WO2010070123A1

WO2010070123A1 - Novel method for the controlled hydrolysis of polysaccharides

Info

Publication number: WO2010070123A1
Application number: PCT/EP2009/067581
Authority: WO
Inventors: Roberto Nervo; Alain Heyraud; Daniel Samain
Original assignee: Kalys; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2008-12-18
Filing date: 2009-12-18
Publication date: 2010-06-24
Also published as: FR2940293B1; FR2940293A1

Abstract

The invention relates to a novel method for the controlled hydrolysis of a polysaccharide in the solid state in the presence of an acidic gas such as NO2. The invention also relates to a novel method for obtaining oligosaccharides by depolymerization of natural polysaccharides. This method is carried out by means of a heterogeneous chemical reaction which takes place in a gaseous medium under the action of nitrogen dioxide (NO2 and N2O4) or other acidic gases. In particular, by controlling various parameters, against all expectations, the reaction results in functionalized or unfunctionalized oligosaccharides of defined and reproducible size and DO (degree of oxidation).

Description

NOVEL PROCESS FOR CONTROLLED HYDROLYSIS OF ACCHARIDE POLYS

The present invention relates to a method of controlled hydrolysis of polysaccharides using an acid in the gaseous state. Polysaccharides are an important family of molecules of interest for human food but also for the industry that transforms polysaccharides by chemical or biological processes.

Apart from starch and its dietary role, most neutral polysaccharides are not metabolizable by mammals and do not have clearly identified biological activities. Their wide use in the food industry is mainly due to the exploitation of their properties as texture agents. Glucomannans, polysaccharides constituting between 60 and 90% of the flour extracted from tubers of species Amorphophallus sp., Are a typical example. This flour, known as Konjac, has been used for hundreds of years in Asia to prepare viscoelastic pasta consumed in times of scarcity. In recent years, this flour has been used in the West as a food additive (E425): either as a thickener, alone or in synergy with other polysaccharides, or as a gelling agent.

In addition to this use for these properties of texturizing agents, other applications can be envisaged in food (substitutes for fat, gelatin, casings, non-fermentable soluble fibers in appetite suppressants or stabilizers to limit water activity), or in other areas (food films, artificial skin after burns, modification of water activity in cosmetics ...). Considering its molecular structure, the glucomannan of Amorphophallus sp. is a high molecular weight polymer (2 × 10 ⁵ to 10 ⁶ g mol ^-1 ) consisting of β- (1-4) -linked D-glucose (G) and D-mannose (M) residues with a non-homogeneous primary structure. The M / G ratio characteristic of this type of polysaccharide is 1.6 and the distribution of residues is random. The residues have C 2, C 3 or C 6 acetyl (3 to 11%) substituents of the mannose and the polymer can be weakly branched (<8%). If these structural details govern the properties in solution and the gelling abilities of glucomannans, on a purely nutritional level, they can also have a preponderant role. Molecular weight, for example, leads to differences in background depending on the application: thus molecules of low molecular weight will show very different macroscopic results, such as solubility, transparency, mouth or taste. In addition, polysaccharides can produce different effects depending on their structural characteristics and their molar mass during their fermentation in the intestine. Some are not metabolized at all but others can be degraded to obtain beneficial or unwanted intermediate molecules. A way of upgrading the polysaccharides is therefore their transformation by chemical reactions and especially their hydrolysis provided that this depolymerization reaction can be controlled to obtain a mixture of oligosaccharides of structure and size perfectly targeted. Controlled hydrolysis of polysaccharides thus leads to a whole series of compounds having many potential applications in various fields.

The conventional routes for obtaining oligosaccharides from polysaccharides are hydrolysis by enzymatic or acid catalysis in an aqueous medium.

The enzymatic hydrolysis of glucomannans such as Konjac has thus been described by Cescutti et al. (2002). EP 0 268 885 describes a process for the hydrolysis of glycosaminoglycans in the presence of hypochloric acid.

However, the hydrolysis in aqueous medium of the polysaccharides leads to several technical problems. The dissolution in solution is very difficult to implement because it requires large amounts of liquid, a reactor of very large volume and in some cases almost impossible because strongly depends on the solubility of the polysaccharides to be hydrolysed. In addition, the polysaccharide solutions quickly become very viscous by limiting the mechanical agitation. Then, the recovery of the products after the reaction is not always easy. There is the need to atomize or lyophilize the liquid hydrolyzate containing the final product. In addition, the distributions of oligosaccharides obtained in particular with enzymatic or acidic hydrolysis in an aqueous medium exhibit an accumulation of mono-, di-, trisaccharides and undesirable molecules. This therefore requires subsequent purification steps by ultrafiltration or liquid chromatography.

An alternative to the hydrolysis of polysaccharides is the synthesis of oligosaccharides. In the industry it produces mainly maltodextrins and oligofructans. In addition, it requires specific glycosyltransferases according to the oligosaccharide to be synthesized.

Another way of exploiting polysaccharides is a well-known reaction allowing the selective chemical modification of polysaccharides to be oxidation. The oxidation of starch with nitrogen dioxide NO ₂ is for example described as early as 1945 in US Pat. No. 2,472,590. The starch is oxidized in an organic solvent comprising NO ₂ or treated with gaseous NO ₂ .

US 5,821,360 describes the oxidation of polysaccharides to obtain polycarboxylates fluidized bed in the presence of NO ₂ gas.

US 5856470, US 5541316 and US 5959101 also disclose processes for preparing polycarboxylates.

DE4426443 also relates to the manufacture of polycarboxylates by the oxidation of polysaccharides. FR2 873 700 relates to a controlled oxidation process of polysaccharides in which the reaction is carried out in an inert gas in the supercritical state.

GB779820 also relates to the oxidation of cellulose in the presence of NO ₂ .

US2003 / 0073663 relates to medical devices composed of oxidized polysaccharides. METHOCEL is dissolved in water, frozen and the oxidation is carried out with gaseous NO ₂ .

Butrim et al. (2001) describe the kinetics of oxidation of starch by N ₂ O ₄ in CCl ₄ in the liquid phase.

WO2006 / 018552 describes a controlled oxidation process of polysaccharides using an oxidizing agent such as NO ₂ in which the oxidation reaction is carried out in a densified fluid which is inert with respect to the oxidizing agents used. artwork.

It has been found that these polysaccharide oxidation reactions, in particular with NO _2, may be accompanied by hydrolysis of the polysaccharides. However, this hydrolysis reaction is not controlled at all, and therefore poorly understood, it is generally considered as a secondary or parallel reaction which is absolutely to be avoided during the oxidation of the polysaccharides.

To date, the oligosaccharides are essentially produced either by synthetic routes or by acid or enzymatic depolymerization in an aqueous or organic liquid medium. However, the existing methods are limited because they require a lot of steps and are not economical. The products do not have a molecular weight distribution that is still suitable for the intended applications. Acidic hydrolysis is difficult to control, DPs are very low, temperatures are quite high, and reactor volume is important, especially for industrial applications. Enzymatic hydrolysis requires a specific enzyme, not always commercial, and difficult to find because of the specific type of glycosidic bond that characterizes the structure of the polysaccharide to be treated.

There is therefore an important need for controlled hydrolysis processes of polysaccharides. It has now been found that it is possible to hydrolyze polysaccharides in a controlled manner in the presence of an acid gas such as NO ₂ , SO ₂ , SO 3 or HCl, for example.

The invention relates to a new process for obtaining oligosaccharides by depolymerization of natural polysaccharides. This process is carried out by a heterogeneous chemical reaction which takes place in a gaseous medium under the action of nitrogen dioxide (NO ₂ and N ₂ O ₄ ) or other acid gases. In particular with the mastery of different parameters (time, temperature, reagents, pressure, humidity) the reaction leads against all expectations to oligosaccharides functionalized or not, size and DS (degree of substitution) defined and reproducible. This reaction, which comprises only one step, makes it possible at the same time to manufacture oligosaccharides and to obtain new structures. It also proves that it makes it possible to overcome the accumulation of mono-, di- and trisaccharides, typical of existing competing methods. The process ends with washing and drying.

The applications envisaged are in fields as varied as the food industry, pharmaceuticals, plant biotechnologies, cosmetics, nutraceuticals and infectious diseases.

By controlling the hydrolysis in the processes according to the present invention, it is possible to obtain an original distribution of the molar mass of the products obtained.

Advantageously, the controlled hydrolysis processes of the polysaccharides according to the invention make it possible to hydrolyze the primary or main chains of the polysaccharides without hydrolyzing the side chains. It is therefore mainly the glycoside bonds of the main chain of the polysaccharide which are hydrolysed in the processes according to the invention. The primary structure of the starting polysaccharide which can be branched or include sugars in side groups or substituents is therefore retained.

The branched primary structure of the starting polysaccharide is thus preserved.

Advantageously, the controlled hydrolysis processes of the polysaccharides according to the invention make it possible to hydrolyze the polysaccharides without modifying the substituent groups carried by the polysaccharides. In particular, substituent groups pyruvate are not affected while these groups are modified during acid hydrolysis in aqueous medium.

Advantageously, the processes according to the invention make use of a technology that does not require prior preparation of the polysaccharide to be modified. In particular, there are no problems of solubilization or problems of dispersion. Any type of product may be modified by the process according to the invention, whether it be a crude or purified product.

The methods according to the invention do not have a high energy requirement to implement stirring (gaseous diffusion), there is little risk of clogging reactors or blocking of moving parts. Obtaining the final product does not require spray drying or lyophilization.

The wide choice of experimental conditions of implementation makes it possible to adapt the reaction to the needs of time, energy, to different commercial targets.

The processes according to the invention make use of a technology that is economical in reagents and therefore quite clean compared to a conventional form in aqueous media.

Obtaining products having the desired degrees of polymerization (DP) makes it possible to dispense with separation steps by ultrafiltration or chromatography.

Oligosaccharides and polysaccharides obtained by controlled depolymerization have many applications in the food field but also in various fields such as the pharmaceutical field, the biomedical field and cosmetics.

Description of the invention

The invention thus relates to a method for controlled hydrolysis of a polysaccharide comprising the following steps:

a polysaccharide in the solid state is available, the initial water content of the polysaccharide is adjusted to a content of between 5% and 30% w / w, the polysaccharide is hydrolysed in the presence of an acid gas at a temperature less than 50ºC,

In a preferred embodiment, the method comprises the following steps:

a polysaccharide in the solid state is available, the initial water content of the polysaccharide is adjusted to a content of between 5% and 30% w / w, the polysaccharide is hydrolysed in the presence of an acid gas at a temperature of less than 50 ° C., the acid gas is eliminated, the hydrolyzed polysaccharide is washed with an alcoholic solvent, the hydrolyzed polysaccharide is neutralized,

the hydrolyzed polysaccharide is dried.

Advantageously, during the hydrolysis step of the polysaccharide in the presence of an acid gas, the water content of the polysaccharide is maintained between 5% and 30% w / w.

Preferably, the initial water content of the polysaccharide is adjusted to a content of between 10% and 20% w / w and / or during the step of hydrolysis of the polysaccharide in the presence of an acidic gas the water content of the polysaccharide is maintained or adjusted between 5% and 30% w / w.

Preferably, the initial water content of the polysaccharide is adjusted and / or during the step of hydrolysis of the polysaccharide in the presence of an acid gas, the water content of the polysaccharide is maintained so as to obtain a hydrolysis product of which the DP is between 5 and 200.

Preferably, the initial water content of the polysaccharide is adjusted and / or during the step of hydrolysis of the polysaccharide in the presence of an acid gas, the water content of the polysaccharide is maintained so as to obtain a hydrolysis product of which the OD is between 20 and 100%.

Advantageously, the hydrolysis step of the polysaccharide in the presence of an acid gas is carried out at a temperature below 40ºC.

Preferably, the hydrolysis is carried out in the presence of an acid gas at a temperature of between 20 ° C. and 40 ° C. Preferably, the molar ratio between the acid gas and the polysaccharide is less than 1.5.

Preferably, the hydrolysis step of the polysaccharide in the presence of an acid gas is carried out at a pressure of less than 10 bars.

Advantageously, the acid gas is NO ₂ . In other embodiments, the acid gas is HCl, SO ₂ , or SO 3

The polysaccharide is for example chosen from glucomannans, dextrans, alginates, cellulose, starch, galactomannans and xanthans.

In a preferred embodiment, the polysaccharide is Konjac glucomannan extracted from tubers of Amophophallus sp. In one embodiment, the acid gas is NO ₂ and the hydrolysis process is accompanied by oxidation of the primary hydroxyls - the polysaccharide.

The invention also relates to a hydrolysis product of a polysaccharide obtained by the process according to the invention. Preferably, this product has a DP of between 6 and 15.

The subject of the invention is also a product for hydrolysis of Konjac glucomannans extracted from tubers of Amophophallus sp. in which more than 50% of the product has a degree of polymerization of between 4 and 30, an oxidation state of between 50 and 100% and a polydispersity index of between 1 and 1.2. The invention also relates to a hydrolysis product of a polysaccharide chosen from glucomannans, galactomannans, dextran, xanthan, an alginate, cellulose, a starch or a polymannane, in which more than 50% of the product has a degree of polymerization of between 4 and 30, an oxidation state of between 50 and 100% and a polydispersity index of between 1 and 2.

The invention therefore relates to a process for controlled hydrolysis or controlled depolymerization of polysaccharides such as glucomannans, dextrans, alginates, cellulose, starch, galactomannans and xanthan.

The hydrolysis reaction of the polysaccharides is carried out according to the following reaction scheme in the presence of the H ⁺ ions which are catalysts (R is a piece of polysaccharide chain):

This hydrolysis reaction may be accompanied by an oxidation reaction of the free primary hydroxyl groups which are optionally present on the polysaccharide. This oxidation reaction is carried out according to the following scheme:

The table below indicates for various polysaccharides the reactions that can be obtained:

By polysaccharide is meant polymers formed of a number of dares (or monosaccharides) linked together by O-osidic bonds. The term oligosaccharide refers to molecules comprising between 2 and 40 monosaccharides. As used herein, the term polysaccharide may refer to both polysaccharides and oligosaccharides.

The polysaccharides may be in linear form or in branched form and may have side groups or substituents.

The methods of the present invention allow the hydrolysis of any type of polysaccharide regardless of the types of glycosidic linkages that form it. The hydrolysis processes of the present invention make it possible to obtain oligosaccharides having a low DP as well as polysaccharides having a degree of polymerization (DP) lower than the starting polysaccharide. The degree of polymerization is defined as the number of bonded monomers forming the main chain of a polysaccharide or oligosaccharide.

Advantageously, the hydrolysis process of the present invention makes it possible to control the DP of the final product obtained in particular by precisely adjusting the water content of the starting polysaccharide. Advantageously, the water content of the polysaccharide is maintained in a range throughout the hydrolysis step. In these embodiments, the water content of the polysaccharide is controlled throughout the hydrolysis in the presence of acid gas. Other parameters such as time, temperature, acid gas to polysaccharide molar ratio, and pressure can also be used in the methods of the present invention to control the hydrolysis reaction.

This reaction, which comprises only one step, makes it possible at the same time to manufacture oligosaccharides and to obtain new structures. It also proves that it makes it possible to overcome the accumulation of mono-, di- and tri-saccharides, typical of the processes of the state of the art.

During the hydrolysis reaction, the oligosaccharides and polysaccharides obtained can also be functionalized (oxidized) or not depending on the presence of primary hydroxyl groups on the starting polysaccharide and according to the reaction conditions used.

The control of the water content of the polysaccharide is also a means of controlling the degree of oxidation (OD) of the polysaccharide obtained after the hydrolysis process when it is associated with an oxidation reaction of the polysaccharide. The degree of oxidation is defined as the number of -COOH groups present in the molecule in relation to the DP (percentage value).

The choice of acid gas is a means of carrying out a hydrolysis and oxidation reaction or a hydrolysis reaction alone depending on the type of polysaccharide being treated. In the presence of an acid gas such as hydrochloric acid (HCl), only the hydrolysis reaction will occur.

The methods of the present invention make it possible to control the hydrolysis and, where appropriate, the oxidation leading to functionalized or non-functionalized oligosaccharides and polysaccharides, defined and reproducible DP and DO. It is then possible to obtain in a single step oligosaccharides and / or polysaccharides which have a distribution of the molar mass represented by much narrower curves around a standard sugar size without the need for separation by chromatography. This new distribution of the hydrolysis products obtained is illustrated in FIG. 9 as well as in the examples.

The hydrolysis processes of the present invention make it possible to obtain oligosaccharides having a low polydispersity index. The polydispersity index is the ratio of the number-average molar mass (M _n ) to the weight (M _w ).

The subject of the invention is therefore a process for the controlled hydrolysis of polysaccharides in the presence of an acid gas. The reaction is carried out in a heterogeneous phase with a polysaccharide in the solid state and an acid in the gaseous state.

Preferably, the acid gas used in the processes of the present invention is NO ₂ . By NO ₂ or nitrogen dioxide is meant NO ₂ of N ₂ O ₄ (nitrogen peroxide) and their mixtures. In another preferred embodiment, the acid gas is hydrochloric acid

(HCl). In the presence of gaseous HCl, only the hydrolysis reaction of the polysaccharide occurs.

In another preferred embodiment, the acid gas is SO ₂ , SO 3 or mixtures thereof. Previously, the initial water content of the polysaccharide is adjusted to a value between 3, 5, 10, 15, 20, 25 and 30% w / w of water. It may also be advantageous to maintain the water content of the polysaccharide between 3, 5, 10, 15, 20, 25 and 30% w / w of water during the hydrolysis step in the presence of an acid gas. The water content of the polysaccharide is therefore adjusted and / or maintained at a content of between 3% -30%, between 5% and 30%, between 8% and 30%, between 10% and 30%, between 15% and 30%. % and more preferably between 10% and 20%.

The polysaccharide is, for example, moistened with water vapor in a closed chamber until the desired water content is obtained. The water content of the polysaccharide is a very important parameter, indeed with the control of the amount of water available as moisture in the polysaccharide, it is possible to limit or promote hydrolysis while controlling perfectly its kinetics .

In order to control the hydrolysis satisfactorily, the reaction with the acid gas is carried out at a temperature below 50 ° C., preferably below 40 ° C. and preferably between 20 ° C.-50 ° C., 25 ° C.-50 ° C., 25 ° C. -40 ° C or between 20 ° C-40 ° C. For the control of the hydrolysis reaction of the polysaccharide, the reaction is typically carried out at a pressure of less than 10, 5, 2 or 1 bar.

Advantageously, the acid gas (in particular NO ₂ ) molar ratio on polysaccharide is less than 1.5, 1.4 1.3, 1.2, 1.1, 1, 0.8, 0.6 or 0.5 in order to better control the hydrolysis. The hydrolysis by the acid gas is carried out in the presence of a stoichiometric amount of gas or in the presence of acid gas slightly in excess relative to the polysaccharide.

The time of the hydrolysis reaction is preferably less than 5, 4, 3 or 2 hours.

The hydrolysis reaction is carried out in contact with the acid gas. The polysaccharide placed in a suitable reactor must be accessible to the acid gas by diffusion phenomena.

After removal of the acid gas, the reaction is stopped by scanning with an inert gas such as for example nitrogen.

Preferably, it is also carried out with an alcoholic solvent such as a mixture of water and isopropanol 50% v / v or with ethanol. It is also possible, if necessary, to perform a second wash to neutralize the product. This washing is carried out for example with sodium ethylate (C ₂ H ₅ NaO). Preferably, a neutralization step and also a final drying step are thus carried out.

Solubilization in water and lyophilization of the final product obtained by the process is also possible.

The method according to the invention makes it possible in particular to obtain a product for hydrolysis of Konjac glucomannans extracted from tubers of Amophophallus sp. having a DP, a DO and a predetermined polydispersity index. In particular, the hydrolysis product of Konjac has a degree of polymerization (DP) of between 4, 5, 10, 15, 20 and 30, preferably of between 5 and 15 and even more preferably of between 6 and 15. advantageously, the DP is equal to 8. The degree of oxidation (OD) is between 50, 60, 70, 80, 90 and 100%, preferably the OD is between 80% and 100% and even more preferably the OD is 100%.

The methods according to the invention make it possible to closely control the DP of the polysaccharides / oligosaccharides obtained by hydrolysis. The Konjac hydrolysis products obtained by the processes according to the invention thus advantageously have a polydispersity index of between 1 and 2, preferably of between 1 and 1.2, and more preferably of a polydispersity index of 1.11. Advantageously, the hydrolysis products of Konjac do not contain mono-, di- and trisaccharides because they do not accumulate in the hydrolysis processes according to the present invention. Konjac has a M / G (D-mannose / D-glucose) ratio of 1.6 characteristic of this type of polysaccharide. Advantageously, the hydrolysis processes of the present invention allow the hydrolysis of Konjac without modifying this M / G ratio. Preferably, the Konjac hydrolysis products therefore have a M / G ratio of 1.6.

More generally, the invention advantageously allows the controlled hydrolysis of polysaccharides and in particular galactomannans without modifying the M / G ratio.

The invention also relates to a hydrolysis product of dextran in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation is between 50 and 100% and the index of The polydispersity is between 1 and 2. Preferably, the hydrolysis products have a branched molecular structure such as the starting dextran.

More generally, the controlled hydrolysis process according to the present invention makes it possible to hydrolyze the main chain of the polysaccharide without hydrolyzing the side chains. Thus, the primary structure of the polysaccharides is retained in the hydrolysis processes of the present invention.

The invention also relates to a product of hydrolysis of xanthan in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation is between 50 and 100% and the index of polydispersity is between 1 and 2. Preferably, the hydrolysis products have the same level of pyruvate groups as the starting xanthan.

More generally, the methods of the present invention allow controlled hydrolysis of polysaccharides without affecting the substituent groups of the polysaccharides. The level of substituent groups is therefore identical before and after hydrolysis. Advantageously, the invention therefore allows the hydrolysis of a polysaccharide without affecting the pyruvate groups carried by this polysaccharide.

The invention also relates to a hydrolysis product of an alginate in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation is between 50 and 100% and the polydispersity index is between 1 and 2.

The invention also relates to a product for the hydrolysis of cellulose in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation is between 50 and 100% and the index polydispersity is between 1 and 2.

The invention also relates to a product for the hydrolysis of starch in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation is between 50 and 100% and the polydispersity index is between 1 and 2. The invention also relates to a hydrolysis product of a polymannane in which more than 50% of the product has a degree of polymerization of between 4 and 30, the degree of oxidation. is between 50 and 100% and the polydispersity index is between 1 and 2.

Other features and advantages of the invention will appear in the examples and figures below. The figures and examples are given in a non-limiting manner.

Figure 1: Effect of Hydration Rate on Oxidation Degree:% COOH as a Function of Time and Hydration Rate (% w / w)

Figure 2: Effect of hydration rate on the Degree of Polymerization: DP as a function of time and hydration rate NO2 / Kjc = 1.35 (mol / mol) Figure 3: Kinetic effect of the ratio NO ₂ / Product on the DP: DP versus time Figure 4: Kinetic effect of NO ₂ ratio / Product on DP: OD versus time Figure 5: Dionex chromatography. Hydrolysis of Konjac M1HCO1 by HCl acid for different reaction times Figure 6: Biogel P2 chromatography. Hydrolysis of Konjac M1HCOl by Cellulase Figure 7: GPC chromatography. Hydrolysis of Konjac M1HCOl in the gas phase Figure 8: Distribution of the molar mass of Konjac M1HCOl oligosaccharides obtained in the gas phase.

FIG. 9: Comparative scheme of the depolymerization according to the invention and the enzymatic or acidic depolymerization in a liquid medium

Examples

Example 1: Description of the necessary equipment

Reactor

The reaction takes place in a simple jacketed reactor for stainless steel chemical reactions of internal volume equal to 250 ml. The reactor thermostated with water in closed circuit, is equipped with a temperature and pressure sensor. It is connected to a bottle of NO ₂ or other gas, by a mechanical injector and it is also connected to a nitrogen circuit.

Gas injection system

NO ₂ in the liquid state is injected by means of a refrigerated double-jacketed injector controlled by compressed air.

Sample holder The sample in powder form is placed in a porous Teflon ^® bag. The powder must be accessible to gas by diffusion phenomena and the sample holder must be chemically inert.

Example 2: Experimental Protocol (EP)

Sample preparation

The sample is moistened beforehand in a closed chamber with the chosen water vapor content. Then it is placed in a bag of Teflon ^® and introduced into the reactor on a support. The temperature of the system must be stable at the chosen value. Injection of the gas

We perform an injection of the selected amount of gas by depression at the opening of the inlet valve in the reactor. The gas is thus deposited at the bottom of the reactor. End of the reaction

To complete the reaction after opening the outlet valve of the reactor we perform a gas scan to empty the reactor and replace up to 4 times the total volume with nitrogen. The reaction stops definitely with the washing step. Washing and neutralization

The washing is carried out with a mixture of water and isopropanol 50/50% v / v, then a second wash with sodium ethanolate is necessary to neutralize the product and simplify the drying step. Sodium ethanolate is prepared from a mixture of ethanol and NaOH / water with an overall concentration of 2 M. In this mixture, water is essential for carrying out the exchange with the Na ⁺ ions. Drying

The powder is allowed to air dry or in a vacuum oven for a few days. To avoid any alcoholic trace in the final product solubilization in water followed by lyophilization or atomization can be carried out. Characterization of the products of the reaction

Once the products have been dried, we can characterize them with the classical physicochemical characterization techniques of the following oligo- and polysaccharides:

• GPC (Molar Mass Distribution, DP and Mean Molar Mass),

• Acid base measurement by conductivity measurement (oxidation degree, OD), • ¹ H NMR, ¹³ C (reaction selectivity, presence of by-products, quality of washing and drying).

Thermogravimetric analysis (ATG or TGA)

Thermogravimetric analysis is a thermal analysis technique that measures the amount and rate of mass variation of a sample as a function of temperature and time. It can be used to evaluate any loss of mass or phase changes when the product breaks down, dehydrates or oxidizes. In our case, ATG was used to evaluate the water content of the osidic substrates, after stabilization in a controlled atmosphere or in ambient conditions. The devices used are a Setaram thermometer TGA-92-12 and SDT Q600. We used about 10 mg of sample and the following temperature cycle:

- rise from 20 to 120 ºC at 3 ° C / min.,

- Bearing at 120 ºC for 10 min, lowering temperature to 3 ° C / min. from 120 to 20 ºC. The moisture content in an initial sample is defined as follows:

with T _e : water content (%) contained in the initial sample, M ₁ : the initial mass of the sample, M _sec : the mass of the dry sample. This equation allows us to access the dry sample mass for a given sample. The water content will depend on the environmental moisture conditions and the water activity of saturated solutions of salts that we used to stabilize powdered polysaccharides. Size Exclusion Chromatography for Determination of Molecular Weight (SEC)

This technique is used to separate molecules with molar masses ranging from a few thousand to 2.10 ⁶ g.mol ^-1 . We chose it to determine the molar masses of polysaccharides or oligosaccharides thanks to a multi-detection system. The samples to be analyzed are solubilized at a concentration ranging from 0.5 to 5 g / L as appropriate in 0.1M sodium nitrate (NaNO ₃ ). Before injection, the solutions are filtered on a Sartorius membrane in nitrate. cellulose with a porosity of 0.22 μm.

The differential refractometer measures the actual concentration C ₁ of the solution at the outlet of the columns at volume V ₁ .

The viscometer is composed of three capillaries; pressure sensors located at the terminals thereof can calculate the ratio between the voltage due to the pressure difference during the passage of the polymer solution and the solvent.

This measures the relative viscosity

for the elution volume V ₁ and APo the value of the pure solvent.

Knowing the measured polymer concentration C, the reduced viscosity

is calculated:

The polymer concentration in the columns is very low, and the calculated reduced viscosity is likened to an intrinsic viscosity.

The multi-angle light scattering detector allows the determination of the radius of gyration and the molar mass M ₁ from the Zimm equation, which presents the evolution of according to the following equation is a version

simplified corresponding to the concentration extrapolation and angle θ null:

This is the concentration of the solution in g.mL ^-1 .

N is the number of Avogadro, λ is the wavelength of the light source, (dn / dc) is the increment of the refractive index n as a function of the concentration.

D is the transmittance; this value is related to the position of the attenuators for each measurement Go made in the device taking into account

which is the signal of the photomultiplier for the scattered light. depend on the characteristics of

the device.

The software used (Astra IV) allows double extrapolation, at zero concentration and angle, to obtain the molar mass and the zero-concentration angular function to obtain the radius of gyration. This software calculates the mass M ₁ and the reduced viscosity of each eluted fraction (J) which can be considered as monodispersed. It also allows the calculation of the average molar masses in number

and in mass

as well as the relationship

that is to say the variation of the radius of gyration

as a function of the molar mass M ₁ . We also obtain the molar mass distribution and the polymolecularity index. High Resolution Nuclear Magnetic Resonance Spectrometry (NMR) NMR is a long wave spectroscopy technique, widely used to determine the structure of polysaccharides and oligosaccharides in solution but also in solid phase. It makes it possible to obtain information on the electronic environment of nuclei whose spins have non-zero magnetic moments, such as the

The sample in solution is placed in a strong magnetic field kept constant, resulting in the maintenance of nuclei in the same state of energy and to regenerate the 2 quantized levels of nuclear spins. In the case of Fourier transform NMR, they are then excited by an electromagnetic pulse of short duration of high power and characteristic frequency depending on the type of core studied. The energy absorption by the nuclei causes their transition to an excited state and their return to the initial state of energy where the relaxation is accompanied by the emission of a resonance frequency characteristic of the chemical environment of the nucleus. core. The NMR spectrum thus obtained thus has as many signals as there are nuclei with different chemical environments and the intensity of each signal is proportional to the number of nuclei with the same chemical environment. The position of the signals relative to a reference substance (TMS) is called chemical shift (measured in ppm) and makes it possible to compare the spectra between them. We can use NMR spectrometry for both its qualitative and quantitative aspect:

to dose the amount of polymer contained in a powder (provided that it is completely soluble),

to characterize the structure (for example the location of the acetyl groups, the type of residue and the substituted carbon).

The experiments were carried out on the spectrometers:

- Bruker AC 300 (300 MHz) equipped with a mixed probe

and a temperature control unit, - Bruker Avance 400 (400 MHz) equipped with a QNP or bbiz probe, 5 mm, a Z gradient and a temperature control unit.

The samples (1 to 30 mg) are solubilized in 0.5 mL of deuterated water (D ₂ O).

Spectrum acquisition and processing is done using NTNMR, XWINNMR and MestReC software. We observed ¹ H and ¹³ C nuclei with one-dimensional analyzes.

Measurement of the degree of oxidation

The degree of oxidation (OD) of the modified polysaccharides, expressed as a percentage

(number of carboxylic functions per 100 sugar units), is determined from an acid / base type assay followed by conductivity measurement

Sample preparation

A mass of 30 mg of polysaccharide is introduced into 5 ml of 0.05 M NaOH giving complete solubilization after 30 minutes. stirring, dilution is carried out to a volume of 250 ml. A first assay is performed (go) with a commercial solution of 0.05 M HCl with additions of 0.1 mL followed by a second assay

(return) with 0.05 M sodium hydroxide.

Calibration of acid and base

The acid and base are calibrated to find the exact correction factor that

we will use for calculations of the degree of oxidation. Calculation of the degree of oxidation

Once the conversion factor is known, the degree of oxidation, in percentage of oxidized residues on the totality, is calculated by the following formula:

with Vd the volume dosed, fc the conversion factor and M _s the dry mass to be determined. Volume V

corresponds to the difference between the total volume and the volume of acid

in excess obtained on return:

The dosed volume (V _d ) is calculated by difference between the excess volume of soda obtained in the first

The second dosage (return) by the soda is performed to verify the correct position of V _a ', the second turn of the curve in excess of acid (go), once the carboxyles dosed, is not clear.

Example 3

2 g of Konjac glucomannan in the form of a fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 5 to 300 minutes. We obtain Konjac oligosaccharides with DPs of 170 to 8 and an OD of 15 to 100%.

Example 4

2 g of Konjac glucomannan in the form of a fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 6% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes.

We obtain Konjac oligosaccharides with DPs from 241 to 21 and an OD of 32 to

82%.

Example 5

2g of Konjac glucomannan in the form of a fine powder are introduced into the reactor

(PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 16% w / w and the temperature of the system is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain Konjac oligosaccharides with DPs of 48 to 17 and an OD of 32 to 82%.

Example 6

2 g of Konjac glucomannan in the form of a fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 22% w / w and the temperature of the system is set at 30ºC. The reaction time varies from 15 to 180 minutes.

We obtain Konjac oligosaccharides with DPs of 26 to 5 and an OD of 49 to 96%. Example 7

2 g of guar in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain oligosaccharides with DP 80 to 48 and OD 15 to 100%.

Example 8 2 g of starch in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain oligosaccharides with DPs of 30 to 10 and an OD of 15 to 100%.

Example 9

2 g of dextran in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain oligosaccharides with DPs of 300 to 15.

Example 10

2 g of Alginate in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain Alginate oligosaccharides with DPs of 83 to 11.

Example 11

2 g of Xanthane in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain oligosaccharides with DPs of 170 to 17 and an OD of 15 to 100%. Example 12

2 g of Tara gum in the form of fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes. We obtain oligosaccharides with DP from 100 to 40 and an OD from 15 to 100%.

Example 13

2 g of cellulose (Cotton Linter) in the form of a fine powder are introduced into the reactor (PE) and 3 g of NO ₂ are injected (PE). The powder is pre-equilibrated with a water content of 10% w / w and the system temperature is set at 30ºC. The reaction time varies from 15 to 180 minutes.

Example 14 Comparative Acid, Enzymatic Hydrolysis and Gas Hydrolysis

The same hydrolysis product with three different methods (acid, enzyme and gas) under optimal conditions shows us that in the case of a hydrolysis according to the invention in the gas phase we obtain a very narrow DP distribution between 8 and 15 as the case may be. (average values) whereas in the case of a conventional depolymerization we observe during the reaction always an accumulation of monomers, mainly dimers and trimers. The comparative results obtained are shown in FIGS. 5 to 7.

REFERENCES

S. M. Butrim et al. Kinetics of Starch Oxidation in the Nitrogen System (IV) Oxide - Tetrachloromethane. Russian Journal of Applied Chemistry, Vol. 74, No. 12, 2001, p. 2106-2110. Translated from Zhurnal Prikladnoi Khimii, Vol. 74, No. 12, 2001, p. 2046-2050.

Paola Cescutti et al. "Structure of the oligomers obtained by enzymatic hydrolysis of the glucomannan produced by the plant Amorphophallus konjac". Carbohydrate Research 337 (2002) p. 2505-2511.

Claims

1. Process for the controlled hydrolysis of a polysaccharide, characterized in that it comprises the following steps: a polysaccharide in the solid state is available, the initial water content of the polysaccharide is adjusted to a content of between % and 30% w / w,

the polysaccharide is hydrolyzed in the presence of an acid gas at a temperature below 50 ° C.

2. Process for the controlled hydrolysis of a polysaccharide according to claim 1, characterized in that it comprises the following steps: a polysaccharide in the solid state is available, the initial water content of the polysaccharide is adjusted to a specific content. between 5% and 30% w / w,

the polysaccharide is hydrolysed in the presence of an acid gas at a temperature below 50 ° C., the acid gas is eliminated, the hydrolyzed polysaccharide is washed with an alcoholic solvent, the hydrolyzed polysaccharide is neutralized, and the hydrolyzed polysaccharide is dried.

3. Process for controlled hydrolysis of a polysaccharide according to one of claims 1-2 characterized in that during the step of hydrolysis of the polysaccharide in the presence of an acid gas the water content of the polysaccharide is maintained between 5% and 30% w / w.

4. Process for the controlled hydrolysis of a polysaccharide according to one of the preceding claims, characterized in that the initial water content of the polysaccharide is adjusted to a content of between 10% and 20% w / w and / or when of the hydrolysis step of the polysaccharide in the presence of an acid gas the water content of the polysaccharide is maintained between 10% and 20% w / w.

5. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that the step of hydrolysis of the polysaccharide in the presence of an acid gas is carried out at a temperature below 40ºC.

6. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that during the step of hydrolysis of the polysaccharide the molar ratio between the acid gas and the polysaccharide is less than 1.5.

7. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that the step of hydrolysis of the polysaccharide in the presence of an acid gas is carried out at a pressure less than 10 bar.

8. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that said acid gas is NO ₂ .

9. A method of controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that said acid gas is SO ₂ , or S O3.

10. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that said acid gas is HCl.

11. A method of controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that the polysaccharide is selected from glucomannans, dextrans, alginates, cellulose, starch, galactomannans and xanthans.

12. Process for controlled hydrolysis of a polysaccharide according to one of the preceding claims characterized in that the polysaccharide is a Konjac glucomannan extracted from tubers of Amophophallus sp.

13. Process for controlled hydrolysis of a polysaccharide according to one of claims 1-8 and 11-12 characterized in that the acid gas is NO ₂ and the hydrolysis process is accompanied by oxidation of hydroxyls. primary -CH ₂ OH polysaccharide.

14. Konjac glucomannan hydrolysis product extracted from tubers of Amophophallus sp. characterized in that more than 50% of the product has a degree of polymerization of between 4 and 30, an oxidation state of 50 to 100% and a polydispersity index of 1 to 1.2.

15. A product for the hydrolysis of a polysaccharide chosen from glucomannans, galactomannans, dextran, xanthan, an alginate, cellulose, a starch or a polymannane in which more than 50% of the product has a degree of polymerization of between 4 and 30, an oxidation state of between 50 and 100% and a polydispersity index of between 1 and 2.