FR2857970A1 - Deacetylation of chitin or chitosan for production of material for use e.g. as a pharmaceutical excipient, involves heating with alkali solution, each heating stage being preceded by at least 6 freeze-thaw cycles under vacuum - Google Patents

Abstract

A method for the deacetylation of chitin and/or chitosan by heating with concentrated sodium hydroxide solution under vacuum, in which each heating stage is preceded by at least 6 freeze-thaw cycles in each of which the reaction mixture is frozen solid, degassed under vacuum and then thawed out by heating. A method for the at least partial deacetylation of chitin and/or chitosan (I) involves heating, in one or more stages, a suspension of (I) in concentrated sodium hydroxide solution at up to 100[deg]C for 20-30 minutes under reduced pressure in the absence of oxygen, each heating stage being preceded by at least 6 freeze-thaw cycles, each cycle of which comprises (a) immersing the reactor in a refrigerating medium until the mixture freezes completely, (b) degassing the reactor to below atmospheric pressure and (c) heating the reactor until the mixture thaws out completely. Independent claims are also included for (1) chitin obtained by this method, using a single boiling stage (2) completely deacetylated chitosan with a mol. wt. of more than 450000 (as determined by steric exclusion chromatography), obtained by the above method followed by neutralisation to pH 8.5, washing several times with water (centrifuging after each wash), freezing and then freeze-drying. ACTIVITY : Antibacterial; Vulnerary; Antilipemic. MECHANISM OF ACTION : None given.

Description

i

PROCESS FOR PRODUCING CHITOSAN

  HIGH TOTALLY DESACETYL MOLECULAR WEIGHT

  The present invention relates to the field of biopolymers, more particularly to that of chitosans; it relates to a process of heterogeneous deacetylation of chitin and / or chitosan, based on a thermomechanical-chemical treatment, to obtain chitosan of high molecular weight totally deacetylated.

  Chitin is, along with cellulose, the most common polysaccharide in nature. Chitin is a copolymer consisting of 13-linked Dglucosamine and N-acetyl D-glucosamine repeating units, (1-> 4). It has a degree of acetylation (DA, which represents the percentage in number, or mole 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 chitin c), or the endoskeleton of cephalopods such as squid (in which this case it is chitin>).

  Chitin, semi-crystalline, consists of a network of organized fibers. Chitin exists mainly in two allotropic forms: - chitin ca, whose polysaccharide chains are arranged antiparallel between successive chain planes, which gives rise to many hydrogen bridges, and, consequently, to a high rigidity and low reactivity of chitin cc with respect to deacetylation; chitin ac is not soluble in aqueous solvents and swells only very weakly in an aqueous medium; - chitin A, whose polysaccharide chains are all parallel to each other; Hydrogen bridges are less numerous, which gives chitin 3 a better reactivity and hydrophilicity: it thus has the capacity to swell strongly in water.

  Chitosan, a deacetylated derivative of chitin, is a linear copolymer of β-linked D-glucosamine and N-acetyl D-glucosamine (1-> 4). It is obtained by partial deacetylation of chitin and has the characteristic, by definition, of being soluble in dilute acids, this in contrast to chitin. This property is acquired, during a heterogeneous deacetylation, for a DA less than 40%.

  Chitosan therefore has a much larger number of free amine groups than chitin. In the crystalline state, chitosan has, moreover, only one allotropic variety.

  Chitosan is used on an industrial scale in various fields: waste treatment, food processing, cosmetology, medicine, biotechnology. Due to its biodegradability, bioabsorbability, 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 principle: hypocholesterolemic, bacteriostatic, healing wounds.

  The effectiveness of chitosan is a function of their molecular weight and degree of acetylation, being due more particularly to the presence of reactive amine groups.

  Chitosan used in the pharmaceutical industry must be characterized in a rigorous manner in terms of: quality, intrinsic properties (purity, molecular weight, degree of deacetylation), physical form.

  The molecular weight and degree of deacetylation of chitosan may influence a number of factors such as its aqueous solubility, swelling capacity, and 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 interest of obtaining a totally deacetylated chitosan also relates to other properties of chitosan, in particular to its electrostatic properties, which are at the root of many interactions. Also, the use of totally deacetylated chitosan makes it possible, among other things, to prepare homogeneous series of chitosan, of the same molecular distribution; in this case, the chitosan will be reacetylated at 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; - IR (infrared) spectroscopy with Fourier transform; - circular dichroism; - elementary analysis; gas chromatography.

  The accuracy of the values obtained by these different methods is, however, highly variable (as reported by several authors, for example T.A. Khan et al., J. Pharm Pharmaceutical Sci., 5 (3): 205-212, 2002). By way of example, the detection threshold of the method of 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.

  Similarly, 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 - size exclusion chromatography; - viscometry; photometry of diffusion of the laser light by means of a laser detector multiculp.

  The high molecular weight chitosan has new mechanical and rheological properties: high viscosity, improved ability to form films, son or physical hydrogels, useful especially for applications of chitosan in medicine and in biotechnology.

  The deacetylation of chitin can be performed using enzymes called chitin-deacetylases, with good yields. However, this method is limited at the laboratory scale because of the high cost of chitin deacetylases and their low productivity. As a result, the conversion of chitin into chitosan by chemical deacetylation is necessary because of its lower cost and simpler implementation. The chemical deacetylation of chitin and / or chitosan takes place during a reaction in a basic medium (for example, with concentrated sodium hydroxide), the consequence of which is the rupture of the bond between the acetyl group and the amine group. linked to the C-2 atom. A wide variety of chemical deacetylation techniques have been tested to achieve a high degree of deacetylation with minimal polymer degradation. Indeed, too aggressive experimental conditions can lead to a strong depolymerization, during which the glycoside bond 3, (1-> 4) is broken. These techniques vary the number of treatments, the treatment temperature, the duration of the treatment, the washing and drying steps interposed between the treatment steps, the use of water-miscible organic solvents as the reaction medium, and the like. The conditions usually used: 35-60% sodium hydroxide solution (by weight), temperatures between 80 and 140 C, treatment time ranging from thirty minutes to several days, inert atmosphere or presence of oxygen binding components, although that allowing to obtain high degrees of deacetylation, 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 of substantially 100%, as measured by 1 H NMR and a very high molecular weight (greater than 4.5 x 10 5 g / cm 2). mol), as measured by size exclusion chromatography, under milder heat treatment conditions: temperatures of less than or equal to 100 ° C., preferably between 20 and 30 minutes, if each cooking step was preceded by several freeze-thaw cycles, during which a suspension of chitin and / or chitosan in 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 deacetylation of chitin and / or chitosan, characterized in that it comprises one or more cooking stages, a suspension of chitin and / or chitosan, in a sodium hydroxide solution. concentrated, at a temperature of less than or equal to 100 C, for a duration of preferably between 20 and 30 min, said firing being carried out in a reactor maintained under reduced pressure and in the absence of oxygen, each firing 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 cooling medium until complete freezing of the reaction medium - b) degassing said reactor, so as to to obtain inside a pressure lower than the atmospheric pressure; c) progressively heating said reactor until complete thawing of said suspension.

  The method of the invention also comprises the following steps - a step of neutralization of the sodium hydroxide of the reaction medium until a pH of about 8.5 is reached; 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 process for the heterogeneous deacetylation of chitin and / or chitosan, in 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 inert atmosphere and at ambient pressure (for equivalent temperatures and reaction times).

  The deacetylation process of chitin and / or chitosan according to the invention consists of one or more steps of cooking a suspension of chitin in a concentrated sodium hydroxide solution, each step being preceded by a succession of gel cycles. -degree said suspension.

  The different steps of the process will be analyzed in detail below. Freeze-thaw Cycles A suspension of finely divided chitin and / or chitosan dispersed in a 50% by weight solution of sodium hydroxide, constituting the reaction medium, is placed in a reactor. This reaction medium is heterogeneous. Each freeze-thaw cycle comprises the following steps: the closed reactor is immersed in a cooling medium, for example liquid nitrogen, until complete freezing of said reaction medium; the pressure inside the reactor is reduced by means of a pump which discharges the air present inside the reactor; the final pressure inside the reactor is below 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 complete thawing.

  The plaintiff has found that under these conditions the crystal lattice of chitin is strongly modified. This phenomenon is all the more marked as the number of cycles is important. A minimum of six cycles of freeze is necessary and sufficient to destructure the crystal lattice of chitin. This amorphous material thus becomes more sensitive to the action of sodium hydroxide during the firing step following the last freeze-thaw cycle.

  We can therefore characterize the transformations undergone by chitin and / or chitosan during the freeze-thaw cycles as being due to a thermomechanical action on their crystal lattice. Cooking The cooking of the reaction medium is done in stages, each step or cooking reaction being preceded by a succession of freeze-thaw cycles (at least six), as described above. The firing reaction takes place at a temperature of less than or equal to 100 ° C., preferably of between 80 ° C. and 100 ° C. The duration of a firing 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 baking reaction takes place at a reduced pressure and at a low oxygen concentration. This is particularly important because, under these cooking conditions, oxygen is involved in the reaction mechanism, which results in the hydrolysis and degradation of the polymer. During the entire duration of the cooking, the reactor is under agitation.

  During the second firing stage and under the action of sodium hydroxide, the crystalline structure of chitin is changed to the crystalline structure of chitosan, which is more stable; it is therefore a thermochemical action which is exerted on chitin and / or chitosan during the deacetylation reaction.

  Starting from the chitin ca, after the first cooking reaction, a recrystallized chitin having a crystalline structure different from that of the chitin ca and close to the crystal structure of the chitin 3 is obtained. This chitin deacetylation technique thus allows to erase the differences in reactivity that exist between chitin-ca and chitin-3 with respect to deacetylation by greatly improving the reaction efficiency with respect to chitin c. This is very advantageous, given the more interesting properties of chitin P3, including its ability to swell in water, useful in galenic applications and for medical devices based on chitin.

  The totally deacetylated chitosan obtained according to this process is not completely amorphous because it recrystallizes partially at the end of cooking and during its isolation.

  At the end of the last baking step, the reactor is immersed in liquid nitrogen, in order to lower the temperature of the reaction medium to room temperature, and then it is opened.

  The method of deacetylation of 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 described deacetylation process is subjected to a neutralization step aimed at reducing the concentration of sodium hydroxide in the reaction medium after its cooling at the end of the reaction.

  This is done using a solution of low concentration hydrochloric acid (0.5 M). The final pH at the end of this washing step should be close to 8.5 to avoid solubilization of the sample.

  Washing A succession of washes follows, which aims to eliminate the salts formed (sodium acetate released during the deacetylation, and sodium chloride produced during the neutralization). These washes are carried out using distilled water containing ammonia, 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 the chitin c is obtained at the end of the first baking reaction a product whose structure resembles that of chitin P. Subject to neutralization steps, washing and lyophilization, this product is presented in the form of a sponge that swells in water (while the raw material, chitin ca, does not swell in water). This makes it possible to transform chitin c, which is less amenable to pharmaceutical or biotechnological applications, into a much more open and easily destructurable structure resembling chitin P, which has more interesting properties, especially the ability 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 method described. Five stages of cooking are necessary to completely deacetylate chitin a or chitin P. In this case, the lyophilization step will be carried out at the end of the process, to obtain a totally deacetylated chitosan.

  The technique of deacetylation 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 solubilized in D20), 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 indeed lower than that of the IR method, for example, which makes the result obtained in the context of the present invention more reliable.

  The method described also makes it possible to obtain a chitosan whose high molecular weight reaches values greater than 4.5 × 10 5 g / mol, as measured by steric exclusion chromatography according to the method described by C. Schatz et al. in Biophysromolecules, 2003, 4 (3): 641-648, which reports the variation in dn / dC depending on the degree of acetylation.

  The invention will be better understood on reading the following example embodiment which illustrates it by way of non-limiting example.

  Preparation of fully desacetylated high molecular weight chitosan a) Deacetylation of chitin Freeze-thaw cycles Chitin (0.45 g), finely divided, is introduced into a reactor equipped with a degassing nozzle (similar to a tube of Schlenk). The sodium hydroxide (50% NaOH (w / v)) is then added at room temperature. The reactor is then closed and immersed in liquid nitrogen until complete solidification of the soda / chitin solution. With 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 less than atmospheric pressure and of the order of 0.01 millibar and will remain so until the end of the reaction. The reactor is then immersed in water at room temperature until the thaw is complete.

  For a second cycle, the reactor is immersed in liquid nitrogen as described above.

  Cooking After several freeze-thaw cycles (minimum six), the first cooking step is carried out. Without reopening the reactor, it is placed in an oil bath heated to the desired temperature and stirred for the desired time.

  b) Neutralization, washing To stop cooking, the reactor is immersed in liquid nitrogen. The reactor is then opened and the final product therein is recovered. The sodium hydroxide still present is neutralized with a 0.5M hydrochloric acid solution. The final product is then washed with demineralized water, the pH of which has been adjusted to 8.5 with ammonia until it is recovered. the conductivity of the demineralised water (washing steps alternating with centrifugation). c) Lyophilization The washed and frozen product is finally lyophilized. he

Claims (7)

  A method of at least partial deacetylation of chitin and / or chitosan, characterized in that it comprises one or more steps of cooking a suspension of chitin and / or chitosan in a concentrated sodium hydroxide solution, at a temperature of temperature of less than or equal to 100 C, for a duration of preferably between 20 and 30 min, said firing being carried out in a reactor at reduced pressure and in the absence of oxygen, each firing step being preceded by a succession of at least at least six freeze-thaw cycles, each of these cycles comprising the following steps: a) immersing said reactor in a cooling medium until complete freezing of the reaction medium; b) degassing said reactor so as to obtain inside a pressure lower than atmospheric pressure; c) progressively heating said reactor until complete thawing of said suspension.   2. Method of deacetylation of chitin according to claim 1, characterized in that it comprises a single step of cooking a suspension of chitin in a concentrated sodium hydroxide solution.   3. Chitin obtained according to the method of claim 2, characterized in that it has a reactivity, with respect to the deacetylation process according to claim 1, close to that of chitin P. 4. Method of 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 about 0.01 milibar.   5. Process according to claim 1 for the complete deacetylation of chitin AC or I, comprising five cooking steps, the measurement of the degree of deacetylation being carried out by 1H NMR.   6. Method according to one of claims 1-2 and 4-5, for the at least partial deacetylation of chitin and / or chitosan, comprising the following additional steps: - a sodium neutralization step of 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.   A totally deacetylated chitosan obtained according to the process of claim 6, characterized by a molecular weight greater than 4.5 x 10 5 g / mol as determined by size exclusion chromatography.