FR3059330A1 - NEW PROCESS FOR DEPOLYMERIZATION OF CHITOSAN - Google Patents

Abstract

The invention relates to a novel process for the depolymerization of a chitosan in which the chitosan is contacted with an Ox mixture comprising peracetic acid and hydrogen peroxide such as the ratio by weight peracetic acid /% by mass peroxide of hydrogen is between 0.1 and 0.9.

Description

Holder (s): RHODIA OPERATIONS, THE NATIONAL CENTER FOR SCIENTIFIC RESEARCH Public establishment, UNIVERSITY OF MONTPELLIER Public establishment, HIGHER NATIONAL SCHOOL OF CHEMISTRY OF MONTPELLIER.

Agent (s): RHODIA OPERATIONS.

(04 / NEW PROCESS FOR DEPOLYMERIZATION OF CHITOSAN.

(57) The invention relates to a new process for depolymerizing a chitosan in which the chitosan is brought into contact with an Ox mixture comprising peracetic acid and hydrogen peroxide such as the ratio by mass of peracetic acid /% by mass hydrogen peroxide is between 0.1 and 0.9.

FR 3 059 330 - A1

NEW PROCESS FOR DEPOLYMERIZATION OF CHITOSAN

The present invention relates to a new process for depolymerizing chitosan to prepare an oligochitosan and the oligochitosan obtained by this process.

Chitosan is a polysaccharide resulting from the deacetylation of chitin. In fact, when the degree of deacetylation of chitin, corresponding to the relative amount of acetylated groups removed with chitin during the preparation of chitosan, becomes greater than 50%, this is called chitosan.

Chitin is one of the most abundant natural polysaccharides, present in particular in the exoskeleton of crustaceans, in the endoskeleton of cephalopods or even in fungi. The molecular mass of chitin can reach hundreds or even millions of kilodaltons (kDa).

Chitosan is therefore a bio-based polymer consisting of glucosamine units and / V-acetylglucosamine units linked by β-1,4 glycosidic bonds, generally having a high molar mass but a low solubility in aqueous solvents, in particular for solutions. aqueous with a pH greater than 6, thus limiting its possible applications.

However, it is soluble in acidic solution but we are then left with very viscous acidic solutions which cannot be used as it is.

In order to increase its solubility, in particular in the aqueous phase, depolymerization of chitosan is conventionally carried out in order to obtain oligomers of chitosan having lower molar masses and having good solubility in water.

To this end, different depolymerization methods can be used to prepare oligochitosans.

Thus, it is known to carry out depolymerization by acid hydrolysis using strong acids (typically sulfuric, hydrochloric or even phosphoric acids). However, these processes are not very environmentally friendly, often very long in terms of reaction time and can lead to the total depolymerization of chitosan in the form of glucosamine, which is not desirable.

It is also possible to carry out an enzymatic hydrolysis. However, even if the use of enzymes makes it possible to efficiently obtain oligochitosans while being respectful of the environment, their high cost does not allow their use on an industrial scale.

Furthermore, another known method consists in depolymerizing chitosan via an oxidation method using at least one oxidizing compound. This depolymerization method is respectful of the environment but does not always make it possible to obtain oligochitosans of relatively low molar mass, in particular from chitosans of very high molar mass. Mention may in particular be made of the use of ozone, or of ascorbic acid with a metal co-reactant, of potassium persulfate or alternatively of hydrogen peroxide as an oxidant known for depolymerizing chitosan.

Depolymerization methods by microwave or ultrasonic irradiation as well as thermally have also been considered. These are preferably coupled to another depolymerization method mentioned above, in particular with the oxidation method.

The aim of the invention is to propose a new process for depolymerizing chitosan and thus to prepare oligochitosans with reduced molar masses and which in particular have improved solubility in the aqueous phase.

This process constitutes an alternative to the known processes for depolymerization of chitosan, and which, advantageously, can make it possible to remedy the drawbacks mentioned above.

More preferably, one of the aims of the present invention consists in providing a process which allows depolymerization of chitosan in a single step, which is more efficient and faster while overcoming the nature of the chitosan to be depolymerized, compared to the processes of preparation of the state of the art using an oxidant.

Another object of the invention preferably consists in reducing the amount of oxidant used during the depolymerization of chitosan by an oxidizing method, in particular with respect to these prior art processes.

In particular for these purposes, one of the objects of the invention is a new process for depolymerizing at least one chitosan, said process comprising bringing said chitosan into contact with an Ox mixture comprising peracetic acid and peroxide d hydrogen such that the ratio by mass peracetic acid / mass% hydrogen peroxide in the medium is between 0.1 and 0.9 preferably between 0.2 and 0.8.

According to the invention, the term chitosan is understood here in its normal sense, that is to say any polysaccharide consisting of glucosamine units and Λ / acetylglucosamine units linked by β-1,4 glycosidic bonds derived from the deacetylation of a chitin and typically having a number-average molar mass M _n greater than 20,000 g / mol, in particular possibly ranging from 30,000 to 2,000,000 g / mol. This chitosan may thus have a degree of polymerization of, for example, between 185 and 12,400. It may in particular have an average size greater than or equal to 1,500 repeating units, and for example a degree of deacetylation of between 80 and 95%, for example of around 90%.

The chitosan used in the process according to the invention can typically have a molar mass of between 20,000 and 2,000,000 g / mol, in particular between 30,000 and 1,000,000 g / mol, a degree of deacetylation of the order of 90%.

For example, the chitosan used can be in solid form, typically in the form of a powder.

The chitosan can for example be prepared in a deacetylation step of the chitin prior to the depolymerization process according to the invention.

According to the invention, the mixture Ox is a mixture based on oxidants comprising peracetic acid and hydrogen peroxide such that the ratio by mass of peracetic acid /% by mass of hydrogen peroxide in the medium is between 0.1 and 0.9, in particular between 0.2 and 0.8.

The Ox mixture can also comprise at least one other oxidant.

However, preferably, the mixture Ox consists only of peracetic acid and hydrogen peroxide, in particular in a ratio by mass of peracetic acid /% by mass of hydrogen peroxide in the medium of between 0.2 and 0.8, especially between 0.4 and 0.7.

According to the invention, the quantity of Ox mixture used is generally such that the ratio by mass of Ox mixture /% by mass of chitosan introduced is between 0.2 and 20, in particular between 0.5 and 15.

The Ox mixture can be used in an M mixture. Generally, the M mixture comprises, in addition to the Ox mixture, a co-product such as for example a weak acid, in particular methanoic acid or else acetic acid.

Preferably, the mixture M comprises acetic acid.

Preferably, the mixture M is in the form of an aqueous solution.

According to the invention, the quantity of mixture M used is such that the ratio of quantity of mixture M / quantity of chitosan introduced is between 60 and 1100% by weight, in particular between 100 and 500% by weight.

In a preferred embodiment, the chitosan is brought into contact with an aqueous solution of mixture M comprising the mixture Ox based on peracetic acid and hydrogen peroxide.

According to a particular characteristic of this embodiment, the amount of chitosan when brought into contact is less than 4% by weight. Preferably, this concentration is between 1 and 2% by weight.

In this case, the mixture M is present in an amount between 2 and 25% by weight, in particular between 3 and 22% by weight, preferably between 4 and 8% by weight. In particular, the amount of mixture Ox is between 1 and 25% by weight, in particular between 2 and 22% by weight, preferably between 2 and 5% by weight.

In a preferred variant of this embodiment, the mixture M is an aqueous solution comprising acetic acid, peracetic acid and hydrogen peroxide.

Preferably, the mixture M used in the invention then comprises 0.5 to 3% by weight of peracetic acid, 1 to 6% by weight of hydrogen peroxide and 1 to 2.5% by weight of acid acetic.

One can in particular use, as mixture M a mixture containing 13.5 to 15.5% of peracetic acid, 21 to 24% of hydrogen peroxide and 15 to 17% of acetic acid or a mixture containing 4 , 5 to 5.4% peracetic acid, 21 to 23% hydrogen peroxide and 8 to 10% acetic acid. One can for example use as mixture M the commercial products of the Proxitane® range, in particular Proxitane® 15:23 which contains 13.5 to 15.5% of peracetic acid, 21 to 24% of peroxide hydrogen and 15 to 17% acetic acid or Proxitane® 5:23 which contains 4.5 to 5.4% peracetic acid, 21 to 23% hydrogen peroxide and 8 to 10% acetic acid .

Preferably, the chitosan and the Ox mixture are brought into contact at a pH (pH of the reaction medium) of between 4 and 6.

The temperature at which this contacting step is carried out can be between 20 and 80 ° C.

This contacting can then for example be implemented over a period ranging from 20 minutes to 11 days, depending on the temperature used.

In a preferred variant of the invention, the chitosan and the Ox mixture are brought into contact at a temperature between 50 and 80 ° C., this temperature being maintained until the end of the depolymerization.

When the chitosan and the Ox mixture are brought into contact at a temperature between 50 and 80 ° C., the depolymerization takes place more quickly, between 20 and 90 minutes. Thus, the depolymerization of chitosan by an oxidation method is associated with thermal degradation allowing a faster depolymerization reaction.

Advantageously, the quantity of Ox mixture introduced is then such that the ratio by mass of Ox mixture /% by mass of chitosan introduced is lowered and is preferably between 3 and 4.

The method according to the invention can also be implemented under microwave irradiation. The depolymerization according to the invention can for example be carried out at a constant power, in particular between 100 and 1500 Watt (W), depending on the volume of solution to be depolymerized, in particular varying from 20 to 2000 ml.

In this case, the temperature at which the depolymerization step of chitosan is carried out generally increases under the activation of the microwaves, for example up to a temperature of 100 ° C.

The duration of this depolymerization is then greatly reduced, typically it can be between 200 and 500 seconds.

The depolymerization process of chitosan according to the invention makes it possible to prepare an oligochitosan.

Indeed, at the end of the step of bringing the Ox mixture and chitosan which has just been described, an oligochitosan is obtained.

Preferably, the oligochitosan obtained is in the form of an aqueous solution with an oligochitosan concentration of between 5 and 25% by weight, in particular between 10 and 20% by weight.

Advantageously, the oligochitosan obtained by the depolymerization process according to the invention has a number-average molar mass of less than 2,000 g / mol, in particular less than 1,500 g / mol.

According to a preferred embodiment of the invention, the oligochitosan obtained after the depolymerization reaction of chitosan is recovered.

The recovery of oligochitosan is carried out by any means known per se.

According to a variant of this embodiment, the oligochitosan can be recovered by lyophilization.

According to a preferred variant of the invention, the oligochitosan is recovered by precipitation, in particular by precipitation from the aqueous solution containing the oligochitosan obtained at the end of the depolymerization process according to the invention.

In general, once precipitation has been carried out, the oligochitosan is then in the form of a gelled paste which is soluble in water at any pH and at room temperature. The oligochitosan is then separated.

This separation may include filtration, followed by washing if necessary. Filtration is carried out by any suitable method, for example by means of a press filter, a band filter, a vacuum filter.

The implementation of the depolymerization process according to the invention using a specific Ox mixture of peracetic acid and hydrogen peroxide makes it possible in particular to obtain in a single step and more quickly, oligochitosans having a molar mass and a degree lower polymerization than that of oligochitosans obtained by processes not using this particular mixture, while overcoming the nature of the chitosan to be depolymerized, and in particular its molar mass, and without obtaining glucosamide. In general, the oligochitosans obtained by the process according to the invention preferably have improved properties, characteristic of the polysaccharides.

Advantageously, the method according to the invention makes it possible to reduce the amount of oxidants necessary for the depolymerization of a chitosan while maintaining good oxidative efficiency and obtaining oligochitosans without altering their properties.

The implementation of the method according to the invention makes it possible to obtain an oligochitosan having particular characteristics, and in particular a relatively low molar mass.

Thus, the oligochitosan prepared by the process according to the invention preferably has an average molar mass in number M _{n of} less than 2,000 g / mol, in particular between 600 and 1,500 g / mol, in particular between 800 and 900 g / mol. This number-average molar mass M _n corresponds to the molar mass of the oligosaccharide.

Likewise, the oligochitosan prepared by the process according to the invention preferably has a weight-average molar mass M _w of between 800 and 2,000 g / mol

The oligochitosan obtained by the process according to the invention advantageously has a number average polymerization degree less than 12, preferably less than 10, in particular less than 8, for example between 4 and 6.

In general, the oligochitosans prepared by the process according to the invention have a low polymolecularity or polydispersity, that is to say that the population of oligochitosans obtained by the depolymerization of chitosan according to the process is relatively homogeneous. The polydispersity can be determined by the polymolecularity index I which depends on the width of the distribution curve. The polymolecularity index I corresponds to the ratio of the average molar mass by weight M _w to the average molar mass by number M _n . This index I gives an indication of the extent of the distribution of the molar masses of the different oligochitosans. For oligochitosans all having the same length and therefore the same molar mass, this index I would be equal to 1.

Preferably, the oligochitosans prepared by the process according to the invention have a polymolecularity index I of between 1.2 and 2, preferably of the order of 1.6.

The oligochitosan obtained by the depolymerization process according to the invention advantageously has the same chemical structure as the native chitosan used in the process according to the invention. Indeed, the depolymerization process according to the invention has no significant influence on the chemical structure of chitosan and therefore makes it possible to obtain an oligochitosan of the same structure.

Likewise, the oligochitosan prepared by the process according to the invention preferably has the same degree of deacetylation as the native chitosan used during the depolymerization according to the invention. Indeed, the method according to the invention has no significant influence on the degree of deacetylation of chitosan and therefore on that of the oligochitosan obtained.

These particular characteristics can in particular be confirmed by infrared or by ¹ H NMR spectroscopy by comparing the spectra obtained.

The number and weight average molar masses M _n and M _w , the degree of polymerization and the polymolecularity index of chitosan and oligochitosan can generally be determined by steric exclusion chromatography.

Steric exclusion chromatography (CES) can be carried out as follows: this steric exclusion chromatography produced by an Agilent PL-GPC 50 plus system is carried out on two analytical columns PL-aquagel-OH (with a range of separation between 2M at 400 g / mol) coupled with a refractive index detector.

The chitosans and oligochitosans samples are dissolved in a buffer mixture (30 ml of acetic acid, 16.5 ml of sodium acetate, 0.2 grams of sodium azide, 1 gram of lithium bromide per 1 liter of water) at a concentration of 5 mg / ml, then filtered through pores of 0.20 pm in diameter before injection. The analyzes are carried out at 25 ° C. with an eluent flow rate of 1.0 ml / min. The buffer mixture mentioned above serves as an eluent. The results are analyzed using Cirrus GPC software. The molar masses are calculated from a calibration curve carried out with a range of pullulans.

Chitosans and oligochitosans can be identified and characterized by mass spectroscopy, in particular by using a MALDI-TOF mass spectrometer (matrix-assisted laser desorption-ionization and time of flight Matrix-assisted laser-desorption ionization time-of-flight).

The analysis by mass spectrometry is carried out on a MALDI-TOF spectrometer of the Bruker UltraFlex III type which uses a nitrogen laser with a wavelength of 337 nm. In positive ion mode, the measurements were made with a voltage between 25 and 26.3 kV. A mixture of peptides was used for the external calibration. The samples of chitosans and oligochitosans are dissolved in a buffer mixture (30 ml of acetic acid, 16.5 ml of sodium acetate, 0.2 gram of sodium azide, 1 gram of lithium bromide for 1 liter of water) at a concentration of 5 mg / ml, then filtered through pores of 0.20 pm in diameter before injection. The matrix used is 2,5hydroxybenzoic acid.

The oligochitosan prepared by the process according to the invention can be used in many applications.

It can be used in particular in the pharmaceutical field, for example in medical devices or even in the cosmetic field, in particular because of its biodegradable, bioresorbable, biocompatible and non-toxic properties.

However, it finds particularly interesting applications, especially when it is functionalized.

The following examples illustrate the invention without, however, limiting its scope.

EXAMPLES

EXAMPLE 1

Depolymerization according to the invention of a number average molecular weight chitosan 30,000 g / mol with microwave

The depolymerization of chitosan is carried out in the following manner.

grams of chitosan having a number-average molar mass of

000 g / mol are dissolved in 100 ml of an aqueous solution containing 2% by weight of acetic acid, 5% by weight of hydrogen peroxide and 1.2% by weight of peracetic acid.

The solution is stirred for 2 hours at room temperature, then is placed in a microwave oven at 400 W for 350 seconds.

The mixture is then analyzed by size exclusion chromatography. The oligochitosan obtained has a number-average molar mass Mn of 1000 g / mol, a degree of polymerization DP _n equal to 6 and a polymolecularity index of 1.8.

EXAMPLE 2 (comparative)

Depolymerization with hydrogen peroxide of a molecular weight chitosan 30,000 g / mol with microwave

Likewise, 2 grams of chitosan having a number average molar mass of 30,000 g / mol are dissolved in 100 ml of an aqueous solution containing 2% by weight of acetic acid and 5% by weight of peroxide. hydrogen.

The solution is stirred for 2 hours at room temperature, then is placed in a microwave oven at 400 W for 350 seconds.

The mixture is then analyzed by steric exclusion chromatography.

The oligochitosan obtained has a number-average molar mass Mn equal to 11000 g / mol, a degree of polymerization DP _n of 68 and a polymolecularity index of 2.7.

The significant difference in molar masses between the oligochitosan obtained by the process according to the invention (Example 1) and the oligochitosan of Comparative Example 2 is clearly visible on the spectrum obtained by steric exclusion chromatography presented in Figure 1 .

EXAMPLE 3

Depolymerization according to the invention of a molar mass chitosan

250,000 g / mol thermally

The depolymerization of chitosan is carried out in the following manner.

grams of chitosan having a number-average molar mass of

250,000 g / mol are dissolved in 11 of an aqueous solution containing 4% by weight of acetic acid, 5% by weight of hydrogen peroxide and 3.4% by weight of peracetic acid.

The solution is stirred for 2 hours at room temperature, then heated at 80 ° C for 30 minutes.

The mixture is then analyzed by size exclusion chromatography. The oligochitosan obtained has a number-average molar mass Mn of 900 g / mol, a degree of polymerization DP _n of approximately 5.5 and a polymolecularity index of 1.5.

EXAMPLE 4 (comparative)

Depolymerization with hydrogen peroxide of a chitosan with a molar mass of 250,000 g / mol by thermal means

Similarly, 20 grams of chitosan having a number average molar mass of 250,000 g / mol are dissolved in 11 of an aqueous solution containing 2% by weight of acetic acid and 5% by weight of hydrogen peroxide .

The solution is stirred for 2 hours at room temperature, then heated at 80 ° C for 30 minutes.

The mixture is then analyzed by steric exclusion chromatography.

The oligochitosan obtained has a number-average molar mass Mn equal to 139,000 g / mol, a degree of polymerization DP _n of 860 and a polymolecularity index of 2.9.

The significant difference in molar masses between the oligochitosan obtained by the process according to the invention (Example 3) and the oligochitosan of Comparative Example 4 is clearly visible on the spectrum obtained by steric exclusion chromatography presented in Figure 2 .

Claims (9)

5 1- Process for depolymerization of at least one chitosan, characterized in that the process comprises bringing said chitosan into contact with an Ox mixture comprising peracetic acid and hydrogen peroxide such as the mass% peracetic acid ratio /% by mass hydrogen peroxide is between 0.1 and 0.9, preferably between 0.2 and 0.8. 2- A method according to claim 1, characterized in that the Ox mixture consists of peracetic acid and hydrogen peroxide. 3- A method according to claim 2, characterized in that the ratio by mass of 15 peracetic acid /% by mass of hydrogen peroxide is between 0.2 and 0.8, in particular between 0.4 and 0.7. 4- Method according to one of claims 1 to 3, characterized in that the ratio% by mass mixture Ox /% by mass chitosan is between 0.2 and 20, in 20 especially between 0.5 and 15. 5- Method according to one of claims 1 to 4, characterized in that the mixture Ox is included in a mixture M comprising acetic acid. 25 6- A method according to claim 5, characterized in that the mixture M comprises 0.5 to 3% by weight of peracetic acid, 1 to 6% by weight of hydrogen peroxide and 1 to 2.5% by weight d 'acetic acid. 7- Method according to one of claims 1 to 6 for preparing an oligochitosan, 30 characterized in that the oligochitosan obtained is obtained at the end of the contacting step. 8- A method according to claim 7, characterized in that the oligochitosan has a number average molar mass of less than 2000 g / me, in 35 particular between 600 and 1500 g / mol. 9- Method according to one of claims 7 and 8, characterized in that the oligochitosan has an average degree of polymerization in number less than 12, preferably less than 10. 5 10-A method according to one of claims 7 to 9, characterized in that the oligochitosan has a polymolecularity index of 1.2 and 2. 1/2