Peaux Sensibles, Peaux Réactives

L Misery

Cosmétologie Cosmétologie des états cutanés frontières

Keywords: Peau sensible, Peau réactive, Neurone, Épiderme, Prurit, Sensitive skin, Reactive skin, Neurone, Epidermis, Pruritus

Les peaux sensibles (ou peaux réactives) se définissent comme un syndrome se manifestant par la survenue de sensations déplaisantes (picotements, brûlures, douleurs, prurits, fourmillements) en réponse à des stimuli qui, normalement, ne devraient pas provoquer de telles sensations. Ces sensations déplaisantes ne peuvent pas être expliquées par des lésions attribuables à une maladie cutanée spécifique. La peau peut apparaître normale ou être accompagnée d'un érythème. Les peaux sensibles peuvent atteindre toute localisation cutanée mais en particulier le visage. Les peaux sensibles sont très fréquentes puisqu'elles concernent environ la moitié de la population, avec une intensité variable. Le diagnostic se fait essentiellement par l'entretien avec le (la) patient(e). La physiopathologie commence à être mieux comprise : il s'agirait d'une hyperréactivité du système nerveux cutané, liée en particulier à l'activation de protéines sensorielles présentes sur les kératinocytes et les terminaisons nerveuses. La prise en charge reste encore assez spéculative.

Introduction

Les termes « peau sensible », « peau réactive », « peau hyperactive », « peau irritable » ou « peau intolérante » sont synonymes. Le terme de peau réactive est le meilleur, car celui de peau sensible entraîne un risque de confusion avec la peau sensibilisée, c'est-à-dire allergique. Toutefois, le terme de peau sensible est de loin le plus fréquemment utilisé. Il a été cité pour la première fois dans la littérature médicale dès 1947 [[1]] puis a été développé dans les années 1970 [[2,3]] avant de devenir un syndrome largement reconnu [[4]] .

Définitions

Les peaux sensibles ont désormais une définition consensuelle internationale, proposée par un groupe d'experts réuni à l'initiative de la société internationale sur le prurit (International Forum for the Study of Itch [IFSI]) [[5]] . Ainsi, les peaux sensibles se définissent comme un syndrome se manifestant par la survenue de sensations déplaisantes (picotements, brûlures, douleurs, prurits, fourmillements) en réponse à des stimuli qui, normalement, ne devraient pas provoquer de telles sensations. Ces sensations déplaisantes ne peuvent pas être expliquées par des lésions attribuables à une maladie cutanée spécifique. La peau peut apparaître normale ou être accompagnée d'un érythème. Les peaux sensibles peuvent atteindre toute localisation cutanée mais en particulier le visage.

Les facteurs déclenchants de ces sensations désagréables sont généralement les cosmétiques et produits de toilette, l'eau, le froid, le chaud ou les variations de température, le vent, la climatisation ou l'air sec, ainsi que les rayons ultraviolets (UV), voire le stress ou les menstruations. Des facteurs très imprécis tels que la pollution sont aussi évoqués par les sujets atteints mais celle-ci ne peut pas être retenue comme étant un facteur déclenchant des peaux sensibles en l'état actuel des connaissances.

La peau irritée est un phénomène différent : une peau irritée présente la même symptomatologie mais en réponse à des facteurs qui sont irritants chez tout le monde (le rasage, le laser ou les détergents par exemple) [[6]] . Quant à la peau sensibilisée ou allergique (Tableau 1), elle relève d'un domaine complètement différent : il s'agit d'une peau qui a été sensibilisée à un allergène précis (parfois plusieurs) et qui est donc le siège d'un eczéma (plus rarement une urticaire de contact ou une dermite de contact aux protéines) au contact de cet allergène. Les peaux sensibles doivent être aussi distinguées des bouffées vasomotrices liées à la rosacée [[7]] .

Diagnostic

Les peaux sensibles étant définies par la survenue de sensations, le diagnostic se fait en interrogeant les personnes atteintes sur les sensations perçues (éventuellement un érythème) et le mode de déclenchement. Les patients consultent rarement pour cette raison-là, probablement parce qu'ils considèrent ces sensations comme étant physiologiques.

Des échelles peuvent aider au diagnostic de peau sensible mais sont plutôt utiles pour mesurer la sévérité et l'évolution. L'échelle sensitive scale permet ainsi en dix questions sur la symptomatologie d'obtenir un score sur 100, avec une moyenne à 44 dans la population générale [[8]] .

Un score spécifique pour le cuir chevelu sensible, défini à partir des réponses au questionnaire 3S sur la symptomatologie spécifique au cuir chevelu, permet d'obtenir un total maximum de 20 [[9]] .

Des tests physiques comme le stinging test (application d'une solution d'acide lactique à 10 % dans un sillon nasogénien comparativement à l'application de sérum physiologique dans l'autre sillon) [[2]] , le test de sensibilité thermique ou le test à la capsaïcine [[10]] sont intéressants eux aussi pour suivre l'évolution, mais pas pour faire le diagnostic car ils ne donnent qu'une appréciation très partielle de la réactivité cutanée tout en étant peu sensibles et peu spécifiques [[11]] . Le stinging test est néanmoins très utilisé et des scores ont été définis : la sensation de picotement est cotée de 0 à 3 et on ajoute les scores obtenus à 3 et 5 min. Le test au laurylsulfate de sodium ne semble pas pertinent car non discriminant entre peau sensible et peau irritée [[12]] .

Le diagnostic différentiel est très simple si l'on s'en tient strictement à la définition [[5]] . Dans la pratique, il est parfois difficile de distinguer ce qui relève d'une peau sensible de ce qui relève d'une pathologie proche, comme les bouffées vasomotrices de la rosacée ou l'alloknésie (perception de multiples sensations comme étant prurigineuses) d'une peau atopique en dehors des poussées. Des sensations désagréables peuvent être associées à de nombreuses dermatoses, en particulier un prurit [[13]] , mais elles peuvent coexister avec une vraie peau sensible. Enfin, lorsque les signes sont très intenses, il faut savoir discuter une neuropathie des petites fibres [[14]] ou une majoration des symptômes dans un contexte anxiodépressif ou même une origine psychogène [[15]] . Évolution L'évolution est chronique avec une grande variabilité au cours du temps. Les peaux sensibles connaissent ainsi des poussées, suivies de rémissions. Ces poussées peuvent être rythmées par le cycle menstruel [[16]] , le stress, où les saisons (plus fréquents en été) [[17]] . La durée d'évolution est donc plus ou moins longue. Les peaux sensibles ont un retentissement sur la qualité de vie, tout au moins dans sa dimension psychologique [[17]] .

Épidémiologie

Les peaux sensibles sont habituellement décrites chez les adultes avec une possible augmentation de fréquence avec le vieillissement [[18]] , mais elles existent aussi probablement chez les enfants.

Les peaux sensibles sont très fréquentes puisqu'elles concerneraient environ la moitié de la population française [[19]] . Les femmes sont plus fréquemment atteintes que les hommes : 60 contre 40 %.

À la connaissance des auteurs, la première étude épidémiologique date de 2001 [[20]] . Cette étude a été réalisée au Royaume-Uni. Environ 50 % des femmes et 40 % des hommes déclaraient avoir une peau sensible. Une étude européenne a montré des variations qui pouvaient être importantes entre huit pays d'Europe [[21]] , avec une fréquence des peaux dites « sensibles » et « très sensibles » allant de 26 % en Belgique à 54 % en Italie.

Aux États-Unis, plusieurs études épidémiologiques utilisant une méthodologie similaire ont été réalisées au cours du temps [[22-25]] et cela semble être en faveur d'une augmentation de la prévalence des peaux sensibles [[25]] , avec une fréquence déclarée augmentant de 50 à 85 % en un peu plus d'une décennie ! Il faut toutefois rester prudent car cette augmentation de prévalence pourrait en fait correspondre simplement à une meilleure connaissance des peaux sensibles par la population.

Des variations importantes ont été observées entre les pays. Au Japon, un peu plus de la moitié des hommes et des femmes (avec des chiffres similaires) sont concernés [[26]] . Au Brésil et en Russie, c'est près de la moitié des femmes et près du quart des hommes [[27]] . Il est difficile de savoir si ces variations sont liées à des facteurs linguistiques ou bien à l'importance de l'image corporelle et de la sensorialité selon les pays ou bien des facteurs liés aux climats (ensoleillement, chaleur, vent) ou bien surtout à l'usage des cosmétiques.

Les variations selon les ethnies ont été discutées [[22,23]] , mais ne sont pas démontrées, ce qui est logique étant donné l'absence de définition scientifique possible du terme d'ethnie. En revanche, il existe clairement des variations selon le phototype avec une fréquence des peaux sensibles d'autant plus importante que le phototype est clair [[23]] . Alors que cela est parfois évoqué, il est désormais bien montré qu'il n'y a pas de différence significative entre les catégories socioprofessionnelles. Il n'y a pas de relation démontrée avec des symptômes psychiques [[17]] ou avec l'état d'hydratation cutané.

Si les peaux sensibles sont essentiellement retrouvées au visage, elles peuvent être présentes en toutes localisations du tégument, en particulier aux mains, au cuir chevelu et au cou [[28]] . Pour ce qui est du cuir chevelu [[18]] , la symptomatologie est un peu différente car l'érythème est rare et le prurit et les picotements sont au premier plan, plutôt que les brûlures et les fourmillements observés au visage. Les facteurs déclenchants sont aussi différents avec un rôle spécifique des shampooings et autres produits capillaires.

Physiopathologie

Les mécanismes physiopathologiques sont débattus mais des recherches récentes permettent de mieux les comprendre. Différentes hypothèses ont été proposées [[11,29-31]] et ne s'excluent pas forcément, mais il apparaît de plus en plus clairement que les peaux sensibles sont liées à une hyperréactivité du système nerveux cutané.

Hypothèse épidermique

La première hypothèse formulée supposait que les peaux sensibles étaient dues à la mise en contact des couches profondes de l'épiderme avec des facteurs avec lesquels elles n'auraient pas dû être en contact du fait d'anomalies de la barrière cutanée, et donc en clair une peau sèche.

Une revue générale récente [[11]] a recensé 11 études comparant la perte en eau transépidermique (TEWL) chez des sujets ayant une peau sensible ou non. En général, il était mis en évidence une perte en eau plus importante chez ceux qui ont une peau sensible, tout au moins en moyenne. Ce même travail a recensé dix études sur l'hydratation cutanée et les résultats sont similaires. Mais ces moyennes sont trompeuses car les patients qui ont une peau sensible peuvent avoir une peau sèche, mixte, grasse ou normale par ailleurs [[21]] . La revue systématique de la littérature [[11]] montre que certaines études mettaient en évidence un niveau de sébum plus important dans les peaux sensibles, mais d'autres un niveau moins important et d'autres un niveau équivalent.

Le pH cutané joue un rôle majeur dans l'efficacité de la barrière épidermique et il était tentant de considérer que des modifications du pH pouvaient conduire à la fois à des anomalies de cette barrière et à des sensations désagréables. Mais, là aussi, les résultats des différentes études sont contradictoires [[11]] .

Hypothèse atopique

Dès les premières études épidémiologiques [[20]] , une proportion importante de personnes présentant un terrain atopique était retrouvée parmi les sujets ayant une peau sensible (de l'ordre d'un tiers). Certains avancent dès lors que l'atopie serait un facteur prédisposant des peaux sensibles [[32]] . Cela peut se comprendre si l'on envisage les peaux sensibles comme étant liées à des anomalies de la barrière cutanée mais aussi parce que les atopiques ont un fonctionnement différent du système nerveux cutané, avec des perceptions parfois altérées comme une alloknésie [[33]] .

A contrario, la grande majorité des sujets ayant une peau sensible ne sont pas atopiques. Et les facteurs déclenchant la sensibilité cutanée, surtout lorsqu'ils sont physiques, ne peuvent pas le faire par des mécanismes immunologiques.

Hypothèse vasculaire

La présence fréquente d'un érythème et la difficulté de séparer peaux sensibles et bouffées vasomotrices de rosacée dans de nombreux cas incitent à penser qu'une hyperréactivité vasculaire pourrait être à l'origine des peaux sensibles. Là aussi, on trouve des résultats contradictoires, certaines études montrant une vasodilatation plus facile chez ceux qui ont une peau sensible, mais d'autres ne trouvant pas de différence et une étude montrant même une vasoconstriction plus importante [[11]] . Quoi qu'il en soit, les symptômes sensitifs sont bien plus fréquents que la rougeur sur les peaux sensibles, qui ne fait d'ailleurs même pas partie de la définition [[5]] .

Hypothèse biochimique

Les facteurs déclenchants sont essentiellement connus par ce que relatent les patients puisque les peaux sensibles ont une symptomatologie essentiellement subjective. Le rôle de facteurs physicochimiques liés aux mouvements de l'air et aux variations de température semble aussi clair que celui de l'exposition à des produits cosmétiques, des produits de toilette ou même à l'eau.

Le déclenchement des peaux sensibles par des facteurs si différents, qui peuvent être physiques, chimiques ou physicochimiques, est surprenant. En réalité, il peut très bien s'expliquer par l'activation ou plutôt l'hyperactivation de protéines présentes à la surface des kératinocytes et des terminaisons nerveuses intraépidermiques, qui ont pour fonction de nous permettre de percevoir de multiples facteurs environnementaux, en particulier de la famille des transient receptor potential (TRP) [[34,35]] mais aussi quelques autres.

Ces protéines ont la particularité de pouvoir être activées aussi bien par des facteurs physiques que chimiques sans que nous soyons capables de faire la différence. Par exemple, le TRP vanilloide 1 (V1) peut être activé aussi bien par la capsaïcine que les protons ou la chaleur alors que le TRP member 8 (M8) peut être aussi bien activé par le froid que par le menthol (Tableau 2).

Dans les peaux sensibles, des protéines sensorielles telles que TRPV1 et acid-sensing ion channel 3 (ASIC3) sont effectivement surexprimées [[36]] .

Hypothèse neurogène

La présence de sensations anormales et éventuellement d'un érythème suggère un rôle du système nerveux et les peaux sensibles apparaissent effectivement comme étant essentiellement liées à une hyperactivation d'un système nerveux cutané altéré.

En réponse au stinging test, qui consiste en l'application d'acide lactique dans le sillon nasogénien, les sujets ayant une peau sensible ont une activation cérébrale dans les deux hémispères, alors que celle-ci est très localisée à une région de l'hémisphère gauche chez les autres, comme le montre une étude en imagerie par résonance magnétique fonctionnelle (IRMf) [[37]] . Les sensations éprouvées dans les peaux sensibles peuvent être assimilées à des douleurs neuropathiques, au moins pour les plus sévères d'entre elles [[38]] .

Une étude physiopathologique (histologique, comparative en double aveugle) a tenté de faire le tour des mécanismes potentiels des peaux sensibles [[39]] . Il n'a pas été retrouvé d'argument en faveur d'une altération de la fonction barrière ou bien d'une inflammation non spécifique ou bien d'anomalie de la résolution de l'inflammation. En revanche, il a été mis en évidence une diminution de la densité intraépidermique en terminaisons nerveuses, ce qui est un élément montrant très clairement que les peaux sensibles sont liées à une altération de ses terminaisons nerveuses, similaires à celles que l'on peut retrouver au cours des neuropathies des petites fibres.

Les peaux sensibles seraient donc l'expression clinique d'une inflammation neurogène. La dégranulation des mastocytes semble aussi être impliquée [[40]] .

Autres hypothèses

Le microbiote cutané pourrait jouer un rôle dans les peaux sensibles. Une étude clinique a montré l'intérêt de probiotiques [[41]] . A contrario, le microbiote cutané ne semble pas modifié au cours des peaux sensibles [[42]] . L'évolution des peaux sensibles est rythmée par les saisons puisqu'elles sont plus fréquentes et intenses en été [[17]] , ce qui suggère un rôle des UV. De même, elle est rythmée par le cycle menstruel [[16]] .

De manière surprenante, une étude en microarray a mis en évidence que les peaux sensibles étaient associées à des anomalies ioniques et des métabolismes des glucides et des lipides mais aussi une dysfonction des fibres musculaires lisses [[43]] . Les mêmes auteurs ont montré plus tard qu'il existait un déficit en adiponectine chez les patients ayant une peau sensible [[36]] . Ces résultats suggèrent la possibilité d'anomalies de la composition des lipides épidermiques et donc du pH, mais cela reste à confirmer.

Synthèse

On peut considérer les peaux sensibles comme une diminution du seuil de tolérance cutanée qui n'est pas liée à des mécanismes allergiques ou immunologiques. Les peaux sensibles sont liées à des anomalies du système nerveux cutané, qui devient hyperréactif, cette hyperréactivité pouvant être modulée par de multiples facteurs (hydratation cutanée, cycle menstruel, facteurs environnementaux, etc.).

Prise en charge

Les cosmétiques et produits de toilette semblent donc bien être les principaux facteurs déclenchants des peaux sensibles et probablement le principal facteur entretenant ce phénomène.

Il faut donc limiter leur usage ou utiliser des produits haute tolérance c'est-à-dire des produits ne contenant que peu ou pas de conservateurs et de tensio-actifs.

Certains cosmétiques contiennent aussi des actifs apaisants qui peuvent être anti-inflammatoires ou inhibiteurs de l'inflammation neurogène et donc peuvent avoir un intérêt, même si des essais comparatifs devraient être réalisés.

De très nombreux produits sont proposés comme dédiés aux peaux sensibles mais il faut savoir se préserver des allégations commerciales faiblement justifiées à ce sujet (et souvent même sans aucun fondement).

Conclusion

Les peaux sensibles sont très fréquentes et correspondent à une réactivité anormale de la peau dont le seuil de tolérance est abaissé. Les mécanismes sont mal connus mais une atteinte des terminaisons nerveuses apparaît de plus en plus clairement comme le facteur principal. Les peaux sensibles seraient donc ainsi liées à une libération excessive de cytokines et de neuromédiateurs, en réponse à une (hyper)activation de protéines sensorielles telles que les TRP. Elles doivent conduire à limiter l'usage des cosmétiques et produits de toilette ou bien à utiliser des produits peu irritants, voire apaisants.

Points essentiels

• Les peaux sensibles (ou peaux réactives) se caractérisent par la survenue de sensations déplaisantes (picotements, brûlures, douleurs, prurits, fourmillements) en réponse à des stimuli qui, normalement, ne devraient pas provoquer de telles sensations. Un érythème peut être présent.

• Les peaux sensibles ne sont pas limitées au visage mais peuvent atteindre toutes les zones du tégument.

• Les peaux sensibles sont très fréquentes puisqu'elles concernent environ la moitié de la population, avec une intensité variable.

• Le diagnostic se fait essentiellement par l'entretien avec le (la) patient(e).

• Il s'agit d'une hyperréactivité du système nerveux cutané.


Bianca C Maniglia
email: bianca.chieregato-maniglia@oniris-nantes.fr

Manoel Dâmaris C Lima

Matta Junior

Patricia Le-Bail

Alain Le-Bail

Pedro E D Augusto

Manoel D Matta

Patricia Le- Bail

Hydrogels based on ozonated cassava starch: Effect of ozone processing and gelatinization conditions on enhancing 3D-printing applications

Keywords: cassava starch, starch modification, ozone, oxidation, 3D printing

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Starches are biodegradable biopolymers obtained naturally from different sources, and widely used in different industries, such as foodstuffs, animal feed, chemicals, petrochemicals, textiles, paper, and in other materials. In the search to increase the application potential of starches for different sectors and discover new functionalities, starch modification is conducted using various methods, including chemical, physical, enzymatic and genetic modifications. Ozone technology is considered an innovative chemical treatment for modifying starch, as it is considered a green technology because ozone is rapidly decomposed into oxygen, without leaving residues in food or in the environment [START_REF] Pandiselvam | Ozone : An Advanced Oxidation Technology for Starch Modification Ozone : An Advanced Oxidation Technology for Starch Modification[END_REF]. Ozone is a very strong oxidizing agent and is an interesting alternative to the traditional oxidation processes used in the starch industry.

Ozone processing c an promote starch modification through the cleavage of the glycosidic bonds of both amylose and amylopectin molecules (by decreasing their sizes and distribution), and it also leads to the replacement of hydroxyl groups by carbonyl and carboxyl groups (by changing their charges and chemical affinity), mainly in the amorphous regions of the granules [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF]. This process can be used safely in both gaseous and aqueous forms [START_REF] Amorim | Effect of ozonation on the sensory characteristics and pasting properties of cassava starch[END_REF].

Ozone treatment has shown good results for starch modification and contributes interesting properties for different applications. [START_REF] Chan | Physicochemical and functional properties of ozone-oxidized starch[END_REF] [START_REF] Chan | Physicochemical and functional properties of ozone-oxidized starch[END_REF] stated that no residues were observed when ozonized starches were introduced into food products. [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF] [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF] treated cocoyam and yam starches with ozone, and [START_REF] Klein | Ozone oxidation of cassava starch in aqueous solution at different pH[END_REF] [START_REF] Klein | Ozone oxidation of cassava starch in aqueous solution at different pH[END_REF] worked with cassava starch, although in systems different from that used in the present work. [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF] [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF] conducted direct reactions of starch with ozone gas, while [START_REF] Klein | Ozone oxidation of cassava starch in aqueous solution at different pH[END_REF] [START_REF] Klein | Ozone oxidation of cassava starch in aqueous solution at different pH[END_REF] showed that the efficiency of ozone oxidation of starch under aqueous solution is dependent on pH. [START_REF] Amorim | Effect of ozonation on the sensory characteristics and pasting properties of cassava starch[END_REF] [3] carried out ozone treatment on cassava starch, without observing changes of color; however, they observed changes of odor. Çatal & İbanoğlu (2014) [START_REF] Çatal | Effect of aqueous ozonation on the pasting, flow and gelatinization properties of wheat starch[END_REF] observed that ozone treatment produced a modified wheat starch with higher cooking stability and lower retrogradation tendencies. These authors also reported different pasting properties, as well as structural changes in starch granules. Matta Junior et al. (2019) [START_REF] Da Matta Junior | Ozone technology as an alternative to fermentative processes to improve the oven-expansion properties of cassava starch[END_REF] conducted a mild ozone treatment on cassava starch, resulting in higher clarity, better oven expansion and a softer texture in the biscuits obtained. [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF] and [START_REF] Castanha | Properties and possible applications of ozone-modified potato starch[END_REF] [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF][START_REF] Castanha | Properties and possible applications of ozone-modified potato starch[END_REF] studied potato starch processed by ozone. They obtained materials with a low peak apparent viscosity and gels stronger than native starch, thus with characteristics interesting for certain applications, such as the 3D printing highlighted in the present proposal.

Among possible applications for starch, 3D printing is gaining attention in the food sector as it can provide new products, such as food structure customization, and customized and digitalized nutrition, thereby broadening the source of food raw materials and shortening the food supply chain [START_REF] Chen | Research on the impact of 3D printing on the international supply chain[END_REF][START_REF] Godoi | 3d printing technologies applied for food design: Status and prospects[END_REF][START_REF] Jia | Investigating the feasibility of supply chain-centric business models in 3D chocolate printing: A simulation study[END_REF]. According to [START_REF] Azam | Study on 3D printing of orange concentrate and material characteristics[END_REF] [START_REF] Azam | Study on 3D printing of orange concentrate and material characteristics[END_REF], starch can improve the rheological properties of food matrixes to achieve higher printability for 3D applications and improve their sensory attributes. The literature comprises many works involving the application of native starches for 3D food printing [START_REF] Azam | Study on 3D printing of orange concentrate and material characteristics[END_REF][START_REF] Chen | Effect of rheological properties of potato, rice and corn starches on their hot-extrusion 3D printing behaviors[END_REF][START_REF] Yang | Physical properties of 3D printed baking[END_REF][START_REF] Yang | LWT -Food Science and Technology Improving 3D printing process of lemon juice gel based on fl uid fl ow numerical simulation[END_REF][START_REF] Liu | Creation of internal structure of mashed potato construct by 3D printing and its textural properties[END_REF]. However, the use of modified starches for this application still needs investigation. Besides its application in the food sector, the 3D printing of starch gels has been evaluated and considered for tissue engineering, as a scaffold [START_REF] Lam | Scaffold development using 3D printing with a starch-based polymer[END_REF] and for cartilage [START_REF] Murphy | The Meniscus in Normal and Osteoarthritic Tissues: Facing the Structure Property Challenges and Current Treatment Trends[END_REF] production. The use of modified starches has not been investigated for 3D printing, though it can expand the range of starch gel applications.

Cassava (Manihot esculenta Crantz), also called tapioca, yucca or manioc, is a perennial shrub which is currently the sixth most important world food crop, providing sustenance for more than 800 million people in tropical and sub-tropical Africa, Asia and Latin America [START_REF] Prochnik | The Cassava Genome: Current Progress, Future Directions[END_REF][START_REF] Tappiban | Recent understanding of starch biosynthesis in cassava for quality improvement: A review[END_REF]. Cassava presents high tolerance to drought and harsh climatic conditions and around-the-year availability, making it a reliable crop for ensuring food security [START_REF] Zhu | Composition, structure, physicochemical properties, and modifications of cassava starch[END_REF]. Cassava starch, the most essential component of cassava, is commonly used as a thickener and stabilizer in food processing to improve the convenience of the process and quality of final products. It is also used as an additive in the textile and papermaking industries [START_REF] Anggraini | Characterization of Cassava Starch Attributes of Different Genotypes[END_REF]. Another advantage of exploiting cassava as a resource is to greatly increase the choice and diversity of staple foods for a large population, especially as a gluten-free food for people with celiac disease [START_REF] Liu | Preparation of starch dough using damaged cassava starch induced by mechanical activation to develop staple foods: Application in crackers[END_REF].

In this context, the aim of this work was to explore the modification of cassava starch by using ozone technology and evaluate the potential of the hydrogels obtained for 3D food printing. To this end, the effect of ozonation was first evaluated in starch structure and properties. Then, the hydrogels obtained from the modified starches were evaluated for their paste and gel properties, including the printability of the gels. The effects of starch concentration, gelatinization temperature and storage period were also evaluated.

Material and methods

Cassava starch and ozone processing

Native cassava starch (Amilogill 1500) was supplied by Cargill Agrícola -Brazil (moisture: 13.2 g/100 g, amylose content: 28.9 g /100 g and amylopectin content: 71.1 g /100 g). All the chemicals were of analytical grade.

The ozone processing of cassava starch was conducted using a method similar to that described by [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF] [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF], shown in Fig. 1. Ozone processing was carried out in a reactor with cassava starch suspended in distilled water (10 g dry starch / 100 g suspension; 1500 mL) under constant stirring using a magnetic stirrer. Ozone was produced in an ozone generator unit (Ozone & Life, O&G Model RM 3.0, São José dos Campos, Brazil) using the coronal-discharge method from industrial oxygen (95% purity). The ozone-rich gas current was directed to a cylindrical glass reactor (2 L capacity, 56 cm high, and 6 cm internal diameter) via a glass tube ending in a porous gas disperser. The gas flow in the reactor was maintained constant at 1.0 L min -1 , and the ozone concentration in the gas current was kept at 43 mg O3 L -1 . The gas flow leaving the reactor was directed to an ozone destructor, which thermally destroys the residual ozone in oxygen. After the ozone treatment, the reactor was supplied with oxygen only for a further 10 min, in order to eliminate the residual ozone. The cassava starch was processed for 15 and 30 min. Native cassava starch, referred to here as "0 min" processing, was also considered in the evaluation.

After processing, the starch suspension was maintained at rest for decanting, and the supernatant liquid was discarded later. The starch was dried in an air circulation oven (MA 035, Marconi, São Paulo, Brazil) at 35 °C until reaching a moisture content of approximately 12%. The dried starch was then macerated, sifted (250 μm) and stored for further analysis in glass containers.

Molecular characteristics of starch: determination of pH, and carbonyl and carboxyl contents

The pH of the starch suspension (10.7 g starch / 100 g suspension) was obtained using a potentiometer (Tecnal, model TEC-5 mode, Piracicaba -Brazil) under constant stirring.

Carbonyl and carboxyl contents were obtained according to the method described by [START_REF] Smith | Production and use of hypochlorite oxidized starches[END_REF] [START_REF] Smith | Production and use of hypochlorite oxidized starches[END_REF] and [START_REF] Chattopadhyay | Optimisation of conditions of synthesis of oxidised starch from corn and amaranth for use in film-forming applications[END_REF] [START_REF] Chattopadhyay | Optimisation of conditions of synthesis of oxidised starch from corn and amaranth for use in film-forming applications[END_REF], respectively, with small modifications.

For carbonyl content determination, 2 g of sample was dispersed in 100 mL of distilled water and heated in a bath with boiling water for 30 min under constant stirring. The sample was cooled to 40 °C, the pH was adjusted to 3.2 using HCl solution (0.1 M), and 15 mL of hydroxylamine chloride solution (25 g of hydroxylamine hydrochloride diluted in 100 mL of NaOH 0.5 M) was added. The samples were incubated at 40 °C for 4 h and then the excess hydroxylamine was titrated with 0.1 M HCl until pH 3.2 using an automatic titrator (Plus Titrino 848, Metrohm, Herisau, Switzerland). A blank was made without the sample but following the same procedures used for the samples. The carbonyl content was calculated as the amount of carbonyl groups in relation to 100 units of glucose (CO/ 100 GU) according to Equation 1: The analyses were done in triplicate.

Starch molecular characteristics: Molecular size distribution

The molecular size distribution profile of the native starch (control) and ozonated starches (15 and 30 min) were determined using a gel permeation chromatography (GPC) system, according to Song & Jane (2000) [START_REF] Song | Characterization of barley starches of waxy, normal, and high amylose varieties[END_REF], with some modifications.

A glass column (diameter: 2.6 cm and high: 70 cm) packed with Sepharose CL-2B gel (Sigma, Sweden) was used. A solution with 0.1 g of starch with 10 mL of Dimethylsulfoxide (DMSO; 90%, Labsynth, Brazil) was prepared. The solution was heated in a bath with boiling water for 1 h and then kept at 25° C for 24 h under constant

stirring. An aliquot of 3 mL of this starch solution was mixed with 10 mL of absolute ethanol and then centrifuged (30 min, 3000 g). The precipitated starch was dissolved in boiling distilled water (9 mL) and heated in a bath with boiling water for 30 min. This starch solution (4 mL) was added in the chromatographic column with an eluent solution (25 mmol•L -1 of NaCl and 1 mmol•L -1 of NaOH), at a rate of 60 mL•h -1 .

Aliquots of 4 mL were collected using a fraction collector (Gilson, model FC203B, Middleton, England) and then analysed by the blue value method [START_REF] Juliano | A simplified assay for milled-rice amylose[END_REF], using a spectrophotometer at a wavelength of 620 nm (Femto, model 600S, São Paulo -Brazil).

Glucose was used as marker to indicate the end of the analysis.

Differential scanning calorimetry (DSC)

The thermal characteristics during starch gelatinization were determined by using a Multi-Cell Differential Scanning Calorimeter (MC-DSC) -(TA Instruments, Lindon, UTAH, USA). Before analysis, the samples were weighed in the ampoules (total mass (starch + water) so that each contained 700 mg, keeping the relation of 10.7

g starch / 100 g). An ampoule with alumina powder was used as reference and three runs for each sample were analyzed. The MC-DSC heating program consisted in going from 30 to 130 °C at a rate of 2 °C/min. The onset temperature (To), the peak temperature (Tp), the conclusion temperature (Tc), and the enthalpy (ΔH) associated with the starch gelatinization interval were calculated with the aid of the Universal Analyzer software (TA Instruments).

Pasting properties

Starch pasting properties were determined using a Rapid Visco Analyzer -RVA- The starch concentration of 10.7 g starch/100 g suspension is a standard concentration conventionally used to determine the pasting properties of the starch under standard conditions of analysis. However, the standard concentration used in the RVA assays is higher than the regular concentration of industrial applications, especially for food products. Therefore, the starch concentrations analyzed (10.7 and 3.6 g g starch/100 g suspension) were used as a tool to compare the behavior of the samples under different conditions, as the standard conditions may not be representative for all industrial applications.

Gel firmness

The hydrogel firmness (strength) was determined in the different starches and conditions of gelatinization, using the gels obtained after the RVA analysis.

Immediately after finishing the RVA analysis, the gels were placed in plastic cups (40 mm diameter × 20 mm height) and stored for different periods (1 and 7 days) in the refrigerator (5 ± 2 °C) before texture evaluation. The cups were kept in a desiccator with water at the bottom to ensure uniform moisture.

The samples were then removed from the refrigerator and analyzed immediately to ensure a constant temperature. Gel firmness was determined by a puncture assay using a texture analyzer TA TX Plus (Stable Micro Systems Ltd., Surrey, UK) with a load cell of 50 kgf (490.3 N). The samples were penetrated until a distance of 4 mm using a cylindrical probe (P/0.5R, 0.5 cm of diameter) at 1 mm•s -1 . The equipment measured the force as a function of penetration depth. Gel firmness was evaluated by the energy required to penetrate the material (calculated by the area below the curve force versus distance of penetration).

Gel structure: Optical microscopy

The gels obtained by the RVA analysis were observed using a light microscope (model L1000, Bioval, Brazil; with a 20 W halogen lamp, the magnification of 40 x) and a microscope digital camera of 5.1 megapixels (ECMOS C-Mount, Sony, Japão).

The material analyzed consisted of the gels obtained immediately after RVA analysis, diluted with distilled water (1:1, v/v) and colored with Lugol´s solution. These samples were placed on a glass slide, covered by a glass cover slip and directly evaluated.

Images were obtained after verifying their representativeness.

Hydrogel application: evaluating printability

The gels stored for 1 and 7 days with different starch concentrations (3.6 and 10.7 g / 100 g suspension) and gelatinization conditions were placed in a plastic syringe with a die diameter of 1.5 mm and extruded over a glass slide for visual evaluation of the gel's consistency and moldability at room temperature. To ensure the reproducibility and veracity of the procedure, manual extrusion using the syringe was carried out ten times (lines with ~ 6 cm), and photographs were taken to identify the most predominant visual aspect of the extruded material. Although simple, this approach allowed evaluating the printability of the hydrogels obtained (Fig. 9), which is the first step toward 3D printing. Based on this approach, several conditions were selected for 3D printing.

The selected gels were processed in a 3D printer Stampante 3D (3DRAG V1.2, Futura Elettronica, Italy). The gels were produced following the same proportion of 10.7 g starch / 100 g. The suspension was stirred and heated until reaching the desired gelatinization temperature, and maintained in this condition for 5 min. The gels obtained were transferred to the printer syringes (60 mL) that were closed using parafilm plastic, and then stored in a refrigerator (5 ± 2 °C) for 7 days. The syringes were then removed from the refrigerator and immediately processed in the 3D printer: a 0.8 mm diameter nozzle was used; the nozzle height was set at 18 mm with a speed of KG, Willich, Germany) and the dimensions of the star shape were 5 cm x 5 cm x 2 mm (Length x Width x Height).

Experimental design and statistical analysis

A completely randomized design was applied with four replicates for each processing condition. To evaluate the differences between the materials analyzed, an analysis of variance (ANOVA) and Tukey's test at a 5% significance level were performed with the software Statistic 13 (StatSoft, USA).

Results and discussion

Ozonated cassava starch characterization

Starch molecular characteristics

According to Oladebeye et al. (2013) [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF], carbonyl and carboxyl are crucial for starch oxidation, as they are responsible for the depolymerization of starch chains and the conversion of hydroxyl to carbonyl and carboxyl. Therefore, considering that ozone is a recognized oxidizing agent, these functional groups were determined to identify the extension of oxidation in cassava starch by ozone processing.

Fig. 2 shows the pH, carbonyl, and carboxyl contents of the cassava native starch ozonated for 15 and 30 min. The pH decreased, and the carbonyl and carboxyl group contents increased as a function of ozonation processing time, confirming the high level of starch oxidation. The reduction of starch pH can be associated with the higher carboxyl content, a functional group acidic nature.

In this work, we produced modified starches with two different oxidation levels.

This strategy was aimed at extending the cassava starch application as the proportion of carbonyl and/or carboxyl groups present in oxidized starches directly influences their physicochemical properties, potentially fitting the products for different industrial uses [START_REF] Vanier | Molecular structure, functionality and applications of oxidized starches: A review[END_REF].

According to [START_REF] Wurzburg | Modified starches-properties and uses[END_REF] [START_REF] Wurzburg | Modified starches-properties and uses[END_REF], two important reactions occur during starch oxidation: (1) the oxidation of hydroxyl groups to carbonyl and carboxyl groups (already mentioned in item 3.1.1), and (2) the de-polymerization of starch molecules by the cleavage of (1 → 4)-α-D and (1 → 6)-α-D glycosidic linkages. In the case of ozone processing, starch de-polymerization is even more substantial and the oxidation of hydroxyl groups to carbonyl and carboxyl occurs simultaneously [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF].

The ozonated cassava starches obtained in this work showed slightly higher contents of carbonyl and carboxyl groups than the potato starch ozonized for 30 min In order to evaluate this potential de-polymerization, the starch molecular size distribution was studied by gel permeation chromatography (Fig. 3). Using this technique, the starch molecules are separated and eluted through a column filled with a gel of known porosity to allow separation based on molecular size: the higher molecular weight fractions are eluted first, since they rarely penetrate the pores, while the lowest molecular weight fractions are retained longer in the column [START_REF] Hizukuri | Starch: analytical aspects[END_REF]. Therefore, as can be seen in Fig. 3, we can observe two peaks: the first consists of molecules of larger size and more ramifications, which can be associated with amylopectins, while the second peak represent molecules of smaller size and a linear structure, which can be associated with amyloses.

The first peak of the ozonated cassava starches decreased, while the second peak increased, according to the increase in ozonation process time. Another observation is Once the ozonation process has changed the molecular structure of the cassava starch, the effects on its properties must be evaluated.

Pasting properties and gel firmness

The RVA curves, firmness, and images of the gel obtained for the native and ozonated starch for 15 and 30 min are shown in Fig. 4. Table 1 shows the pasting properties of native and ozonated cassava starches. We can observe that the ozonation process affected the pasting properties of the cassava starch differently when different processing times were applied. The peak apparent viscosity of the ozonated starch for 30 min decreased significantly when compared with native and ozonated starch for 15 min (Fig. 4C). Castanha et al. (2017) [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF] attributed this result to the partial cleavage of the glycosidic bonds promoted by the ozone treatment, as observed in Fig. 3. Therefore, the decrease in the apparent viscosity might reflect the weakening of starch granules, implying a reduction of shear resistance to the point where granule integrity cannot be maintained [START_REF] Obadi | Effects of ozone treatment on the physicochemical and functional properties of whole grain flour[END_REF]. [START_REF] Chan | Molecular structure, rheological and thermal characteristics of ozone-oxidized starch[END_REF] [34] also found a reduction of the peak apparent viscosity for tapioca starch processed with ozone in the gaseous phase.

The trough, final, and setback apparent viscosities showed similar tendencies to the peak viscosity, decreasing significantly (p < 0.05) as the exposure time to ozone increased. On the other hand, the pasting temperature increased significantly as a function of ozonation process time. Castanha et al. (2017) [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF] associated this behavior with the presence of smaller size molecules in the ozonated starches (molecular depolymerization), which need more energy to gelatinize than large size molecules.

The Relative Breakdown (RBD) (calculated by the ratio between the values of Breakdown (BD) and the Peak Apparent Viscosity (PAV)) is a parameter which can be associated with the facility of starch granule disruption [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF]. The ozonated cassava starches showed higher values for the RBD than the native starch. This can be associated with the fact that the ozone treatment confers a new ability of intermolecular association with the polymers due to the replacement of the hydroxyl group by carbonyl and carboxyl groups (Fig. 2) and a change in molecular size distribution (Fig. 3) [START_REF] Castanha | Potato starch modification using the ozone technology[END_REF][START_REF] Chan | Molecular structure, rheological and thermal characteristics of ozone-oxidized starch[END_REF][START_REF] Zhou | Potato starch oxidation induced by sodium hypochlorite and its effect on functional properties and digestibility[END_REF].

Gel firmness was higher for the cassava starch ozonated for 30 min and lower for the cassava starch ozonated for 15 min, when compared with the native cassava starch (Fig. 4B). This result is very interesting since both stronger and weaker gels can be obtained by changing the process conditions, which can cover different industrial applications. This behavior is once again linked to the changes in both molecular size distribution and chemical affinity, including charges (by oxidation of hydroxyl groups to carbonyl and carboxyl), in molecular interaction and gel formation. Specific molecular size distribution results in different rearrangements, with stronger or weaker interactions, as can be see for the difference between the ozonated samples for 15 and 30 min. Besides having a smaller molecular size than the other treatments, the starches ozonated for 30 min are composed of a higher fraction of molecules with similar sizes (Fig. 3), which may have promoted better pairing to form the network in gel. This behavior can be observed in Fig. 4(C andD) which shows the visual consistency of these gels and the energy required to deform them, respectively, in line with the results obtained.

From these results, we can see that the paste obtained from ozonated starch is less consistent, and the gel obtained from the starch ozonated for 30 min is the strongest, displaying good behavior for a material intended for 3D printing. However, different aspects such as gelatinization temperature and storage time can change the hydrogel's behavior, making it necessary to carry out a further evaluation.

Thermal properties

The DSC curves are shown in Figure 6 and the gelatinization properties of the samples (native and cassava starches modified by ozone) are summarized in Table 2. The To, Tp, and Tf increased as the ozonation processing time increased, which indicates the ozone processing resulted in starch with higher thermal stability. The results obtained in this study suggest that the process of gelatinization, which involves the destruction of starch crystallite and the loss of helical conformation [START_REF] Oladebeye | Functional, thermal and molecular behaviours of ozone-oxidised cocoyam and yam starches[END_REF], was affected by the ozonation. Regarding the peak temperature (Tp), the values and behavior were similar to those found for the pasting temperature obtained in the RVA analysis (Table 1). The enthalpy of gelatinization represents the amount of thermal energy involved in the gelatinization process and can provide an indirect measure of the number of crystalline regions [START_REF] Wootton | Application of Differential Scanning Calorimetry to Starch Gelatinization. III. Effect of Sucrose and Sodium Chloride[END_REF][START_REF] Kurdziel | Changes in the physicochemical properties of barley and oat starches[END_REF] and the structure of double helices of the starch broken during heating [38]. The smallest gelatinization enthalpy value (ΔH) found for the starch ozonated for 30 min clearly corroborated the hypothesis that in addition to the depolymerization of starch chains, some double helices disassociated within the crystalline and non-crystalline regions of the starch, leading to a decrease in crystallinity [START_REF] Wootton | Application of Differential Scanning Calorimetry to Starch Gelatinization. III. Effect of Sucrose and Sodium Chloride[END_REF][START_REF] Kurdziel | Changes in the physicochemical properties of barley and oat starches[END_REF].

Effect of gelatinization temperature, starch concentration and storage time on hydrogel properties

Pasting properties

Fig. 6 shows the RVA curves for the different starch concentrations at gelatinization temperatures of 65, 75, 85, and 95 o C.

We can observe in Fig. 6 that the starch concentration at 3.6% suspension resulted in much lower apparent viscosity (~ 20 x) than when using a 10.7% suspension, as expected. This behavior is related to the smallest interaction between the swollen granules and the continuous phase, as usually occurs at low concentrated suspensions.

As the concentration increases, starch-starch interactions become dominant and a larger number of water molecules are immobilized, resulting in an additional increase of apparent viscosity [START_REF] Bertuzzi | Physicochemical characterization of starch based films[END_REF].

The difference of the apparent viscosity between the modified starches in the lowest starch concentration was not as evident as in the highest concentration. For the starch concentration of 3.6%, the ozonated starches always presented a lower apparent viscosity than the native one, followed by the cassava starch ozonated for 15 min and for 30 min. For the starch concentration of 10.7%, the cassava starch ozonated for 15 min showed results similar to those of the native starch when gelatinized at 85 and 95 o C.

By analyzing the sample's behavior at different gelatinization temperatures, it can be observed that there is an increase in the apparent viscosity when the gelatinization temperature changes from 65 to 75 o C. However, it did not vary significantly between 75 and 95 o C, except for the starch ozonated for 15 min at the higher starch concentration, which showed an increase in apparent viscosity when the temperature changed from 75 to 85 o C. When the starch granules under suspension are heated, the apparent viscosity increases due to the gelatinization process, which occurs through the absorption of a large amount of water, resulting in swelling and increased granule size [START_REF] Liu | Effect of soybean soluble polysaccharide on the pasting, gels, and rheological properties of kudzu and lotus starches[END_REF]. The notable change of viscosity between 65 and 75 o C can be associated with the temperature zone for which the starches present the highest increase in swelling power, though for temperatures above 75 o C there were no significant changes to this property. Fig. 7 shows the optical microscopy images used to understand how the starch granules are changed to a gel during the RVA analysis.

According to Atkin et al. (1998) [START_REF] Ren | Effect of modified tapioca starches on the gelling properties of whey protein isolate[END_REF], starch gelatinization is accompanied by water uptake, swelling, the irreversible disruption of internal molecular order through the dissociation of double helices and the melting of crystallites, the leaching of polysaccharides, the disintegration of swollen granules, and the formation of a paste or gel.

At 65 o C, the microscopies of all the starch gels showed swollen granules for both starch concentrations. At 75 o C, native and cassava starch ozonated for 15 min showed apparently swollen starch granules but cassava starch ozonated for 30 min showed less defined granule shapes (less marked surfaces), indicating a more significant breakdown of these structures.

At the lowest starch concentration at 85 and 95 o C, the cassava starches presented only "ghosts"; "Ghosts" is name given to "empty shell" fragments of starch granules found in the medium [START_REF] Atkin | The events leading to the formation of ghost remnants from the starch granule surface and the contribution of the granule surface to the gelatinization endotherm[END_REF]. At the highest starch concentration, at 85 and 95 o C, native starch still presented apparent swollen starch granules but the ozonated cassava starches presented only "ghosts". The same behavior was observed by Castanha et al. (2019) [START_REF] Castanha | Properties and possible applications of ozone-modified potato starch[END_REF], when comparing native potato starch with ozonated potato starches.

These authors explained that unmodified granules require more energy to disrupt the bonds of the crystalline region, a finding confirmed by the lower RBD value calculated for the native starch when compared with the ozonated starches. This means that native starches show greater resistance to starch granule disruption.

For all the samples, the increasing temperature was positive for the granular rupture and greater amylose leaching, mainly between 65 and 75 o C. As can be seen for the pasting properties, the transition from 65 to 75 o C was more decisive for granule swelling and rupture.

Therefore, we obtained starches with different rheological behaviors that may lead to diverse applications. In the case of 3D food printing, the ideal viscosity of the material should be low enough to permit easy extrusion through a small nozzle and high enough to be cohesive without deformation of the layers deposited previously [START_REF] Lille | Applicability of protein and fiber-rich food materials in extrusion-based 3D printing[END_REF].

Thus, the starch ozonated for 30 min can be a good alternative to form a gel with lower viscosity at the temperatures evaluated.

Gel firmness

This criterion is used to evaluate the strength of the hydrogels produced after RVA. After the starch undergoes gelatinization in the presence of water, the subsequent cooling causes retrogradation, i.e. a process in which the disordered macromolecular components of starch are reorganized (amylose or amylopectin) [START_REF] Wang | Starch Retrogradation: A Comprehensive Review[END_REF]. Thus, gel firmness depends on the reorganization and network of these structures.

Fig. 8 shows the strength of the different hydrogels obtained. Firstly, it is important to compare the order of magnitude of axis y for the starch suspension of 3.6 g/ 100 g and 10.7 g /100 g. By increasing the starch content 3 times, a significant increase (~ 2 until 40 x) in gel strength was reached for all the starches (native and ozonated). The higher starch concentration led to an increase in the number of starch molecules per unit volume, and increased the probability of intermolecular hydrogen bonding, leading to a more compact network structure with increased gel strength [START_REF] Yang | Physical properties of 3D printed baking[END_REF].

The storage period had a small effect on the gel strength, which can be interesting when considering industrial product storage. On the other hand, the effect of gelatinization temperature was considerable (although to different extents as a function of the different treatments).

For the gels produced with the lowest starch concentration (3.6%), the increase in gelatinization temperature had a small effect on gel strength, with the exception of the native starch from 75ºC to 85 ºC, which increased ~30%. On the other hand, by increasing the gelatinization temperature, stronger gels were obtained at 10.7%, in particular in the case of the cassava starch ozonated for 30 min. The strongest gel was obtained with 10.7%, with ozonation for 30 min and gelatinization at 95 ºC. Its strength was ~4 times higher than the stronger gel obtained from the native starch (gelatinization at 65 ºC) and ~10 times the gel obtained from the native starch at the same temperature.

This indicates that a specific molecular size distribution and charges were reached in this treatment to ensure better rearrangement, promoting better pairing to form the network in the gel. It is worth mentioning that this result is remarkable from the industrial standpoint.

Finally, using three variables: starch type, starch concentration and gelatinization temperature, we observed different behaviors of the gel strength obtained, thus extending the possible applications of gels. Their printability was then evaluated.

Evaluating the printability of the gels

Printability, or the potential of a material to be used for printing, requires that the dimensional stability of the material in question prevents it spreading on surfaces, and that it is capable of supporting its own weight during layer-by-layer deposition [START_REF] Godoi | 3d printing technologies applied for food design: Status and prospects[END_REF].

The gels produced with the lowest starch concentration (3.6 %) were not able to form a straight line when extruded and spread on a surface by a syringe. Therefore, the evaluation was conducted with the gels produced with the highest concentration of starch (10.7%). Fig. 9 shows images of the simulated printing with the gels stored for 1 and 7 days at this concentration, considering different ozonation times and gelatinization temperatures.

In Fig. 9, we can see that it was possible to extrude all the starch gels produced in a straight line and with a defined shape. However, it can be noticed that the line produced by the cassava starch ozonated for 15 min resulted in less defined and regular lines, for all the gelatinization temperatures employed. On the other hand, the gels produced with the native starch and starch ozonated for 30 min show a regular structure, especially the ozonated starch. Furthermore, the gels stored for 7 days show a more regular structure than gels stored for 1 day. Indeed, these results agree with the gel strengths obtained (and also demonstrate the important of evaluating gel strength with respect to printability).

At a gelatinization temperature of 65 o C, the gels produced by native cassava starch and cassava starch ozonated for 30 min resulted in good candidates for use in 3D

printing. For process temperatures up to 75 o C, the cassava ozonated starch for 30 min was the best candidate for 3D printing.

To evaluate if the ozone treatment really improves the printability of these hydrogels, 3D stars were printed with selected gels. Fig. 10 shows images of the stars produced based on gels obtained from the control (native) starch and those processed for 30 min with ozone, at gelatinization temperatures of 65 and 95 o C, and a storage period of 7 days in a refrigerator (5 ± 2 °C).

Fig. 10 shows that when the gels were produced using the lowest gelatinization temperature (65 o C), it was possible to print stars with both gels. However, it is notable that the star produced with the ozonated starch showed better resolution (well-defined angles). For the gels produced using the highest gelatinization temperature (95 o C), the native starch did not present good printability and spread over the surface. On the other hand, the hydrogels produced with starch ozonated for 30 min continued to exhibit good printability, resulting in a star with better resolution when compared with the others printed in this work.

Consequently, this work successfully developed ozonated starches for use as ingredients with enhanced properties for 3D printing.

Conclusion

This work demonstrated that ozone treatment is a relevant strategy for modifying cassava starch intended for 3D printing. The effect of ozonation processing was evaluated on the starch biopolymer, paste and gel properties. Then, the obtained hydrogels were evaluated by also considering different starch concentrations, gelatinization temperatures and storage times. The results obtained were correlated, in order to better understand the changes made to the paste, gel strength, structure and printability properties of the ozonated starches, when compared to the native one. This work also revealed that starch concentration was an important parameter for the gelling stage and that the gelatinization temperature was changed for the native and ozonated starches. Ozone processing promoted starch oxidation, reduced starch molecular size (depolymerization), and the starch ozonated for 30 min exhibited a lower peak apparent viscosity. The lowest starch concentrations studied were not capable of forming consistent gels with the requisite characteristics of printability. On the other hand, the highest starch concentration formed stronger and more printable gels. The stabilization of the gels for 7 days was found to be preferable, resulting in a lower propensity for spreading and better consistency thanks to the retrogradation of starch biopolymers. The gels produced by native and starches ozonated for 30 min presented good printability when the gelatinization temperature used was 65 o C, but up to this temperature, starch ozonated for 30 min produced more printable gels. The gels produced by starch ozonated for 15 min did not present good printability. Finally, ozone processing can be an alternative for obtaining starches with new and potentially interesting properties, thereby expanding the potential of using cassava starch in 3D printing.

  volume of NaOH required for the ozonated samples; Vb = the volume of NaOH used in the blank; M = the molarity of NaOH; W = the sample mass in dry basis. To determine the carboxyl content, 2 g of sample was dispersed in 25 mL of HCl solution (0.1 M) in water under stirring (25 o C) for 30 min and centrifuged. The residue was filtrated using a vacuum system with a qualitative filter paper (205 μm), and then washed with 400 mL of distilled water. Next, 300 mL of distilled water was added and the dispersion was heated in a bath with boiling water under continuous stirring for 15 min. While it was still hot and under stirring, the sample was titrated with 0.01 M NaOH until reaching pH 8.3 using an automatic titrator (Plus Titrino 848, Metrohm, Herisau, Switzerland). A blank was made with the non-modified starch but following the same procedures used for the other samples. Carboxyl content was expressed as the amount of carboxyl groups in relation to 100 units of glucose (COOH / 100 GU), volume of HCl used in the blank; Vs = the volume of HCl required for the sample; M = the molarity of HCl; W = the sample mass in dry basis.

4 (

 4 Newport Scientific Pvt. Ltd., Australia, with Thermocline for Windows software, version 3.0). In the second part of this work (characterization of the modified starches obtained), the standard conditions for starch characterization were employed, using a suspension of 10.7 g starch / 100 g (correct to 14% moisture basis) and following the procedure: temperature held at 50 o C for 1 min, then increased to 95 o C (6 o C.min -1 and maintained for 5 min), followed by cooling to 50 o C (6 o C.min -1 ), and finally holding at 50 o C for 2 min. In the third part of this work (evaluation of the hydrogels obtained), the RVA behavior of starch suspensions was characterized using different concentrations (3.6 and 10.7 g / 100 g, correct to 14 % moisture basis) and at different gelatinization temperatures (65, 75, 85, and 95 o C). The procedure consisted in keeping the starch suspension at 50 o C for 1 min, then heating it to the specific gelatinization temperature (at 6 o C.min -1 ), maintaining it at these temperatures for 5 min, followed by cooling to 50 o C (6 o C.min -1 ), and finally maintaining it at 50 o C for 2 min.

  20 mm/s and an extrusion rate of 30 mm/s at 20 o C. The physical model (star) was created using the Repetier Host V2.0.1 and Slic3r software (Hot-World GmbH & Co.

( 0 .

 0 11 and 0.04 per 100 g glucose unit for carbonyl and carboxyl contents, respectively) in the work of Castanha et al. (2017) [2]. The ozone action mechanism on the starch molecule proposed was schematized by Castanha et al. (2017) [2]. Briefly, the starch molecule undergoes two main transformations: (1) the depolymerization of both amylose and amylopectin, resulting in smaller molecules with a different distribution; and (2) oxidation in the hydroxyl groups resulting in carbonyl and carboxyl groups, promoting different interactions between the molecules.

  that the second peak moved to the right (i.e. eluting later) as a function of the increase in ozonation process time. These results indicate the ozone treatment reduced the size of the molecules, attributable to the cleavage of the glycosidic bonds of the ozonated starch molecules[START_REF] Castanha | Potato starch modification using the ozone technology[END_REF].[START_REF] Sandhu | Ozone gas affects physical and chemical properties of wheat (Triticum aestivum L.) starch[END_REF] [START_REF] Sandhu | Ozone gas affects physical and chemical properties of wheat (Triticum aestivum L.) starch[END_REF] also obtained similar results when treating wheat starch with ozone gas.

Fig. 6

 6 Fig.6shows endothermic peaks, indicating the transition from ordered to disordered structures for all the samples. Regarding the gelatinization parameters in Table 2 (To, Tp, Tf and gelatinization enthalpy (ΔH)), significant differences (p < 0.05) between the control and the ozonated starches were found. Matta Junior et al. (2019)[START_REF] Da Matta Junior | Ozone technology as an alternative to fermentative processes to improve the oven-expansion properties of cassava starch[END_REF] observed similar values for certain gelatinization parameters of native cassava starch (Tp = 68.1 o C and ΔH = 13.3 J/g).

Figure 1 .

 1 Figure 1. Schematic representation of the ozone processing system.

Figure 2 .

 2 Figure 2. pH and contents of carbonyl, and carboxyl groups of control (native) and cassava starches ozonated for 15 and 30 min. Carboxyl groups in native starches are considered as negligible and thus zero. The dots are the mean values and the vertical bars are the standard deviations.

Figure 3 .

 3 Figure 3. Molecular size distribution determined by the blue value (BV) during gel permeation chromatography of control (native) and cassava starches ozonated for 15 and 30 min.

Figure 4 .Figure 5 .

 45 Figure 4. (A) RVA curves, (B) Gel firmness, (C) images of the gels obtained, and (D) the energy required in a puncture assay of the control (native) and cassava starches ozonated for 15 and 30 min gels (10.7 g starch / 100 g suspension)

Figure 6 .

 6 Figure 6. RVA curves for the different starch concentrations at the gelatinization temperatures: 65, 75, 85, and 95 o C.

Figure 7 .Figure 8 .Figure 9 .Figure 10 .

 78910 Figure 7. Optical microscopy for the gels produced after the RVA analysis.

Table 1 .

 1 Pasting properties of native and ozonated cassava starches (average ± standard deviation) Peak Apparent Viscosity (PAV), Through Apparent Viscosity (TAV), Relative Breakdown (RBD), Final Apparent Viscosity (FAV), Setback (SB) and Pasting Temperature (PT). a -c: different small caps in the same column indicate significant difference among treatments, as revealed by Tukey's test, p < 0.05.

	Processing Time (min)	PAV (mPa.s)	TAV (mPa.s)	RBD (mPa.s)	FAV (mPa.s)	SB (mPa.s)	PT (ºC)
	0	4308.50 ± 61.39 a	1264.25 ± 19.07 a	70.54 ± 0.96 c	2111.50 ± 71.71 a 847.25 ± 56.96 a	65.99 ± 0.79 c
	15	4314.60 ± 106.45 a	90.40 ± 14.12 b	97.89 ± 0.90 a	164.80 ± 21.86 b	74.60 ± 10.85 b	70.14 ± 0.26 b
	30	3414.60 ± 167.05 b	40.40 ± 1.52 c	99.03 ± 1.07 b	64.60 ± 2.88 c	24.20 ± 1.92 c	71.47 ± 0.37 a

Table 2 .

 2 Gelatinization properties of native and ozonated cassava starches (average ± standard deviation) -c: different small caps in the same column indicate significant difference among treatments, as revealed by Tukey's test, p < 0.05. To = onset temperature, Tp = peak temperature, Tf = final temperature and ΔH = enthalpy

	Processing time (min)	To ( o C)	Tp ( o C)	Tf ( o C)	ΔH (J/g)
	0	55.30±0.40 c	65.77±0.08 c	81.14±0.56 b	12.61±0.22 a
	15	56.41±0.46 b	67.98±0.26 b	81.57±0.30 b	12.22±0.32 a
	30	59.36±0.62 a	69.06±0.26 a	84.26±0.79 a	11.03±0.29 b

a

Acknowledgments

The authors are grateful to the Région Pays de la Loire / RFI "FOOD 4 TOMORROW" for funding the Post-doctoral fellowship "STARCH-3D" of B. Maniglia, to the São Paulo Research Foundation (FAPESP, Brazil) for funding project n° 2016/18052-5, and to the National Council for Scientific and Technological Development (CNPq, Brazil) for funding the productivity grant of P.E.D. Augusto (306557/2017-7). This study was funded in part by the "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES)" -Finance Code 001, through the D.C. Lima Ph.D. scholarship.


familles multiplexes où la transmission se fait manifestement sur un mode autosomique dominant et un cas sporadique ont été analysés par une technique assez classique de séquençage d'exome, après élimination de la présence de mutations des gènes déjà incriminés dans cette affection. De façon un peu inattendue deux mutations faux-sens Les analyses en cryomicroscopie électroniques indiquent que les 2 substitutions identifiées affectent probablement directement l'activité de régulation des efflux de cations par le canal transmembranaire TRPM4. En effet, les études électrophysiologiques montrent une hyperactivité importante chez les mutants, comme en témoignent une activité de base prononcée, une sensibilité accrue au Ca+ intracellulaire et un potentiel de membrane de repos élevé, ce qui indique qu'il s'agit de mutations avec gain de fonction ce qui rend compte de la transmission autosomique dominante. Des études in vitro ont enfin démontré une prolifération accrue des kératinocytes surexprimant l'un ou l'autre des mutants, à mettre en parallèle d'une augmentation de l'expression des marqueurs de prolifération et de différenciation des kératinocytes dans les tissus cutanés affectés. Il semble donc bien que TRPM4 soit un acteur important dans la pathogenèse des canalopathies cutanées et une cible potentiellement intéressante dans le traitement des troubles de la différenciation avec hyperkératose. Xu Z, Lee BH, Vu S, Hu L, Lee M et al.Gain-of Function Mutations in TRPM4 Activation Gate Cause Progressive Symmetric Erythrokeratodermia. J Invest Dermatol. 2019;139:1089-1097

	Chronique génomique
	ERYTHROKERATODERMIE PROGRESSIVE ET SYMETRIQUE :
	MUTATIONS ACTIVATRICES DE TRPM4
	PROGRESSIVE SYMMETRIC ERYTHROKERATODERMIA:
	ACTIVATING MUTATIONS OF TRPM4
	O Dereure
	Adresse postale : Département de dermatologie et Unité INSERM U1058, hôpital Saint-Eloi,
	Université Montpellier I, 80 avenue Augustin-Fliche, 34295 Montpellier cedex 5, France Référence :
	1. Wang H,
	Cliniquement, l'érythrokératodermie peut être
	divisé en deux groupes principaux, la forme dite variable et la forme progressive et symétrique
	(EKPS) malgré l'existence de fréquents chevauchements phénotypiques entre ces deux entités.
	(c.3099C> G et c.3119T> C) de le protéine TRPM4 ont été identifiées, codant pour les protéines
	mutantes p.Ile1033Met et p.Ile1040Thr et toutes deux situées dans le domaine
	transmembranaire S6 de la protéine (1). TRPM4 est un canal ionique voltage et calcium-
	dépendant qui régule les mouvements transmembranaires de cations monovalents sous
	l'influence de divers stimuli chimiques et physiques. De nombreux canaux similaires de
	différents sous-types sont présents dans l'épiderme et jouent un rôle essentiel dans
	l'homéostasie fonctionnelle de la peau.

Adresse e-mail : o-dereure@chu-montpellier.fr On désigne sous le terme d'érythrokératodermie un groupe de troubles de la kératinisation caractérisé par des plaques érythémateuses et hyperkeratosiques, habituellement bien délimitées, souvent transitoires ou migratoires. L'EKPS se caractérise par un début précoce, une progression lente, des plaques cutanées érythémateuses symétriques, principalement sur les faces d'extension des extrémités avec ou sans kératodermie palmoplantaire. Des mutations de plusieurs gènes (LOR, GJB4, GJA1, KDSR, ELOVL4 et KRT83 codant respectivement pour la loricrine, la connexine 30.3, la connexine 43, la 3-cétodihydrosphingosine réductase, une protéine probablement impliquée dans l'élongation de longues chaines d'acides gras et la kératine 83 respectivement) ont été liées à l'EKPS. Afin d'avancer dans la connaissance de cette affection probablement assez hétérogène, des patients chinois issus de deux