WO2005019272A1 - Method of producing totally-desacetylated chitosan with a high molecular weight - Google Patents

Method of producing totally-desacetylated chitosan with a high molecular weight Download PDF

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Publication number
WO2005019272A1
WO2005019272A1 PCT/FR2004/001982 FR2004001982W WO2005019272A1 WO 2005019272 A1 WO2005019272 A1 WO 2005019272A1 FR 2004001982 W FR2004001982 W FR 2004001982W WO 2005019272 A1 WO2005019272 A1 WO 2005019272A1
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chitin
chitosan
reactor
deacetylation
cooking
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PCT/FR2004/001982
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French (fr)
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Guillaume Lamarque
Marion Cretenet
Christophe Viton
Alain Domard
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Universite Lyon 1 Claude Bernard
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Publication of WO2005019272A1 publication Critical patent/WO2005019272A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof

Definitions

  • the present invention relates to the field of biopolymers, more particularly to that of chitosans; it relates to a heterogeneous deacetylation process for chitin and / or chitosan, based on a thermo-mechanical-chemical treatment, in order to obtain chitosan of high molecular weight which is totally deacetylated.
  • Chitin is, with cellulose, the most widespread polysaccharide in nature. Chitin is a copolymer consisting of D-glucosamine and N-acetyl D-glucosamine repeating units linked to ⁇ , (l-> 4).
  • chitin is extracted from exoskeletons of arthropods such as lobster, crab, shrimp (in this case it is ⁇ chitin), or from the endoskeleton of cephalopods such as squid (in this case it is chitin ⁇ ).
  • Chitin semi-crystalline, is made up of a network of organized fibers. Chitin mainly exists in two allotropic forms:
  • chitin ⁇ is not soluble in aqueous solvents and swells only very slightly in an aqueous medium; ⁇ chitin, the polysaccharide chains of which are all mutually parallel; the hydrogen bridges are less numerous, which gives the chitin ⁇ a better reactivity and hydrophilicity: it thus exhibits the capacity to swell strongly in water.
  • Chitosan a deacetylated derivative of chitin, is a linear copolymer of D-glucosamine and N-acetyl D-glucosamine linked ⁇ , (l-> 4). It is obtained by partial deacetylation of chitin and has the particularity, by definition, of being soluble in dilute acids, this in contrast to chitin. This property is acquired, during a heterogeneous deacetylation, for an AD of less than 40%. Chitosan therefore has a much greater number of free amino groups than chitin. In the crystalline state, chitosan also has only one allotropic variety.
  • Chitosan is used on an industrial scale in various fields: waste treatment, food processing, cosmetology, medicine, biotechnology. Thanks to its biodegradability, bioresorbability, biocompatibility and non-toxicity properties, it is widely used in many pharmaceutical applications: as an excipient in conventional pharmaceutical devices; as a controlled drug delivery system; as active ingredient: cholesterol-lowering, bacteriostatic, wound healing.
  • the effectiveness of chitosans is a function of their molecular weight and their degree of acetylation, being more particularly due to the presence of reactive amino groups.
  • the chitosan used in the pharmaceutical industry must be rigorously characterized in terms of: quality, intrinsic properties
  • the molecular weight and the degree of deacetylation of chitosan can influence a good number of factors such as its solubility in aqueous medium, its swelling capacity and its rate of enzymatic degradation. These parameters influence not only the physico-chemical properties of chitosan, but also: its biodegradability, its immunological activity and its mechanical or rheological properties. In the case of many applications in pharmacy and cosmetology, a deacetylation rate close to 100% is necessary.
  • the advantage of obtaining a totally deacetylated chitosan also relates to other properties of chitosan, in particular to its electrostatic properties, which are the basis of numerous interactions.
  • the use of chitosan fully deacetylated allows, among other things, to prepare homogeneous series of chitosan, with the same molecular distribution; in this case, the chitosan will be reacetylated to different degrees of acetylation, rigorously characterized, this being particularly useful and important in pharmaceutical applications but also in many other applications.
  • the detection threshold of the method for measuring the degree of deacetylation is close to 1% for IR spectroscopy, while it is less than 0.1% in the case of proton NMR spectroscopy.
  • the estimation of the average molecular weight of chitosan depends on the measurement method used.
  • the average molecular weight of a polymer can be measured by one of the following methods: - steric exclusion chromatography; - viscosimetry; - laser light scattering photometry using a multi-angle laser detector.
  • High molecular weight chitosan has new mechanical and rheological properties: high viscosity, improved capacities to form films, wires or physical hydrogels, useful in particular for applications of chitosan in medicine and biotechnology.
  • the deacetylation of chitin can be carried out using enzymes called chitin deacetylases, with good yields.
  • this method is limited to the laboratory scale due to the high cost of chitin deacetylases and their low productivity. Therefore, the transformation of chitin into chitosan by chemical deacetylation is essential, thanks to its lower cost and easier implementation.
  • the chemical deacetylation of chitin and / or chitosan takes place during a reaction in basic medium (for example, with concentrated sodium hydroxide), the consequence of which is the breaking of the bond between the acetyl group and the amino group linked to the atom C-2.
  • the conditions usually used sodium hydroxide solutions at 35-60% (by weight), temperatures between 80 and 140 ° C, treatment time ranging from thirty minutes to several days, inert atmosphere or presence of components which fix oxygen, although allowing high degrees of deacetylation to be obtained, however, lead to a significant alteration of the macromolecular structure of chitosan.
  • the invention relates to a process for at least partial heterogeneous deacetylation of chitin and / or chitosan, characterized in that it comprises one or more stages of cooking, of a suspension of chitin and / or chitosan, in a solution of concentrated soda, at a temperature less than or equal to 100 ° C., for a duration preferably between 20 and 30 min, said cooking being carried out in a reactor maintained under reduced pressure and in the absence of dioxygen, each cooking step being preceded by a succession of at least six freeze-thaw cycles, each of these cycles comprising the following steps: a) immersing said reactor in a refrigerating medium until the reaction medium is completely frozen; - b) degassing said reactor, so as to obtain inside a pressure below atmospheric pressure; - c) gradually heating said reactor until thawing of said suspension is complete.
  • the process of the invention also comprises the following stages: a stage of neutralization of the sodium hydroxide from the reaction medium until a pH of approximately 8.5 is obtained; - a washing step with demineralized water consisting of several washes interspersed with centrifugations; - a freeze-drying step after freezing.
  • the present invention relates to a heterogeneous deacetylation process for chitin and / or chitosan, under milder conditions than those known in the prior art: lower cooking temperatures, shorter cooking times, reduced numbers of successive cooking compared to a conventional process under an inert atmosphere and at ambient pressure (for equivalent temperatures and reaction times).
  • the process for deacetylation of chitin and / or chitosan in accordance with the invention consists of one or more stages of cooking a suspension of chitin in a concentrated sodium hydroxide solution, each stage being preceded by a succession of gel cycles - thawing of said suspension.
  • the different stages of the process will be analyzed in detail below.
  • Gel-deoel cycles A suspension of finely divided chitin and / or chitosan dispersed in a 50% by weight sodium hydroxide solution, constituting the reaction medium, is placed in a reactor. This reaction medium is therefore heterogeneous.
  • Each freeze-thaw cycle comprises the following stages: the closed reactor is immersed in a refrigerating medium, for example liquid nitrogen, until said reaction medium is completely frozen; - the pressure inside the reactor is reduced by means of a pump which evacuates the air present inside the reactor; the final pressure inside the reactor is lower than atmospheric pressure and will remain so until the end of the reaction; - The reaction medium is gradually heated by immersion of said reactor in water at room temperature until it is completely thawed.
  • the applicant has found that, under these conditions, the crystal lattice of chitin is greatly modified. This phenomenon is all the more marked the greater the number of cycles. A minimum of six freeze-thaw cycles is necessary and sufficient to deconstruct the crystal lattice of chitin.
  • This amorphous material thus becomes more sensitive to the action of soda, during the cooking step following the last freeze-thaw cycle.
  • Cooking The reaction medium is cooked in stages, each stage or cooking reaction being preceded by a succession of freeze-thaw cycles. (at least six), as described above.
  • the cooking reaction takes place at a temperature less than or equal to 100 ° C, preferably between 80 and 100 ° C.
  • the duration of a cooking reaction is preferably between 20 and 30 min. Without opening it, the reactor is placed in an oil bath heated to the desired cooking temperature.
  • the cooking reaction takes place at reduced pressure and at a low oxygen concentration. This is particularly important because, under these cooking conditions, the oxygen intervenes in the reaction mechanism, which then results in hydrolysis and degradation of the polymer.
  • the reactor is stirred.
  • ⁇ chitin After the first cooking reaction, a recrystallized chitin is obtained which has a crystal structure different from that of ⁇ chitin and close to the crystal structure of ⁇ chitin.
  • This chitin deacetylation technique therefore makes it possible to erase the differences in reactivity which exist between ⁇ chitin and ⁇ chitin with respect to deacetylation by greatly improving the efficiency of the reaction with regard to ⁇ chitin.
  • This is very advantageous, given the more interesting properties of ⁇ chitin, in particular its capacity to swell in water, useful in galenical applications and for medical devices based on chitin.
  • the totally deacetylated chitosan obtained according to this process is not completely amorphous, because it partially recrystallizes at the end of the cooking and during its isolation.
  • the reactor is immersed in liquid nitrogen, in order to lower the temperature of the reaction medium to room temperature, then it is opened.
  • the process for deacetylating chitin and / or chitosan of the invention comprises, in addition to the steps described above, additional steps of neutralization, washing and lyophilization.
  • Neutralization The final product obtained by the deacetylation process described is subjected to a neutralization step aimed at reducing the concentration of sodium hydroxide in the reaction medium after it has cooled down at the end of the reaction. This is done using a low concentration hydrochloric acid solution (0.5 M).
  • the final pH at the end of this washing step must be close to 8.5 to avoid solubilization of the sample.
  • Lava ⁇ es A series of washes follows, which aims to eliminate the salts formed (sodium acetate released during deacetylation, and sodium chloride produced during neutralization). These washes are carried out using distilled water containing ammonia, this in order to raise the pH of the washing water to 8.5. Each wash lasts a minimum of 30 minutes and is followed by centrifugation to recover the deacetylated product. Lyophilization The final product present in a solution of pH 8.5 is subjected to lyophilization. This gives totally deacetylated chitosan. The deacetylation process of chitin and / or chitosan according to the invention can be stopped after one or more cooking steps.
  • chitin ⁇ a product is obtained at the end of the first cooking reaction, the structure of which resembles that of chitin ⁇ . Subjected to the stages of neutralization, washing and lyophilization, this product is in the form of a sponge which swells in water (whereas the raw material, chitin ⁇ , does not swell in water). This therefore makes it possible to transform the chitin ⁇ , which lends itself less to pharmaceutical or biotechnology applications, into a much more open and easily destructurable structure resembling in this the chitin ⁇ , which has more interesting properties, in particular the capacity to swell in water.
  • the cooking reactions are continued according to the process described. Five cooking steps are necessary to completely deacetylate the chitin ⁇ or the chitin ⁇ . In this case, the lyophilization step will be carried out at the end of the process, making it possible to obtain a totally deacetylated chitosan.
  • the deacetylation technique of chitin and / or chitosan of the invention makes it possible to obtain a chitosan with a very high degree of deacetylation, practically equal to 100%, as measured by 1 H NMR (when the sample can be dissolved in D 2 0), according to the technique described, for example, by Hirai, A, Odani, H. and Nakajima, A. in "Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy" Polym. Bull. 1991, 26, 87-94.
  • the detection threshold of this technique is in fact lower than that of the IR method, for example, which makes the result obtained in the context of the present invention more reliable.
  • the pressure inside the reactor thus becomes lower at atmospheric pressure and of the order of 0.01 millibar and it will remain so until the end of the reaction.
  • the reactor is then immersed in water at room temperature until total thaw.
  • the reactor is immersed in liquid nitrogen as described above.

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Abstract

The invention relates to a method for the at least partial desacetylation of chitin and/or chitosan. The inventive method comprises one or more steps consisting in boiling a suspension of chitin and/or chitosan in a concentrated soda solution at a temperature of less than or equal to 100 °C for a period of preferably between 20 and 30 min, said boiling being performed in a reduced pressure reactor in the absence of dioxygen. Moreover, each boiling step is preceded by a series of at least six freeze/thaw cycles, each of said cycles comprising the following steps consisting in: a) submerging the reactor in a cooling medium until the reaction medium is fully frozen; b) degassing the reactor such as to produce therein a pressure that is lower than atmospheric pressure; and c) gradually heating the reactor until the suspension has completely thawed.

Description

PROCEDE DE PRODUCTION DE CHITOSANE DE HAUT POIDS MOLECULAIRE TOTALEMENT DESACETYLE La présente invention se rapporte au domaine des biopolymères, plus particulièrement à celui des chitosanes ; elle porte sur un procédé de desacetylation hétérogène de la chitine et/ou du chitosane, basé sur un traitement thermo- mécano-chimique, afin d'obtenir du chitosane de haut poids moléculaire totalement désacétylé. La chitine est, avec la cellulose, le polysaccharide le plus répandu dans la nature. La chitine est un copolymère constitué d'unités de répétition D- glucosamine et N-acétyl D-glucosamine liées β,(l->4). Elle présente un degré d'acétylation (DA, qui représente le pourcentage en nombre, ou fraction molaire, des résidus N-acétyl glucosamine dans le polymère), généralement supérieur à 80%, ce qui lui confère la propriété d'être complètement insoluble en milieu aqueux. Industriellement, la chitine est extraite d'exosquelettes d'arthropodes tels que le homard, le crabe, la crevette (dans ce cas, il s'agit de la chitine α), ou de l'endosquelette de céphalopodes tels que le calamar (dans ce cas il s'agit de la chitine β). La chitine, semi-cristalline, est constituée d'un réseau de fibres organisées. La chitine existe principalement sous deux formes allotropiques : The present invention relates to the field of biopolymers, more particularly to that of chitosans; it relates to a heterogeneous deacetylation process for chitin and / or chitosan, based on a thermo-mechanical-chemical treatment, in order to obtain chitosan of high molecular weight which is totally deacetylated. Chitin is, with cellulose, the most widespread polysaccharide in nature. Chitin is a copolymer consisting of D-glucosamine and N-acetyl D-glucosamine repeating units linked to β, (l-> 4). It has a degree of acetylation (DA, which represents the percentage by number, or molar fraction, of the N-acetyl glucosamine residues in the polymer), generally greater than 80%, which gives it the property of being completely insoluble in aqueous medium. Industrially, chitin is extracted from exoskeletons of arthropods such as lobster, crab, shrimp (in this case it is α chitin), or from the endoskeleton of cephalopods such as squid (in this case it is chitin β). Chitin, semi-crystalline, is made up of a network of organized fibers. Chitin mainly exists in two allotropic forms:
- la chitine α, dont les chaînes polysaccharidiques sont disposées de façon antiparallèle entre plans de chaîne successifs, ce qui donne naissance à de nombreux ponts hydrogène, et, par conséquent, à une rigidité importante et une faible réactivité de la chitine avis à vis de la desacetylation ; la chitine α n'est pas soluble dans les solvants aqueux et ne gonfle que très faiblement en milieu aqueux ; la chitine β, dont les chaînes polysaccharidiques sont toutes parallèles entre elles ; les ponts hydrogène y sont moins nombreux, ce qui confère à la chitine β une meilleure réactivité et hydrophilie : elle présente ainsi la capacité de gonfler fortement dans l'eau. Le chitosane, dérivé désacétylé de la chitine, est un copolymère linéaire de D-glucosamine et N-acétyl D-glucosamine liés β,(l->4). Il est obtenu par desacetylation partielle de la chitine et présente la particularité, par définition, d'être soluble dans les acides dilués, ceci contrairement à la chitine. Cette propriété est acquise, lors d'une desacetylation hétérogène, pour un DA inférieur à 40 %. Le chitosane possède donc un nombre beaucoup plus important de groupes aminé libres que la chitine. A l'état cristallin, le chitosane ne possède, de plus, qu'une seule variété allotropique. Le chitosane est utilisé à l'échelle industrielle dans des domaines variés : traitement des déchets, transformation des aliments, cosmétologie, médecine, biotechnologie. Grâce à ses propriétés de biodégradabilité, biorésorbabilité, biocompatibilité et de non toxicité, il est utilisé largement dans de nombreuses applications pharmaceutiques : comme excipient dans des dispositifs pharmaceutiques conventionnels ; comme système de libération contrôlée de médicaments ; comme principe actif : hypocholestérolémiant, bactériostatique, cicatrisant des plaies. L'efficacité des chitosanes est fonction de leur poids moléculaire et de leur degré d'acétylation, étant due plus particulièrement à la présence de groupes aminé réactifs. Le chitosane utilisé dans l'industrie pharmaceutique doit être caractérisé d'une manière rigoureuse en termes de : qualité, propriétés intrinsèques- α chitin, whose polysaccharide chains are arranged in an antiparallel manner between successive chain planes, which gives rise to numerous hydrogen bridges, and, consequently, to significant rigidity and low reactivity of chitin deacetylation; chitin α is not soluble in aqueous solvents and swells only very slightly in an aqueous medium; β chitin, the polysaccharide chains of which are all mutually parallel; the hydrogen bridges are less numerous, which gives the chitin β a better reactivity and hydrophilicity: it thus exhibits the capacity to swell strongly in water. Chitosan, a deacetylated derivative of chitin, is a linear copolymer of D-glucosamine and N-acetyl D-glucosamine linked β, (l-> 4). It is obtained by partial deacetylation of chitin and has the particularity, by definition, of being soluble in dilute acids, this in contrast to chitin. This property is acquired, during a heterogeneous deacetylation, for an AD of less than 40%. Chitosan therefore has a much greater number of free amino groups than chitin. In the crystalline state, chitosan also has only one allotropic variety. Chitosan is used on an industrial scale in various fields: waste treatment, food processing, cosmetology, medicine, biotechnology. Thanks to its biodegradability, bioresorbability, biocompatibility and non-toxicity properties, it is widely used in many pharmaceutical applications: as an excipient in conventional pharmaceutical devices; as a controlled drug delivery system; as active ingredient: cholesterol-lowering, bacteriostatic, wound healing. The effectiveness of chitosans is a function of their molecular weight and their degree of acetylation, being more particularly due to the presence of reactive amino groups. The chitosan used in the pharmaceutical industry must be rigorously characterized in terms of: quality, intrinsic properties
(pureté, poids moléculaire, degré de desacetylation), forme physique. Le poids moléculaire et le degré de desacetylation du chitosane pourront influencer un bon nombre de facteurs tels que sa solubilité en milieu aqueux, sa capacité de gonflement et sa vitesse de dégradation enzymatique. Ces paramètres influencent non seulement les propriétés physico-chimiques du chitosane, mais aussi : sa biodégradabilité, son activité immunologique et ses propriétés mécaniques ou rhéologiques. Dans le cas de nombreuses applications en pharmacie et en cosmétologie, un taux de desacetylation voisin de 100% est nécessaire. L'intérêt d'obtenir un chitosane totalement désacétylé se rapporte également à d'autres propriétés du chitosane, notamment à ses propriétés électrostatiques, qui sont à la base de nombreuses interactions. Aussi, l'utilisation du chitosane totalement désacétylé permet entre autre de préparer des séries homogènes de chitosane, de même distribution moléculaire ; dans ce cas, le chitosane sera réacétylé à des degrés d'acétylation différents, rigoureusement caractérisés, ceci étant particulièrement utile et important dans les applications pharmaceutiques mais aussi dans de nombreuses autres applications. Il existe plusieurs méthodes pour déterminer le degré de desacetylation du chitosane : - la spectroscopie de résonance magnétique nucléaire RMN ; - la titration potentiométrique linéaire (titration volumétrique) ; - la spectrophotométrie UV (ultra-violet) à partir de la dérivée première ; - la spectroscopie IR (infra-rouge) à transformée de Fourrier ; - le dichroïsme circulaire ; - l'analyse élémentaire ; - la chromatographie en phase gazeuse. La précision des valeurs obtenues par ces différentes méthodes est, cependant, très variable (comme rapporté par plusieurs auteurs, par exemple T. A. Khan et al. dans J. Pharm. Pharmaceut Se/. 5(3) :205-212, 2002). A titre d'exemple, le seuil de détection de la méthode de mesure du degré de desacetylation est proche de 1% pour la spectroscopie IR, alors qu'il est inférieur à 0,1% dans le cas de la spectroscopie RMN du proton. De même, l'estimation du poids moléculaire moyen du chitosane dépend de la méthode de mesure utilisée. Le poids moléculaire moyen d'un polymère peut être mesuré par une des méthodes suivantes : - la chromatographie d'exclusion stérique; - la viscosimétrie ; - la photométrie de diffusion de la lumière laser à l'aide d'un détecteur laser mlultiangles. Le chitosane de haut poids moléculaire présente des propriétés mécaniques et rhéologiques nouvelles : viscosité élevée, capacités améliorées de former des films, des fils ou des hydrogels physiques, utiles notamment pour les applications du chitosane en médecine et dans les biotechnologies. La desacetylation de la chitine peut être réalisée à l'aide d'enzymes appelés chitine-désacétylases, avec de bons rendements. Cependant, cette méthode est limitée à l'échelle du laboratoire en raison du coût élevé des chitines-désacétylases et de leur faible productivité. De ce fait, la transformation de la chitine en chitosane par desacetylation chimique s'impose, grâce à son coût plus réduit et une mise en œuvre plus simple. La desacetylation chimique de la chitine et/ou du chitosane a lieu au cours d'une réaction en milieu basique (par exemple, avec de la soude concentrée), dont la conséquence est la rupture de la liaison entre le groupe acétyl et le groupe aminé lié à l'atome C-2. Une grande variété de techniques de desacetylation chimique a été testée, afin d'obtenir un degré élevé de desacetylation, avec une dégradation du polymère réduite au minimum. En effet, des conditions expérimentales trop agressives peuvent entraîner une forte dépolymérisation, au cours de laquelle la liaison glycosidique β,(l->4) est rompue. Ces techniques font varier le nombre de traitements, la température du traitement, la durée du traitement, les étapes de lavage et séchage intercalées entre les étapes de traitement, l'utilisation de solvants organiques miscibles à l'eau comme milieu réactionnel, etc. Les conditions habituellement utilisées : solutions de soude à 35-60% (en poids), températures comprises entre 80 et 140°C, temps de traitement allant de trente minutes à plusieurs jours, atmosphère inerte ou présence de composants qui fixent l'oxygène, bien que permettant d'obtenir des degrés de desacetylation élevés, conduisent cependant à une altération importante de la structure macromoléculaire du chitosane. Le demandeur a constaté que, de manière surprenante, il était possible d'obtenir du chitosane présentant un degré de desacetylation pratiquement de 100%, comme mesuré par RMN ^ et un poids moléculaire très élevé (supérieur à 4,5 x 105 g/mol), comme mesuré par chromatographie d'exclusion stérique, dans des conditions de traitement thermique plus douces : températures inférieures ou égales à 100°C, temps de cuisson compris de préférence entre 20 et 30 min, si chaque étape de cuisson était précédée de plusieurs cycles de congélation-décongélation, au cours desquels une suspension de chitine et/ou de chitosane dans une solution de soude concentrée était congelée, dégazée puis décongelée sous vide à température ambiante. L'invention concerne un procédé de desacetylation hétérogène au moins partielle de la chitine et/ou du chitosane, caractérisé en ce qu'il comprend une ou plusieurs étapes de cuisson, d'une suspension de chitine et/ou chitosane, dans une solution de soude concentrée, à une température inférieure ou égale à 100°C, pour une durée de préférence comprise entre 20 et 30 min, ladite cuisson étant réalisée dans un réacteur maintenu sous pression réduite et en l'absence de dioxygène, chaque étape de cuisson étant précédée par une succession d'au moins six cycles de gel-dégel, chacun de ces cycles comprenant les étapes suivantes : - a) immerger ledit réacteur dans un milieu réfrigérant jusqu'à congélation complète du milieu réactionnel ; - b) dégazer ledit réacteur, de façon à obtenir à l'intérieur une pression inférieure à la pression atmosphérique; - c) chauffer progressivement ledit réacteur jusqu'à décongélation complète de ladite suspension. Le procédé de l'invention comprend également les étapes suivantes : - une étape de neutralisation de la soude du milieu réactionnel jusqu'à l'obtention d'un pH de 8,5 environ ; - une étape de lavage à l'eau déminéralisée consistant en plusieurs lavages entrecoupés de centrifugations ; - une étape de lyophilisation après congélation. La présente invention concerne un procédé de desacetylation hétérogène de la chitine et/ou du chitosane, dans des conditions plus douces que celles connues dans l'art antérieur : températures de cuisson moins élevées, temps de cuisson plus courts, nombres réduits de cuissons successives comparé à un procédé classique sous atmosphère inerte et à pression ambiante (pour des températures et des durées de réaction équivalentes). Le procédé de desacetylation de la chitine et/ou du chitosane conformément à l'invention consiste en une ou plusieurs étapes de cuisson d'une suspension de chitine dans une solution de soude concentrée, chaque étape étant précédée d'une succession de cycles de gel-dégel de ladite suspension. Les différentes étapes du procédé vont être analysées en détail ci-après. Cycles de gel-déoel Une suspension de chitine finement divisée et/ou de chitosane dispersée dans une solution de soude à 50% en poids, constituant le milieu réactionnel, est placée dans un réacteur. Ce milieu réactionnel est donc hétérogène.(purity, molecular weight, degree of deacetylation), physical form. The molecular weight and the degree of deacetylation of chitosan can influence a good number of factors such as its solubility in aqueous medium, its swelling capacity and its rate of enzymatic degradation. These parameters influence not only the physico-chemical properties of chitosan, but also: its biodegradability, its immunological activity and its mechanical or rheological properties. In the case of many applications in pharmacy and cosmetology, a deacetylation rate close to 100% is necessary. The advantage of obtaining a totally deacetylated chitosan also relates to other properties of chitosan, in particular to its electrostatic properties, which are the basis of numerous interactions. Also, the use of chitosan fully deacetylated allows, among other things, to prepare homogeneous series of chitosan, with the same molecular distribution; in this case, the chitosan will be reacetylated to different degrees of acetylation, rigorously characterized, this being particularly useful and important in pharmaceutical applications but also in many other applications. There are several methods for determining the degree of deacetylation of chitosan: - NMR nuclear magnetic resonance spectroscopy; - linear potentiometric titration (volumetric titration); - UV spectrophotometry (ultraviolet) from the first derivative; - Fourrier transform IR (infrared) spectroscopy; - circular dichroism; - elementary analysis; - gas chromatography. The precision of the values obtained by these different methods is, however, very variable (as reported by several authors, for example TA Khan et al. In J. Pharm. Pharmaceut Se /. 5 (3): 205-212, 2002). For example, the detection threshold of the method for measuring the degree of deacetylation is close to 1% for IR spectroscopy, while it is less than 0.1% in the case of proton NMR spectroscopy. Likewise, the estimation of the average molecular weight of chitosan depends on the measurement method used. The average molecular weight of a polymer can be measured by one of the following methods: - steric exclusion chromatography; - viscosimetry; - laser light scattering photometry using a multi-angle laser detector. High molecular weight chitosan has new mechanical and rheological properties: high viscosity, improved capacities to form films, wires or physical hydrogels, useful in particular for applications of chitosan in medicine and biotechnology. The deacetylation of chitin can be carried out using enzymes called chitin deacetylases, with good yields. However, this method is limited to the laboratory scale due to the high cost of chitin deacetylases and their low productivity. Therefore, the transformation of chitin into chitosan by chemical deacetylation is essential, thanks to its lower cost and easier implementation. The chemical deacetylation of chitin and / or chitosan takes place during a reaction in basic medium (for example, with concentrated sodium hydroxide), the consequence of which is the breaking of the bond between the acetyl group and the amino group linked to the atom C-2. A wide variety of chemical deacetylation techniques have been tested in order to obtain a high degree of deacetylation, with degradation of the polymer minimized. Indeed, too aggressive experimental conditions can lead to a strong depolymerization, during which the glycosidic link β, (l-> 4) is broken. These techniques vary the number of treatments, the temperature of the treatment, the duration of the treatment, the washing and drying stages interspersed between the treatment stages, the use of organic solvents miscible with water as reaction medium, etc. The conditions usually used: sodium hydroxide solutions at 35-60% (by weight), temperatures between 80 and 140 ° C, treatment time ranging from thirty minutes to several days, inert atmosphere or presence of components which fix oxygen, although allowing high degrees of deacetylation to be obtained, however, lead to a significant alteration of the macromolecular structure of chitosan. The applicant has found that, surprisingly, it is possible to obtain chitosan having a degree of deacetylation practically 100%, as measured by NMR 1 and a very high molecular weight (greater than 4.5 × 10 5 g / mol), as measured by size exclusion chromatography, under milder heat treatment conditions: temperatures less than or equal to 100 ° C, cooking time preferably between 20 and 30 min, if each cooking step was preceded by several freeze-thaw cycles, during which a suspension of chitin and / or chitosan in a concentrated sodium hydroxide solution was frozen, degassed and then thawed under vacuum at room temperature. The invention relates to a process for at least partial heterogeneous deacetylation of chitin and / or chitosan, characterized in that it comprises one or more stages of cooking, of a suspension of chitin and / or chitosan, in a solution of concentrated soda, at a temperature less than or equal to 100 ° C., for a duration preferably between 20 and 30 min, said cooking being carried out in a reactor maintained under reduced pressure and in the absence of dioxygen, each cooking step being preceded by a succession of at least six freeze-thaw cycles, each of these cycles comprising the following steps: a) immersing said reactor in a refrigerating medium until the reaction medium is completely frozen; - b) degassing said reactor, so as to obtain inside a pressure below atmospheric pressure; - c) gradually heating said reactor until thawing of said suspension is complete. The process of the invention also comprises the following stages: a stage of neutralization of the sodium hydroxide from the reaction medium until a pH of approximately 8.5 is obtained; - a washing step with demineralized water consisting of several washes interspersed with centrifugations; - a freeze-drying step after freezing. The present invention relates to a heterogeneous deacetylation process for chitin and / or chitosan, under milder conditions than those known in the prior art: lower cooking temperatures, shorter cooking times, reduced numbers of successive cooking compared to a conventional process under an inert atmosphere and at ambient pressure (for equivalent temperatures and reaction times). The process for deacetylation of chitin and / or chitosan in accordance with the invention consists of one or more stages of cooking a suspension of chitin in a concentrated sodium hydroxide solution, each stage being preceded by a succession of gel cycles - thawing of said suspension. The different stages of the process will be analyzed in detail below. Gel-deoel cycles A suspension of finely divided chitin and / or chitosan dispersed in a 50% by weight sodium hydroxide solution, constituting the reaction medium, is placed in a reactor. This reaction medium is therefore heterogeneous.
Chaque cycle de gel-dégel comprend les étapes suivantes : - le réacteur fermé est plongé dans un milieu réfrigérant, par exemple de l'azote liquide , jusqu'à congélation complète dudit milieu réactionnel ; - la pression à l'intérieur du réacteur est réduite au moyen d'une pompe qui évacue l'air présent à l'intérieur du réacteur ; la pression finale à l'intérieur du réacteur est inférieure à la pression atmosphérique et elle le restera jusqu'à la fin de la réaction ; - le milieu réactionnel est chauffé progressivement par immersion dudit réacteur dans de l'eau à température ambiante jusqu'à sa décongélation complète. Le demandeur a constaté que, dans ces conditions, le réseau cristallin de la chitine est fortement modifié. Ce phénomène est d'autant plus marqué que le nombre de cycle est important. Un minimum de six cycles de gel-dégel est nécessaire et suffisant pour déstructurer le réseau cristallin de la chitine. Ce matériau amorphe devient ainsi plus sensible à l'action de la soude, au cours de l'étape de cuisson suivant le dernier cycle de gel-dégel. On peut donc caractériser les transformations subies par la chitine et/ou le chitosane au cours des cycles de gel-dégel comme étant dues à une action thermo-mécanique sur leur réseau cristallin. Cuisson La cuisson du milieu réactionnel se fait par étapes, chaque étape ou réaction de cuisson étant précédée d'une succession de cycles de gel-dégel (au minimum six), comme décrit plus haut. La réaction de cuisson a lieu à une température inférieure ou égale à 100°C, de préférence comprise entre 80 et 100°C. La durée d'une réaction de cuisson est de préférence comprise entre 20 et 30 min. Sans l'ouvrir, le réacteur est placé dans un bain d'huile chauffé à la température de cuisson désirée. La réaction de cuisson a lieu à une pression réduite et à une concentration en dioxygène faible. Ceci est particulièrement important, car, dans ces conditions de cuisson, le dioxygène intervient dans le mécanisme réactionnel ayant alors pour conséquence l'hydrolyse et la dégradation du polymère. Pendant toute la durée de la cuisson, le réacteur se trouve sous agitation. Au cours de la deuxième étape de cuisson et sous l'action de la soude, on passe de la structure cristalline de la chitine à la structure cristalline du chitosane, plus stable; c'est donc une action thermo-chimique qui s'exerce sur la chitine et/ou le chitosane pendant la réaction de desacetylation. En partant de la chitine α on obtient, après la première réaction de cuisson, une chitine recristallisée présentant une structure cristalline différente de celle de la chitine α et proche de la structure cristalline de la chitine β. Cette technique de desacetylation de la chitine permet donc d'effacer les différences de réactivité qui existent entre la chitine α et la chitine β vis à vis de la desacetylation en améliorant fortement l'efficacité de la réaction en ce qui concerne la chitine α. Ceci est très avantageux, compte tenu des propriétés plus intéressantes de la chitine β, notamment sa capacité de gonfler dans l'eau, utile dans des applications galéniques et pour les dispositifs médicaux à base de chitine. Le chitosane totalement désacétylé obtenu selon ce procédé n'est pas complètement amorphe, car il recristallise partiellement à la fin de la cuisson et lors de son isolement. A la fin de la dernière étape de cuisson, le réacteur est immergé dans l'azote liquide, afin d'abaisser la température du milieu réactionnel à température ambiante, puis il est ouvert. Le procédé de desacetylation de la chitine et/ou du chitosane de l'invention comprend, en plus des étapes décrites ci-dessus, des étapes supplémentaires de neutralisation, lavage et de lyophilisation. Neutralisation Le produit final obtenu par le procédé de desacetylation décrit est soumis à une étape de neutralisation visant à réduire la concentration en soude dans le milieu réactionnel après son refroidissement en fin de réaction. Ceci est réalisé à l'aide d'une solution d'acide chlorhydrique de faible concentration (0,5 M). Le pH final à la fin de cette étape de lavage doit être proche de 8,5 pour éviter la solubilisation de l'échantillon.Each freeze-thaw cycle comprises the following stages: the closed reactor is immersed in a refrigerating medium, for example liquid nitrogen, until said reaction medium is completely frozen; - the pressure inside the reactor is reduced by means of a pump which evacuates the air present inside the reactor; the final pressure inside the reactor is lower than atmospheric pressure and will remain so until the end of the reaction; - The reaction medium is gradually heated by immersion of said reactor in water at room temperature until it is completely thawed. The applicant has found that, under these conditions, the crystal lattice of chitin is greatly modified. This phenomenon is all the more marked the greater the number of cycles. A minimum of six freeze-thaw cycles is necessary and sufficient to deconstruct the crystal lattice of chitin. This amorphous material thus becomes more sensitive to the action of soda, during the cooking step following the last freeze-thaw cycle. We can therefore characterize the transformations undergone by chitin and / or chitosan during freeze-thaw cycles as being due to a thermo-mechanical action on their crystal lattice. Cooking The reaction medium is cooked in stages, each stage or cooking reaction being preceded by a succession of freeze-thaw cycles. (at least six), as described above. The cooking reaction takes place at a temperature less than or equal to 100 ° C, preferably between 80 and 100 ° C. The duration of a cooking reaction is preferably between 20 and 30 min. Without opening it, the reactor is placed in an oil bath heated to the desired cooking temperature. The cooking reaction takes place at reduced pressure and at a low oxygen concentration. This is particularly important because, under these cooking conditions, the oxygen intervenes in the reaction mechanism, which then results in hydrolysis and degradation of the polymer. During the entire cooking time, the reactor is stirred. During the second stage of cooking and under the action of soda, one passes from the crystal structure of chitin to the crystal structure of chitosan, more stable; it is therefore a thermochemical action which is exerted on the chitin and / or the chitosan during the deacetylation reaction. Starting from α chitin, after the first cooking reaction, a recrystallized chitin is obtained which has a crystal structure different from that of α chitin and close to the crystal structure of β chitin. This chitin deacetylation technique therefore makes it possible to erase the differences in reactivity which exist between α chitin and β chitin with respect to deacetylation by greatly improving the efficiency of the reaction with regard to α chitin. This is very advantageous, given the more interesting properties of β chitin, in particular its capacity to swell in water, useful in galenical applications and for medical devices based on chitin. The totally deacetylated chitosan obtained according to this process is not completely amorphous, because it partially recrystallizes at the end of the cooking and during its isolation. At the end of the last cooking step, the reactor is immersed in liquid nitrogen, in order to lower the temperature of the reaction medium to room temperature, then it is opened. The process for deacetylating chitin and / or chitosan of the invention comprises, in addition to the steps described above, additional steps of neutralization, washing and lyophilization. Neutralization The final product obtained by the deacetylation process described is subjected to a neutralization step aimed at reducing the concentration of sodium hydroxide in the reaction medium after it has cooled down at the end of the reaction. This is done using a low concentration hydrochloric acid solution (0.5 M). The final pH at the end of this washing step must be close to 8.5 to avoid solubilization of the sample.
Lavaαes Une succession de lavages suit, ce qui a pour but d'éliminer les sels formés (acétate de sodium relargué lors de la desacetylation, et chlorure de sodium produit lors de la neutralisation). Ces lavages sont réalisés à l'aide d'eau distillée contenant de l'ammoniac, ceci afin de remonter le pH de l'eau de lavage à 8,5. Chaque lavage dure un minimum de 30 minutes et est suivi d'une centrifugation destinée à récupérer le produit désacétylé. Lyophilisation Le produit final présent dans une solution de pH 8,5 est soumis à une lyophilisation. On obtient ainsi du chitosane totalement désacétylé. Le procédé de desacetylation de la chitine et/ou du chitosane selon l'invention peut être arrêté après une ou plusieurs étapes de cuisson. Comme mentionné plus haut, en partant de la chitine α on obtient à la fin de la première réaction de cuisson un produit dont la structure ressemble à celle de la chitine β. Soumis aux étapes de neutralisation, lavage et de lyophilisation, ce produit se présente sous forme d'une éponge qui gonfle dans l'eau (alors que la matière première, la chitine α, ne gonfle pas dans l'eau). Ceci permet donc de transformer la chitine α, qui se prête moins à des applications pharmaceutiques ou de biotechnologie, en une structure beaucoup plus ouverte et facilement destructurable ressemblant en ceci à la chitine β, qui possède des propriétés plus intéressantes, notamment la capacité de gonfler dans l'eau. Si, par contre, on souhaite obtenir un chitosane totalement désacétylé, on poursuit les réactions de cuisson selon le procédé décrit. Cinq étapes de cuisson sont nécessaires pour désacétyler complètement la chitine α ou la chitine β. Dans ce cas, l'étape de lyophilisation sera effectuée à la fin du procédé, permettant d'obtenir un chitosane totalement désacétylé. La technique de desacetylation de la chitine et/ou du chitosane de l'invention permet d'obtenir un chitosane avec un degré de desacetylation très élevé, pratiquement égal à 100%, comme mesuré par RMN 1H (quand l'échantillon peut être solubilisé dans D20), selon la technique décrite, par exemple, par Hirai, A, Odani, H. et Nakajima, A. dans « Détermination of degree of deacetylation of chitosan by 1H NMR spectroscopy » Polym. Bull. 1991, 26, 87-94. Le seuil de détection de cette technique est en effet inférieur à celui de la méthode IR, par exemple, ce qui rend plus fiable le résultat obtenu dans le cadre de la présente invention. Le procédé décrit permet également d'obtenir un chitosane dont le haut poids moléculaire atteint des valeurs supérieures à 4,5 x 105 g. mol"1, comme mesuré par chromatographie d'exclusion stérique selon la méthode décrite par C. Schatz et al. dans « Typical physicochemical behaviors of chitosan in aqueous solution » Biomacromolecules, 2003, 4(3) : 641-648, qui fait état de la variation du dn/dC selon le degré d'acétylation. L'invention sera mieux comprise à la lecture de l'exemple de réalisation suivant qui l'illustre à titre non limitatif.Lavaαes A series of washes follows, which aims to eliminate the salts formed (sodium acetate released during deacetylation, and sodium chloride produced during neutralization). These washes are carried out using distilled water containing ammonia, this in order to raise the pH of the washing water to 8.5. Each wash lasts a minimum of 30 minutes and is followed by centrifugation to recover the deacetylated product. Lyophilization The final product present in a solution of pH 8.5 is subjected to lyophilization. This gives totally deacetylated chitosan. The deacetylation process of chitin and / or chitosan according to the invention can be stopped after one or more cooking steps. As mentioned above, starting from chitin α, a product is obtained at the end of the first cooking reaction, the structure of which resembles that of chitin β. Subjected to the stages of neutralization, washing and lyophilization, this product is in the form of a sponge which swells in water (whereas the raw material, chitin α, does not swell in water). This therefore makes it possible to transform the chitin α, which lends itself less to pharmaceutical or biotechnology applications, into a much more open and easily destructurable structure resembling in this the chitin β, which has more interesting properties, in particular the capacity to swell in water. If, on the other hand, it is desired to obtain a totally deacetylated chitosan, the cooking reactions are continued according to the process described. Five cooking steps are necessary to completely deacetylate the chitin α or the chitin β. In this case, the lyophilization step will be carried out at the end of the process, making it possible to obtain a totally deacetylated chitosan. The deacetylation technique of chitin and / or chitosan of the invention makes it possible to obtain a chitosan with a very high degree of deacetylation, practically equal to 100%, as measured by 1 H NMR (when the sample can be dissolved in D 2 0), according to the technique described, for example, by Hirai, A, Odani, H. and Nakajima, A. in "Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy" Polym. Bull. 1991, 26, 87-94. The detection threshold of this technique is in fact lower than that of the IR method, for example, which makes the result obtained in the context of the present invention more reliable. The process described also makes it possible to obtain a chitosan whose high molecular weight reaches values greater than 4.5 × 10 5 g. mol "1 , as measured by size exclusion chromatography according to the method described by C. Schatz et al. in" Typical physicochemical behaviors of chitosan in aqueous solution "Biomacromolecules, 2003, 4 (3): 641-648, which reports of the variation of dn / dC according to the degree of acetylation The invention will be better understood on reading the following embodiment which illustrates it without implied limitation.
Préparation de chitosane de haut poids moléculaire totalement désacétylé a) Desacetylation de la chitine Cycles de gel-dégel La chitine (0,45 g), finement divisée, est introduite dans un réacteur muni d'un bec de dégazage (semblable à un tube de Schlenk). La soude (NaOH 50% (w/v)) est ensuite ajoutée, à température ambiante. Le réacteur est ensuite fermé et immergé dans l'azote liquide jusqu'à solidification complète de la solution de soude/chitine. Grâce au bec de dégazage, on relie le réacteur à une pompe à palette et on tire sous pression réduite (temps de dégazage : 1 min). La pression à l'intérieur du réacteur devient ainsi inférieure à la pression atmosphérique et de l'ordre de 0.01 millibar et elle le restera jusqu'à la fin de la réaction. Le réacteur est ensuite immergé dans l'eau à température ambiante jusqu'au dégel total. Pour un deuxième cycle, on immerge le réacteur dans l'azote liquide comme décrit ci-dessus.Preparation of fully deacetylated high molecular weight chitosan a) Deacetylation of chitin Freeze-thaw cycles The finely divided chitin (0.45 g) is introduced into a reactor fitted with a degassing spout (similar to a Schlenk). The sodium hydroxide (NaOH 50% (w / v)) is then added, at room temperature. The reactor is then closed and immersed in liquid nitrogen until the soda / chitin solution has solidified completely. Thanks to the degassing nozzle, the reactor is connected to a vane pump and pulled under reduced pressure (degassing time: 1 min). The pressure inside the reactor thus becomes lower at atmospheric pressure and of the order of 0.01 millibar and it will remain so until the end of the reaction. The reactor is then immersed in water at room temperature until total thaw. For a second cycle, the reactor is immersed in liquid nitrogen as described above.
Cuisson Après plusieurs cycles de gel-dégel (minimum six), on procède à la première étape de cuisson. Sans rouvrir le réacteur, on le place dans un bain d'huile chauffé à la température désirée et on agite pendant le temps souhaité. b) Neutralisation, lavages : Pour arrêter la cuisson, le réacteur est immergé dans de l'azote liquide. Le réacteur est ensuite ouvert et le produit final qui s'y trouve est récupéré. La soude encore présente est neutralisée par une solution d'acide chlorhydrique 0,5 M. Ensuite, le produit final est lavé à l'eau déminéralisée dont le pH a été ajusté à 8,5 grâce à de l'ammoniac jusqu'à retrouver la conductivité de l'eau déminéralisée (étapes de lavages alternant avec des centrifugations). c) Lyophilisation Le produit lavé puis congelé est enfin lyophilisé. Cooking After several freeze-thaw cycles (minimum six), we proceed to the first cooking step. Without reopening the reactor, it is placed in an oil bath heated to the desired temperature and stirred for the desired time. b) Neutralization, washes: To stop the cooking, the reactor is immersed in liquid nitrogen. The reactor is then opened and the final product therein is recovered. The soda still present is neutralized with a 0.5 M hydrochloric acid solution. Then, the final product is washed with demineralized water, the pH of which has been adjusted to 8.5 using ammonia until it finds the conductivity of demineralized water (washing steps alternating with centrifugations). c) Lyophilization The washed and then frozen product is finally lyophilized.

Claims

REVENDICATIONS
1. Procédé de desacetylation hétérogène au moins partielle de la chitine et/ou du chitosane, caractérisé en ce qu'il comprend une ou plusieurs étapes de cuisson d'une suspension de chitine et/ou du chitosane dans une solution de soude concentrée, à une température inférieure ou égale à 100°C, pour une durée de préférence comprise entre 20 et 30 min, ladite cuisson étant réalisée dans un réacteur à pression réduite et en l'absence de dioxygène, chaque étape de cuisson étant précédée par une succession d'au moins six cycles de gel-dégel, chacun de ces cycles comprenant les étapes suivantes : - a) immerger ledit réacteur dans un milieu réfrigérant jusqu'à congélation complète du milieu réactionnel ; - b) dégazer ledit réacteur de façon à obtenir à l'intérieur une pression inférieure à la pression atmosphérique ; - c) chauffer progressivement ledit réacteur jusqu'à décongélation complète de ladite suspension.1. Process for at least partial heterogeneous deacetylation of chitin and / or chitosan, characterized in that it comprises one or more stages of cooking a suspension of chitin and / or chitosan in a concentrated sodium hydroxide solution, a temperature less than or equal to 100 ° C., for a duration preferably between 20 and 30 min, said cooking being carried out in a reactor at reduced pressure and in the absence of dioxygen, each cooking step being preceded by a succession of '' at least six freeze-thaw cycles, each of these cycles comprising the following stages: - a) immersing said reactor in a refrigerating medium until the reaction medium is completely frozen; b) degassing said reactor so as to obtain inside a pressure below atmospheric pressure; - c) gradually heating said reactor until thawing of said suspension is complete.
2. Procédé de desacetylation de la chitine selon la revendication 1, caractérisé en ce qu'il comprend une seule étape de cuisson d'une suspension de chitine α dans une solution de soude concentrée.2. A process for deacetylation of chitin according to claim 1, characterized in that it comprises a single step of cooking a suspension of α chitin in a concentrated sodium hydroxide solution.
3. Chitine obtenue selon le procédé de la revendication 2, caractérisée en ce qu'elle présente une réactivité, vis à vis du procédé de desacetylation selon la revendication 1, proche de celle de la chitine β.3. Chitin obtained according to the method of claim 2, characterized in that it has a reactivity with respect to the deacetylation method according to claim 1, close to that of β chitin.
4. Procédé de desacetylation de la chitine et/ou du chitosane selon la revendication 1, caractérisé en ce que la pression à l'intérieur du réacteur est réduite au moyen d'une pompe jusqu'à une valeur de 0,01 milibar environ. 4. Process for deacetylation of chitin and / or chitosan according to claim 1, characterized in that the pressure inside the reactor is reduced by means of a pump to a value of approximately 0.01 milibar.
5. Procédé selon la revendication 1 pour la desacetylation complète de la chitine α ou β, comprenant cinq étapes de cuisson, la mesure du degré de desacetylation étant réalisée par RMN 1H.5. Method according to claim 1 for the complete deacetylation of chitin α or β, comprising five stages of cooking, the measurement of the degree of desacetylation being carried out by 1 H NMR.
6. Procédé selon l'une des revendications 1-2 et 4-5, pour la desacetylation au moins partielle de la chitine et/ou du chitosane, comprenant les étapes suivantes additionnelles : - une étape de neutralisation de la soude du milieu réactionnel jusqu'à un pH de 8,5 ; - une étape de lavage à l'eau déminéralisée consistant en plusieurs lavages entrecoupés de centrifugations ; - une étape de lyophilisation après congélation.6. Method according to one of claims 1-2 and 4-5, for at least partial deacetylation of chitin and / or chitosan, comprising the following additional steps: - a step of neutralizing the sodium hydroxide from the reaction medium up to '' at a pH of 8.5; - a washing step with demineralized water consisting of several washes interspersed with centrifugations; - a freeze-drying step after freezing.
7.Chitosane totalement désacétylé obtenu selon le procédé de la revendication 6, caractérisé par un poids moléculaire supérieur à 4,5 x 105 g/mol, comme déterminé par chromatographie d'exclusion stérique. 7. A totally deacetylated chitosan obtained according to the method of claim 6, characterized by a molecular weight greater than 4.5 × 10 5 g / mol, as determined by steric exclusion chromatography.
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CN107056986A (en) * 2017-03-30 2017-08-18 池州学院 A kind of chitosan-based high hydroscopic resin process for cleanly preparing
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CN107056986B (en) * 2017-03-30 2020-10-09 池州学院 Clean production process of chitosan-based super absorbent resin
CN109467617A (en) * 2018-11-14 2019-03-15 黄河三角洲京博化工研究院有限公司 A kind of clean preparation method of chitosan with high deacetylation degree
CN109467617B (en) * 2018-11-14 2020-10-23 黄河三角洲京博化工研究院有限公司 Clean production method of chitosan with high deacetylation degree
CN111675773A (en) * 2020-07-20 2020-09-18 济南格莱威医疗科技有限公司 Preparation method and application of chitosan with controllable molecular weight range
CN112898601A (en) * 2021-02-08 2021-06-04 浙江大学 Inorganic nanoparticle reinforced chitosan-based hydrogel with high strength and excellent biocompatibility and preparation method thereof

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