 ± 0.006 1.913 ± 0.010 0.814 ± 0.002	2	0.290
	V4-hydroxybenzoic acid a V6IV P212121 orthorhombic 1.550 ± 0.001 2.836 ± 0.001 0.790 ± 0.001	2	0.572
	V(-)-borneol a	V7I	P1	orthorhombic 1.526 ± 0.017 2.643 ± 0.029 0.803 ± 0.008	2	0.200
	V7	V7II	P212121 orthorhombic 2.813 ± 0.013 2.973 ± 0.011 0.797 ± 0.001	4	0.258
	V1-naphthol a	V7III	P1	orthorhombic 1.663 ± 0.001 2.518 ± 0.001 0.856 ± 0.001	2	0.280
	Vquinoline a	V7IV P212121 orthorhombic 2.702 ± 0.001 3.291 ± 0.001 0.786 ± 0.001	4	0.358
	Vsalicylic acid a	V8I	P21	monoclinic b 3.245 ± 0.006 3.246 ± 0.005 0.793 ± 0.001	4	0.232
	V8	V8II	P43212	tetragonal	2.313 ± 0.006 2.313 ± 0.006 0.790 ± 0.001	2	0.485
	a new allomorphs
	b γ = 116.62 ± 0.13°

  Summary of the allomorphs observed for the crystalline complexes of V-amylose with linear fatty acids as a function of the number of carbon atoms in the fatty chain and comparison with previous works.

		Fatty acid			Previous works		This work
	propionic / propanoic acid (C3)		V6I a,b , V7II a,b		V6I, V7II
	butanoic / butyric acid (C4)		V6I b , V7II a,b	V6I, V6II, V7II
	Complexing agent pentanoic / valeric acid (C5) C (%) 25 ethanol 40-50 n. t. hexanoic / caproic acid (C6)	40 V6I	Incubation temperature (°C) V7II a,b V6I, V6II, V7II 50 60 75 Cooling V6I V6I V6I n. t. V6I a , V6II c V6I, V6II, V7II
	1-propanol octanoic / caprylic acid (C8) 20-25 n. t.	V6II		V6II	-	-	n. t.
	" decanoic / capric acid (C10) 30 n. t.	V6I > V6II V6I > V6II	V6I > V6II	-	n. t.
	1-butanol dodecanoic / lauric acid (C12) * n. t.	V6II		V6II	-	-	n. t.
	1-pentanol tetradecanoic / myristic acid (C14) * n. t.	V6II		V6II V6I a,b	V6I > V6II V6I, V6II, V7II -	n. t.
	n-alcohols (8-16 carbons) hexadecanoic / palmitic acid (C16) * n. t. octadecanoic / stearic acid (C18)	V6II		V6II	V6I > V6II	V6I	n. t.
	n-amines (2-4 carbons) icosanoic / arachidic acid (C20)* 10-60 n. t.	-		-	-	-	n. t.
	1-hexylamine	*	n. t.	V6II		V6II	V6I > V6II	-	n. t.
	n-amines (7-12 carbons)	*	n. t.	V6II		V6II > V6I	V6I > V6II	V6I	n. t.
	dicarboxylic acids (2-6 carbons)	*	n. t.	-		-	-	-	n. t.
	dicarboxylic acids (9-12 carbons)	*	n. t.	V6II		V6II > V6I	V6I > V6II	V6I	n. t.
	octanal	*	n. t.	V6II		V6II	V6I > V6II	V6I	n. t.
	stearamide	*	n. t.	V6II		V6II	V6I > V6II	V6I	n. t.
	1,2-ethanediol	30	-	-		-	-	-	n. t.
	"	40	V6I	-		-	-	-	n. t.
	"	50	V6I	V6I		-	-	-	n. t.
	"	60	V6I	V6I		V6I	-	-	n. t.
	"	80	V6I	V6I		V6I	V6I	V6I	n. t.
	1,4-butanediol	10	V6II	-		-	-	-	V6II
	"	15	V6II	-		-	-	-	V6II
	"	20	V6II	-		-	-	-	V6II
	"	25	V6II	V6II		-	-	-	V6II
	"	30	V6II > V6I V6I > V6II	-	-	-	V6I > V6II
	"	35	V6I	V6I		V6I	-	-	V6I
	"	40	V6I	V6I		V6I	V6I	-	V6I
	"	50-60	V6I	V6I		V6I	V6I	V6I	V6I
	1,6-hexanediol	0.5	-	-		n. t.	-	-	-
	"	1	V6II	V6II		-	-	-	V6II
	"	2.5	n. t.	V6II		V6II	-	-	V6II
	"	5.0	n. t.	V6II		V6II	V6II	-	V6II
	"	10.0	n. t.	V6II		V6II	V6II > V6I	-	V6II
	"	15.0	n. t.	V6II		V6II	V6I > V6II	V6I	V6II
	"	20.0	n. t.	V6II		V6II	V6I > V6II	V6I	V6I > V6II

* The complexing agent was used at saturated; n. t.: not tested; -: no precipitate was observed Table V.2. a Takeo et al. (1973), b Helbert (1994), c Biais (2006); * The crystal type of Vicosanoic acid has not previously been reported.

Table V .

 V 3. Allomorphic type of Vfatty acid crystals formed in water at different fatty acid concentrations (Cfatty acid) and temperatures. The amylose solutions and fatty acids were mixed at 90 °C, then kept at a given crystallization temperature, or allowed to slowly cool down to room temperature in a Dewar container. For mixtures, the major and minor allomorphs are indicated.

	Fatty acids	Physical state of the fatty acid at 25 °C	Cfatty acid	40	50	Crystallization temperature (°C) 60 75	90	Slow cooling
	C3	water-miscible liquid	30 vol%	V7II

Table V .

 V 4. Allomorphic type of Vfatty acid crystals formed in the presence of 2 vol% DMSO at different fatty acid concentrations (Cfatty acid) and temperatures. The amylose solutions and fatty acids were mixed at 90 °C, then kept at a given crystallization temperature. For mixtures, the major and minor allomorphs are indicated. Allomorphic type of Vfatty acid crystals formed in the presence of 10 vol% DMSO at different fatty acid concentrations (Cfatty acid) and temperatures. The amylose solutions and fatty acids were mixed at 90 °C, then kept at a given crystallization temperature, or allowed to slowly cool down to room temperature in a Dewar container. For mixtures, the major and minor allomorphs are indicated.

	Table V.5. Fatty acid	Cfatty acid	Fatty acid 40	Cfatty acid 50	Crystallization temperature (°C) Crystallization temperature (°C) 60 75		90	Slow cooling
	C3	30 vol%	-	-	40	-	60	75 -	90	-	-
	"	40-50 vol%	C3 V6I	n. t.	n. t.		n. t.	n. t.	n. t.
			C4	n. t.	n. t.		n. t.	n. t.	n. t.
			C5	2.5 vol%	V7II		V7II	-	-
			"	3 vol%	V7II > V6II		V7II	-	-
			"	4-5 vol%	V6II		V7II	-	-
			C6	*	V6II	V6I > V7II V6I > V7II	-
			C8-C20	*	n. t.		n. t.	V6I > V7II V6I > V7II
		C3: propanoic acid, C4: butanoic acid, C5: pentanoic acid, C6: hexanoic acid, C8: octanoic acid,
		C10: decanoic acid, C12: dodecanoic acid, C14: tetradecanoic acid, C16: hexadecanoic acid,
		C18: octadecanoic acid, C20: icosanoic acid.
		*: the fatty acid was used at saturation
		-: no precipitate was observed
		n. t.: not tested

Table V

 V

	acetate, butyl acetate and pentyl acetate), branched monoesters (isopentyl acetate, isopropyl
	myristate) and a diester (diethyl malonate). The complexes were prepared in aqueous solution
	having different concentrations of native amylose (0.1-0.5%) by incubation at different
	temperature (25-75 °C) or slowly cooling down to room temperature in a Dewar bottle.
	The results are summarized in Table V.7 and TEM images of typical crystals are shown
	in Figure V.6. Apart from propyl acetate, all linear monoesters, isopropyl myristate and diethyl
	malonate could form V6II. Different from the results of Arvisenet et al. (2002), we did not
	observe the formation of V7II with ethyl hexanoate. Besides, the complexes of ethyl butyrate
	were successfully prepared although this molecule was previously reported to have no
	complexing ability
	.6. Allomorphic type of V1,3-butanediol crystals as a function of crystallization
		temperature and concentration of 1,3-butanediol.
	Concentration		Incubation temperature (°C)
	(%vol)	25	40	60	75	Slow cooling
	20	-	-	-	-	-
	25	V6II > V7II	-	-	-	V6II > V7II
	30	V6II > V7II	V7II	-		V7II > V6II
	35	V6II > V7II, V6I	V7II	-		V6I > V7II, V6II
	40	V6I	V7II > V6I	-	-	V6I > V7II
	45	V6I	V6I > V7II	-	-	V6I > V7II
	50	V6I	V6I	V6I, V7I	-	V6I >V7I
	60	V6I, V7I	V6I, V7I	V7I	V7I	V7I > V6I
	V.5. Complexes with esters
	In this study, several esters were selected for investigating the complexing ability with
	amylose including: linear monoesters (ethyl hexanoate, ethyl valerate, ethyl caproate, propyl

Table V .

 V 8. Allomorphic type of the V-amylose complexed with bicyclic compounds as a function of incubation temperature.

	Complexing agent	25	40	Incubation temperature (°C) 50 60 75 100	115
	trans-Decalin	V7II V7II	-	-	-	-	-
	β-Pinene	V7II V7II	-	-	-	-	-
	cis-Decahydro-1-naphthol	V7II V7II V7II V7II V7II V7I, V7II	-
	Decahydro-2-naphthol	V7II V7II V7II V7II V7II V7I, V7II	V7I, V7II

Table V .

 V 11. Allomorphic type of the Vsalicylic acid as a function of degree of polymerization (DP) of amylose, incubation temperature and DMSO concentration (CDMSO).

	Amylose	CDMSO (vol%)	25	Incubation temperature (°C) 40 60	75
	DP86	0	V8II	V8II	-	-
		10	V8II	V8II > V8I	-	-
		15	V8II > V8I	V8I > V8II	-	-
		25	V8II > V8I	V8I > V8II	-	-
	DP130	0	V8II	V8I > V8II	V8I	-
	DP200	0	V8I > V8II	V8I > V8II	V8I	-
		10	V8I > V8II	V8I > V8II	V8I	-
		15	V8I > V8II	V8I	V8I	-
		25	V8I > V8II	V8I	V8I	-
	DP6500	0	V8I > V8II	V8I > V8II	V8I	V8I
		10	V8I > V8II	V8I > V8II	V8I	V8I
		15	V8I > V8II	V8I	V8I	V8I
		25	V8I > V8II	V8I	V8I	V8I

Table V .

 V 12. Allomorphic types of V-amylose complexes with different complexing agents. "-" indicates that no complex was formed.

	1-Propanol (n-propanol)			POL	V6I, V6II
	1-Butanol (n-butanol)				BU	V6II
	1-Pentanol (n-pentanol)			PENO	V6I, V6II
	1-Octanol (n-octanol)				OCTO	V6I, V6II
	1-Dodecanol (n-dodecanol)			DODO	V6I, V6II
	1-Tetradecanol (n-tetradecanol)		TEDO	V6I, V6II
	1-Hexadecanol (n-hexadecanol)		HEDO	V6I, V6II
		Straight-chain unsaturated alcohols
	cis-3-Hexen-1-ol				HENOL	V6I, V6II
		Straight-chain saturated primary diols
	Complexing agent 1,2-Ethanediol (ethylene glycol) 1,3-Propanediol Hexane 1,4-Butanediol Decane 1,5-Pentanediol Hexadecane 1,6-Hexanediol 2-(2-Hydroxyethoxy)ethan-1-ol (diethylene glycol) Alkanes Straight-chain saturated monocarboxylic acid Abbreviation EG PDIOL HAN BDIOL DEAN PEDIOL HEDAN HDIOL DEG Straight-chain secondary alcohols	V6I V6I, V6II V-type V6I, V6II -V6I, V6II -V6I, V6II -V6I
	Propanoic acid (propionic acid) 2-Propanol (isopropanol)		PA IP	V6I, V7II V6I, V6II, V7II
	Butanoic acid (butyric acid) 2-Butanol (sec-butanol)			BA SB	V6I, V6II, V7II V7II
	Pentanoic acid (valeric acid)	VA Straight-chain secondary diols	V6I, V6II, V7II
	Hexanoic acid (caproic acid) 1,2-Propanediol (propylene glycol)		COA PG	V6I, V6II, V7II V6I, V7II
	Octanoic acid (caprylic acid) 1,3-Butanediol			OA BBOL	V6I, V6II, V7II V6I, V6II, V7I, V7II
	Decanoic acid (capric acid)		Triols	CIA	V6I, V6II, V7II
	Dodecanoic acid (lauric acid) 1,2,3-Propanetriol (glycerol)			LA GOL	V6I, V6II, V7II V6I, V6III
	Tetradecanoic acid (myristic acid)	Halogeno-alcohols	MYA	V6I, V6II, V7II
	Hexadecanoic acid (palmitic acid) 11-Bromo-1-undecanol		PMA BUN	V6I, V6II, V7II V6I
	Octadecanoic acid (stearic acid)	Straight-chain esters	SA	V6I, V6II, V7II
	Icosanoic acid (arachidic acid) Ethyl butanoate (ethyl butyrate)		ARA EB	V6I, V6II, V7II V6II
	Straight-chain unsaturated monocarboxylic acids Ethyl pentanoate (ethyl valerate) EV V6II
	Oleic acid Ethyl hexanoate (ethyl caproate)		OLAN EC	V6I, V6II, V7II V6II
	Linoleic acid Propyl acetate				LINA PRAT	V6I, V6II, V7II -
	Butyl acetate	Straight-chain saturated dicarboxylic acids BAT	V6II
	Ethanedioic acid (oxalic acid) Pentyl acetate			OXA PAT	-V6II
	Propanedioic acid (malonic acid) Lysophosphatidylcoline		MAA LYS	-V6I
	Hexanedioic acid (adipic acid)	Alkyl sulfate	ADA	-
	Nonanedioic acid (azelaic acid) Sodium octadecyl sulfate		AZA SODS	V6I, V6II -
	Dodecanedioic acid				DODA	V6I, V6II
		Straight-chain saturated primary alcohols
	Ethanol				ET	V6I

Table VI . 1 .

 VI1 Maximum crystallization temperature Tc of VIBU in solution in the presence of saturated ibuprofen as a function of DPw of amylose (0.1 wt% solutions).

	Fraction	DP60	DP130 DP192 DP600 DP6500
	Tc (°C)	≤ 60	≤ 60	≤ 75	≤ 75	≤ 75
	Table VI.2. Yield of complexation (%) of a 0.1 wt% DP6500 amylose solution as a function
	of incubation temperature and concentration of ibuprofen (Cibuprofen). The results are expressed
	as a mean value ± standard deviation from 3 independent assays.
	Cibuprofen (wt%)			Incubation temperature (°C)
		25	40		60	75
	0.0025	-		-		-	-
	0.0050	37.7 ± 4.5	23.3 ± 1.2		0.0 ± 0.0	0.0 ± 0.0
	0.0100	84.0 ± 3.6	78.7 ± 5.9		22.5 ± 4.9	0.0 ± 0.0
	0.0200	93.3 ± 0.6*	93.7 ± 1.5		90.7 ± 4.0	10.0 ± 1.3
	0.0300	97.0 ± 2.6*	96.7 ± 1.5*	91.3 ± 1.2*	66.3 ± 3.1

Table VI .

 VI 3. Average thickness and roughness of VIBU lamellar crystals measured form height profiles in AFM images. The roughness was calculated over 100 × 100 nm 2 regions. The theoretical thickness has been estimated from the DPw of amylose, assuming a non-folded 7-fold single helix with a pitch of c = 0.80 nm.

	Fraction	DP60	DP130	DP192	DP6500

Table VI .

 VI 4. Solubility of ibuprofen in different dissolution media at 37 °C.

	Medium	pH	Solubility (mg.L -1 )
	0.1 M HCl	1.2	75.28 ± 4.39
	0.1 M phosphate buffer	5.5	427.74 ± 6.60
	0.05 M phosphate buffer	6.8	3071.32 ± 18.65
	0.05 M phosphate buffer	7.2	4609.90 ± 45.84
	Figure VI.13. Partial dissolution of pure ibuprofen in 0.1 M HCl at pH 1.2, and phosphate
	buffers at pH 5.5 (0.1 M), pH 6.8 (0.05 M) and 7.2 (0.05 M) at 37 °C.

Figure VI.14. Release of ibuprofen from VIBU complexes in 0.1 M HCl at pH 1.2, and phosphate buffers at pH 5.5 (0.1 M), pH 6.8 (0.05 M) and 7.2 (0.05 M) at 37 °C.

Table S .

 S III.2. Fractional coordinates and thermal parameters of independent atoms in the V1-butanol unit cell. Uiso is the temperature factor.

	O32_1 H034_1 C1_2	0.18(12) 0.0514 0.0(3)	0.22(19) -0.1354 0.1(4)	-0.3(10) -0.232 0(3)	0.355 0.426 0.355
	H032_1 O15_1 H11_2	0.183 0.01(11) 0.0449	0.2424 0.09(17) 0.1094	-0.2935 0.1(8) 0.3254	0.426 0.355 0.426
	Atom O13_1 C15_1 H12_2	x 0.12(13) 0.05(11) 0.0562	y 0.13(10) 0.12(12) 0.0576	z -0.1(8) 0.2(7) 0.3992	Uiso (Å 2 ) 0.355 0.355 0.426
	O11_1 C13_1 H15_1 H13_2	-0.13(7) 0.17(13) -0.0357 0.0192	0.14(6) 0.12(10) -0.1581 0.0929	-0.5(5) -0.1(7) 0.1888 0.4925	0.355 0.355 0.426 0.426
	C11_1 H13_1 O55_1 C2_2	0.15(10) 0.192 0.06(15) 0.0(4)	0.19(7) 0.1478 0.11(13) 0.1(12)	-0.5(5) -0.0921 0.4(7) 0(2)	0.355 0.426 0.355 0.355
	H11_1 O53_1 C55_1 H12_2	-0.1823 0.2(2) 0.08(12) -0.0263	0.1911 0.11(10) 0.06(12) 0.0324	-0.4545 0.1(7) 0.4(8) 0.3422	0.426 0.355 0.355 0.426
	O51_1 C53_1 H55_1 H22_2	0.13(11) 0.15(16) -0.0546 -0.0403	0.19(16) 0.06(11) -0.0392 0.085	-0.3(5) 0.1(7) 0.3176 0.28	0.355 0.355 0.426 0.426
	C51_1 H53_1 C65_1 C3_2	0.07(11) 0.1165 0.08(15) 0.0(5)	0.19(10) 0.0578 0.0(2) 0.0(10)	-0.3(5) 0.1083 0.6(8) 0(2)	0.355 0.426 0.355 0.355
	H51_1 C63_1 H65A_1 H13_2	-0.0573 0.16(17) -0.079 0.0339	0.1553 0.05(11) -0.0143 0.065	-0.2981 0.3(7) 0.5767 0.0816	0.426 0.355 0.426 0.426
	C61_1 H63A_1 H65B_1 H23_2	0.06(11) 0.1961 -0.104 0.0107	0.20(15) 0.0594 -0.0649 0.0128	-0.1(5) 0.3407 0.6186 0.105	0.355 0.426 0.426 0.426
	H61A_1 H63B_1 O65_1 C4_2	-0.0935 0.1407 0.0(2) 0.0(2)	0.1918 0.0776 -0.1(4) 0.1(7)	-0.0268 0.3713 0.6(7) 0(2)	0.426 0.426 0.355 0.355
	H61B_1 O63_1 H065_1 H14_2	-0.0345 0.1(10) -0.0323 -0.0655	0.1874 0.0(3) -0.0687 0.0375	-0.0471 0.4(9) 0.7294 -0.0078	0.426 0.355 0.426 0.426
	O61_1 H063_1 C45_1 H24_2	-0.1(3) 0.1417 0.13(13) -0.0485	0.25(16) -0.0108 0.06(10) 0.0925	-0.1(8) 0.2854 0.3(7) -0.0077	0.355 0.426 0.355 0.426
	H061_1 C43_1 H45_1 O_Bu1_2	-0.0457 0.17(13) -0.155 0.0(9)	0.2621 0.02(10) -0.076 0.1(15)	-0.0039 0.0(7) 0.3472 0(2)	0.426 0.355 0.426 0.355
	C41_1 H43_1 C35_1 H04_2	0.04(10) 0.2026 0.13(14) 0.0141	0.23(14) 0.0021 0.07(13) 0.0543	-0.4(6) 0.0754 0.1(7) -0.1791	0.355 0.426 0.355 0.426
	H41_1 C33_1 H35_1 C1_3	-0.0478 0.19(12) -0.1061 0.3(2)	0.2574 0.03(10) -0.0487 0.2(2)	-0.3377 -0.2(8) 0.0432 0.0(10)	0.426 0.355 0.426 0.355
	C31_1 H33_1 C25_1 H11_3	0.07(11) 0.1606 0.10(12) 0.3408	0.23(13) 0.0161 0.12(12) 0.1404	-0.6(6) -0.2299 0.1(7) 0.1085	0.355 0.426 0.355 0.426
	H31_1 C23_1 H25_1 H12_3	-0.055 0.19(14) -0.1211 0.3564	0.2006 0.08(11) -0.1469 0.1954	-0.632 -0.2(7) 0.1421 0.1192	0.426 0.355 0.426 0.426
	C21_1 H23_1 O25_1 H13_3	0.12(11) 0.23 -0.1(2) 0.3699	0.23(9) 0.0882 -0.1(2) 0.1648	-0.6(6) -0.1991 -0.1(8) -0.0401	0.355 0.426 0.355 0.426
	H21_1 O23_1 H025_1 C2_3	-0.1323 0.2(3) -0.1177 0.3(2)	0.2597 0.09(15) -0.1412 0.2(3)	-0.4944 -0.4(7) -0.1194 0.0(10)	0.426 0.355 0.426 0.355
	O21_1 H023_1 O35_1 H12_3	-0.1(2) 0.1492 0.17(18) 0.2744	0.2(2) 0.1005 -0.1(3) 0.1558	-0.7(7) -0.3681 0.0(9) -0.0497	0.355 0.426 0.355 0.426
	H021_1 O33_1 H035_1 H22_3	-0.1379 0.2(2) -0.1705 0.3014	0.2618 0.00(19) -0.0863 0.1989	-0.7561 -0.2(12) -0.0643 -0.1428	0.426 0.355 0.426 0.426
	O31_1 H033_1 O16_1 C3_3	0.05(18) 0.2364 0.14(16) 0.3(2)	0.27(19) -0.0199 0.01(11) 0.2(2)	-0.6(7) -0.1496 0.3(9) 0.1(14)	0.355 0.426 0.355 0.355
	H031_1 O14_1 C16_1 H13_3	-0.0507 0.13(15) 0.19(14) 0.2317	0.2697 0.02(12) 0.01(10) 0.2108	-0.7437 0.0(8) 0.4(8) 0.0865	0.426 0.355 0.355 0.426
	O12_1 C14_1 H16_1 H23_3	0.01(10) 0.14(12) -0.2142 0.2815	0.21(16) 0.07(12) -0.0227 0.2222	-0.4(7) 0.1(8) 0.3394 0.1852	0.355 0.355 0.426 0.426
	C12_1 H14_1 O56_1 C4_3	0.04(10) 0.1794 -0.2(2) 0.3(2)	0.25(13) -0.0715 0.02(9) 0.3(3)	-0.3(6) 0.0981 0.5(7) 0(3)	0.355 0.426 0.355 0.355
	H12_1 O54_1 C56_1 H14_3	0.0289 0.12(15) 0.16(11) 0.2941	0.2816 0.06(18) 0.06(8) 0.2915	-0.3445 0.2(7) 0.6(6) 0.0518	0.426 0.355 0.355 0.426
	O52_1 C54_1 H56_1 H24_3	0.04(14) 0.07(14) -0.1249 0.2744	0.24(15) 0.07(13) 0.055 0.2706	-0.1(6) 0.3(7) 0.5507 -0.1189	0.355 0.355 0.426 0.426
	C52_1 H54_1 C66_1 O_Bu2_3	0.06(10) 0.0526 -0.2(2) 0.2(3)	0.20(11) -0.0384 0.07(17) 0.3(3)	-0.1(6) 0.2427 0.8(5) 0.0(11)	0.355 0.426 0.355 0.355
	H52_1 C64_1 H66A_1 H04_3	0.0386 0.06(16) -0.2033 0.206	0.1701 0.08(19) 0.0519 0.2831	-0.1283 0.4(7) 0.784 0.1032	0.426 0.355 0.426 0.426
	C62_1 H64A_1 H66B_1 OW1_4	0.1(2) 0.0908 -0.145 0.2(4)	0.2(2) -0.0934 0.047 0.3(3)	0.1(6) 0.4967 0.8168 -0.2(10)	0.355 0.426 0.426 0.355
	H62A_1 H64B_1 O66_1 HW1_4	0.0482 0.0503 -0.2(3) 0.1(9)	0.1654 -0.0517 0.1(2) 0.3(5)	0.1466 0.5063 0.8(8) 0(3)	0.426 0.426 0.355 0.426
	H62B_1 O64_1 H066_1 HW2_4	0.0833 0.02(15) -0.1947 0.18(19)	0.2115 0.12(19) 0.1281 0.32(18)	0.1561 0.5(8) 0.7748 -0.3(12)	0.426 0.355 0.426 0.426
	O62_1 H064_1 C46_1 OW2_5	0.0(2) -0.0059 -0.18(7) 0.2(4)	0.2(3) -0.1038 0.11(6) 0.4(4)	0.1(8) 0.4576 0.5(4) 0.0(13)	0.355 0.426 0.355 0.355
	H062_1 C44_1 H46_1 HW1_5	0.0195 0.04(11) -0.2055 0.2(5)	0.2562 0.11(14) 0.1208 0.4(19)	0.1433 0.1(7) 0.5132 0(2)	0.426 0.355 0.426 0.426
	C42_1 H44_1 C36_1 HW2_5	0.11(11) 0.0425 0.18(15) 0.2(9)	0.19(10) -0.139 0.10(13) 0.35(16)	-0.1(7) 0.1866 0.3(5) 0.1(18)	0.355 0.426 0.355 0.426
	H42_1 C34_1 H36_1 OW3_6	0.1371 0.06(13) -0.1521 0.2(4)	0.2025 0.10(13) 0.0914 0.1(4)	-0.0664 0.0(7) 0.2231 0(11)	0.426 0.355 0.426 0.355
	C32_1 H34_1 C26_1 HW1_6	0.13(12) 0.0555 0.22(14) 0.2(8)	0.20(13) -0.0725 0.05(14) 0.1(9)	-0.3(7) -0.0828 0.3(8) 1(3)	0.355 0.426 0.355 0.426
	H32_1 C24_1 H26_1 HW2_6	0.1199 0.12(13) -0.2495 0.3(11)	0.176 0.11(12) 0.0543 0.1(11)	-0.3964 0.0(8) 0.2994 0(21)	0.426 0.355 0.426 0.426
	C22_1 H24_1 O26_1 OW4_7	0.10(10) 0.1296 -0.2(3) 0.2(2)	0.25(14) -0.1396 0.0(2) 0.1(3)	-0.4(6) 0.0337 0.1(8) 1.2(9)	0.355 0.426 0.355 0.355
	H22_1 O24_1 H026_1 HW1_7	0.1127 0.1(2) -0.1969 0.2(5)	0.2757 -0.1(3) 0.0171 0.1(6)	-0.3336 -0.2(9) 0.0607 1.1(10)	0.426 0.355 0.426 0.426
	O22_1 H024_1 O36_1 HW2_7	0.1(2) 0.1562 -0.2(3) 0.3(3)	0.3(3) -0.0805 0.13(18) 0.1(4)	-0.6(7) -0.1957 0.2(8) 1(4)	0.355 0.426 0.355 0.426
	H022_1 O34_1 H036_1	0.1286 0.04(19) -0.2297	0.2566 -0.1(2) 0.1468	-0.5823 -0.1(9) 0.2749	0.426 0.355 0.426

Table S .

 S IV.1. Observed and calculated diffraction angles (2θ) and corresponding Miller indices of V6I of myristic acid (MYA), V6II of 1,3-butanediol (BBOL), V6III of glycerol (GOL), and the new allomorph of V4-hydroxybenzoic acid (V6IV (HBA)).

		V6I (MYA) a			V6II (BBOL) b			V6III (GOL) c			V6IV (HBA) d
	h k l	2θobs (°)	2θcal (°)	h k l	2θobs (°)	2θcal (°)	h k l	2θobs (°)	2θcal (°)	h k l	2θobs (°)	2θcal (°)
	0 1 0	7.42	7.41	2 2 0	6.65	6.65	0 2 0	9.30	9.28	0 2 0	6.23	6.23
	1 1 0	12.86	12.86	1 2 0	7.31	7.30	2 2 0	13.07	13.14	1 1 0	6.49	6.50
	1 1 1	16.85	16.87	2 2 0	9.31	9.31	1 3 0	14.65	14.69	1 2 0	8.44	8.45
	0 2 1	18.47	18.45	3 2 0	11.93	11.93	0 4 0	18.62	18.62	1 3 0	10.97	10.96
	1 2 0	19.70	19.69	1 4 0	13.45	13.45	3 3 0	19.80	19.76	2 0 0	11.42	11.42
	0 3 0	22.36	22.36	3 4 0	16.44	16.44	2 4 0	20.85	20.84	2 2 0	13.05	13.02
	0 3 1	24.91	24.93	1 4 1	17.45	17.45				1 3 1	15.70	15.70
	1 3 0	26.95	26.95	5 2 0	17.93	17.92				1 5 0	16.63	16.64
				0 6 0	19.59	19.59				2 4 0	16.93	16.95
				1 6 0	19.90	19.88				3 1 0	17.45	17.45
				2 6 0	20.71	20.71				3 2 0	18.29	18.28
				4 5 0	21.11	21.12				2 5 0	19.47	19.39
				3 6 0	22.03	22.03				3 3 0	19.47	19.58
				7 1 0	23.69	23.67				3 4 0	21.25	21.28
				5 6 0	25.82	25.84				2 6 0	22.03	22.03
				7 4 0	26.89	26.90				4 1 0	23.23	23.17

Table S .

 S IV.2. Observed and calculated diffraction angles (2θ) and corresponding Miller's index of the new allomorph of V(-)-borneol (V7I (BOR)), V7 of butanoic acid (V7II (BA)), the new allomorph of V1-naphthol (V7III (INAP)), and the new allomorph of Vquinoline (V7IV (QN)).

		V7III (INAP) c			V7IV (QN) d
	h k l 2θobs (°) 2θcal (°)	h k l	2θobs (°) 2θcal (°)
	1 1 0	6.37	6.37	1 2 0	6.27	6.29
	0 2 0	7.03	7.02	2 0 0	6.55	6.54
	1 2 0	8.81	8.81	0 4 0	10.76	10.75
	2 0 0	10.64	10.64	3 2 0	11.20	11.20
	V7I (BOR) a 11.21 h k l 2θobs (°) 2θcal (°) 2 1 0 11.20 1 0 1 11.61 11.63 1 1 0 6.68 6.65 1 3 0 11.81 11.81 2 0 0 11.51 11.53 2 2 0 12.75 12.76 0 2 1 12.74 12.79 1 4 0 15.04 15.05 2 0 1 15.92 15.90 3 1 0 16.37 16.37 1 5 0 17.63 17.65 2 4 0 17.68 17.67 3 3 0 20.03 20.03 1 5 0 18.40 18.41 0 6 1 22.91 22.87 2 5 0 20.60 20.62 4 2 0 24.14 24.13 0 0 2 20.76 20.75 3 4 0 21.36 21.35 4 2 0 22.52 22.52 3 5 0 23.87 23.87	1 0 1 2 4 0 h k l 2θobs (°) 2θcal (°) 11.70 11.72 V7II (BA) b 12.59 12.60 1 1 0 4.35 4 0 0 13.12 13.11 4.35 2 0 0 6.35 4 1 0 13.41 13.38 6.34 2 1 0 6.99 3 4 0 14.58 14.58 7.01 2 2 0 8.71 2 3 1 15.34 15.34 8.71 1 3 0 9.52 0 6 0 16.16 16.16 9.52 3 1 0 9.97 1 6 0 16.49 16.49 9.97 2 3 0 10.99 4 4 0 16.98 16.98 10.99 1 4 0 12.39 5 2 0 17.27 17.27 12.39 2 0 1 12.78 0 5 1 17.55 17.57 12.76 4 1 0 13.05 1 5 1 17.95 17.88 13.04 1 5 0 15.32 3 6 0 18.95 18.94 15.31 5 2 0 17.01 5 4 0 19.65 19.66 16.98 3 5 0 17.77 5 2 1 20.63 20.66 17.77 5 3 0 18.27 0 8 0 21.59 21.60 18.27 6 1 0 19.32 4 5 1 21.99 21.98 19.32 6 2 0 20.00 6 4 0 22.52 22.51 20.02 5 5 0 21.88 5 6 0 23.10 23.11 21.90 2 7 0 21.98 7 2 0 23.68 23.68 21.98 4 7 0 24.64 1 3 2 24.28 24.27 24.64 7 4 0 25.38 3 1 2 24.83 24.85 25.39 6 6 0 26.37 26.35 4 8 0 25.34 25.35
				2 9 0	27.90	27.90

Table S .

 S IV.3. Observed and calculated diffraction angles (2θ) and corresponding Miller indices of the two allomorphs of Vsalicylic acid (V8I (SAL) and V8II (SAL)).

		V8I (SAL) a			V8II (SAL) b
	h k l	2θobs (°) 2θcal (°)	h k l	2θobs (°) 2θcal (°)
	0 2 0	6.09	6.09	1 1 0	5.43	5.42
	2 2 0	6.40	6.40	0 2 0	7.68	7.67
	-2 2 0	10.37	10.38	2 2 0	10.88	10.85
	4 2 0	10.93	10.92	0 1 1	11.86	11.86
	4 0 0	12.23	12.20	1 3 0	12.15	12.14
	0 2 1	12.73	12.73	1 1 1	12.45	12.47
	4 4 0	12.81	12.82	0 2 1	13.63	13.60
	-4 2 0	15.92	15.92	2 3 0	13.90	13.84
	2 6 0	16.53	16.52	2 1 1	14.11	14.14
	-1 5 0	16.87	16.87	0 4 0	15.39	15.37
	-3 4 0	18.27	18.28	2 2 1	15.66	15.63
	7 4 0	19.32	19.32	3 3 0	16.31	16.31
	4 8 0	21.85	21.82	1 3 1	16.60	16.55
	5 8 0	22.33	22.34	2 4 0	17.21	17.20
	6 8 0	23.13	23.14	2 3 1	17.84	17.85
	8 0 0	24.52	24.54	4 1 1	19.42	19.45
	8 1 1	25.89	25.91	4 2 1	20.56	20.58
	8 7 1	26.84	26.82	4 4 0	21.78	21.80
				3 5 0	22.54	22.48
				6 0 0	23.13	23.14
				2 6 0	24.46	24.41
				3 5 1	25.18	25.19
				1 6 1	26.04	26.08
				6 2 1	26.93	26.94

Table S .

 S VI.4. Lamellar thickness of V-amylose crystals measured from atomic force microscopy images.

	Allomorph	Complexing agent	Thickness (nm)
	V6I	1,3-butanediol	9.2 ± 0.9
	V6I	octadecanoic acid	9.8 ± 0.6
	V6II	dodecanoic acid*	8.4 ± 1.2 10.5 ± 0.9
	V6II	hexadecanoic acid	7.8 ± 1.1
	V6II	octadecanoic acid	10.2 ± 0.5
	V6II	dodecanedoic acid	9.9 ± 0.7
	V7 a	ibuprofen	7.0 ± 0.5
	V7 a	cis-decahydro-1-naphthol	7.1 ± 1.2
	V7 a	decahydro-2-naphthol	8.6 ± 0.5
	V7 a	1,3-butanediol	8.7 ± 0.6
	V7III b	1-naphthol	10.3 ± 0.6

a corresponding to V7II in the new nomenclature.

Table S .

 S VI.1. Observed and calculated diffraction angles 2θ in XRD patterns recorded on hydrated and dry Vibuprofen complexes, and corresponding Miller indexes.

		Hydrated complexes a		Dry complexes b
	h k l	2θ obs (°)	2θ cal (°)	h k l	2θ (°) obs	2θ (°) cal
	1 1	4.325	4.320	0 1 0	6.93	6.89
	2 0	6.256	6.259	1 1 0	11.95	11.94
	1 2	6.732	6.732	0 1 1	13.07	13.13
	2 1	6.934	6.933	1 1 1	16.46	16.37
	2 2	8.656	8.646	1 2 0	18.27	18.29
	1 3	9.467	9.477	1 3 0	24.99	25.01
	3 1	9.856	9.857
	2 3	10.93	10.923
	3 2	11.110	11.132
	1 4	12.336	12.341
	2 0	12.724	12.724
	4 1	12.894	12.888
	3 4	15.208	15.210
	5 2	16.831	16.795
	3 5	17.685	17.671
	5 3	18.080	18.087
	6 1	19.056	19.090
	6 2	19.785	19.788
	5 5	21.731	21.725
	3 7	23.022	23.017
	4 7	24.485	24.500
	7 4	25.131	25.127

Acknowledgements

thank my supervisors Dr. Jean-Luc Putaux, ProfThanks to Yoshi Nishiyama and Karim Mazeau for teaching me modeling, for their encouragement and scientific support. Thanks to Annabelle Gèze for supervising the in vitro release experiments conducted at Département de Pharmacochimie Moléculaire (DPM) and to Véronique Blanc-Marquis for her technical support. Thanks to Florent Grimaud and Gabrielle Véronèse for the reception at Laboratoire d', and for illuminating discussions. Thanks to Christine Lancelon-Pin, Marie-France Métral, and Pierre Sailler for sample preparation and technical support.

Conclusions and perspectives

Conclusions and perspectives

Twenty four years after the work of William Helbert (PhD in CERMAV in 1994) and 12 years after that of Benoit Biais (PhD in INRA Nantes in 2006), we have carried out an extensive study on the fascinating property of amylose to form crystalline inclusion complexes with a large variety of small molecules. One of our objectives was to propose convincing molecular models of these complexes in order to better understand the interaction of amylose with the guest molecules, and if possible, be able to predict the structure that would result from the crystallization of amylose with a given complexing agent.

Since, so far, and as is generally the case with polymers, it has not be possible to grow single crystals large enough to allow their analysis by X-ray crystallography, our general approach has been to prepare model lamellar crystals from dilute aqueous solutions and characterize their morphology and structure by combining TEM and ED of these single crystals with XRD of hydrated powders.

The first part of this work has thus been devoted to optimizing the crystallization protocols by investigating the impact of several parameters (DP and concentration of amylose, nature of the solvent, crystallization temperature and time) on the morphology and structure of the crystals. The study of the complexes prepared with a homologous series of straight-chain saturated fatty acids (from C3 to C20) has brought an unexpected new result: a given fatty acid could induce the formation of different allomorphs containing 6-and 7-fold helices, and up to 3 allomorphs in some cases. This observation has prompted us to test more homologous series of molecules differing by their chemical structure (diols, esters, amines, ketones, aromatic compounds, etc.).

The second part of our work consisted in characterizing the large number of crystals prepared in various conditions and analyzing the significant amount of data collected using different techniques. Several conclusions could be drawn. Within the range of selected crystallization conditions, among the 121 agents that were tested, 28 did not yield any crystals and 43 induced the formation of 2 to 4 allomorphs (Table V.11).

Ten different allomorphic families of lamellar V-amylose crystals containing 6-, 7-or 8-fold helices were identified, among which 5 corresponded to structures reported by previous authors and 5 were new allomorphs described for the first time. In order to distinguish these allomorphs, we have augmented the previous nomenclature of V-amylose. The 10 allomorphs were named based on the helical conformation of amylose and relative interhelical space of unit Annex 1

Supplementary material

Chapter III