WO2003099870A2 - Methods for obtaining oligomannuronates et guluronates, products obtained and use thereof - Google Patents

Abstract

The invention relates to a method for obtaining saturated or unsaturated oligo-mannuronates, characterized in that it consists in carrying out the following steps : a) brown algae are dispersed in an acidic medium in a heated state, having a pH of less than or equal to 2; b) the insolube fraction is separated by filtering from the soluble fraction obtained in step a), said insoluble fraction containing algal cellulose from said algae and the A-L guluronic (G) and B-D-mannuronic (M) blocks; the pH of said insoluble fraction is adjusted to approximately 2.8 by adding a base; d) the insoluble fraction is separated by filtering from the soluble fraction obtained in step c), said soluble fraction containing B-D mannuronic acid (M) blocks; e) the soluble fraction obtained from step d) is depolymerized acidically or enzymatically or acidically/enzymatically in order to obtain saturated and/or unsaturated oligomannuronates.

Description

 PROCESSES FOR OBTAINING OLIGO-MANNURONATES AND GULURONATES, THE PRODUCTS OBTAINED AND THEIR USES

The present invention relates to methods for obtaining oligomnnuronates and oligo-guluronates.

It also relates to the oligo-mannuronates and the oligoguluronates obtained by the implementation of this process. Finally, it relates to uses of these oligo-mannuronates and oligo-guluronates.

Alginates are present in the walls of brown algae or Pheophyceae. Alginate biosynthesis is not only carried out by brown algae but also by bacteria (Azotobacter sp and Pseudomonas sp) which produce acetylated alginates in the extracellular medium.

The alginates are extracted industrially, mainly, from species belonging to the orders of the laminaria and fucales, listed in Table I below:

TABLE I

Les principaux alginates commercialisés le sont sous forme de sels de sodium. Ils sont répartis entre alginates gélifiants et épaississants en fonction de leur structure chimique. La production mondiale annuelle s'élève à plus de 30 000 tonnes extraites à partir de 100 000 tonnes d'algues brunes (équivalent sec). Pour chaque type d'alginate, différents grades sont disponibles en fonction de leur viscosité.

The main alginates marketed are in the form of sodium salts. They are divided between gelling alginates and thickeners according to their chemical structure. Annual global production amounts to more than 30,000 tonnes extracted from 100,000 tonnes of brown algae (dry equivalent). For each type of alginate, different grades are available depending on their viscosity.

In addition to sodium alginates, potassium, magnesium, ammonium, calcium, sodium / triethanolamine alginates are marketed. propylene glycol and alginic acid. They are used mainly in the food industry for their thickening, gelling and stabilizing properties, but also in the pharmaceutical, cosmetic and textile sectors.

The term "alginates" is a generic term designating a whole family of linear polysaccharides consisting of units of α-L-guluronic acid (G) and of β-D-mannuronic acid (M) linked in l- »4. These units are organized along the macromolecule in homopolymeric zones of the α-L-guluronic (G) and β-D-mannuronic (M) acids and in the alternate zone of these two acids, as shown in the following sequence:

... MMMMMMMGGGGGGGGGMGMGMGMMGGM ^ ..

Each alginate is characterized not only by a proportion of α-L-guluronic acid (G) and β-D-mannuronic acid (M) commonly called the M / G ratio but also by the length of the homopolymeric blocks and by the distribution of these blocks along the macromolecule.

This structure depends on the species, the age of the alga, the tissue considered, the harvest period. It directly conditions the properties of the macromolecule: calcium gelation, reaction with divalent or multivalent ions, sensitivity to acid hydrolysis or to enzymatic depolymerization, hydrodynamic volume, biological properties. As a result, homopolymeric blocks of α-L-guluronic acid (G) and β-D-mannuronic acid (M) were produced from alginate in order to express specific properties. International application WO-A-98/51710 describes a process for dividing commercial alginates into two types of homopolymeric blocks these fractions are used for specific applications such as rheology modulators, stabilizers of colloidal systems and stimulants of the immune system (blocks M). This technique is largely inspired by the work of Haug and

Smidsrod (1966). A.Haug and O. S idsrod (1966, “A study of the constitution of alginic acid by partial hydrolysis «Acta Chem Scand; 20 183-190) and (A.Haug, B Larsen and O.Smidsrod (1967) "Studies of the sequence of uronic acid residues in alginic acid" Acta Chem Scand 21, 691-704).

The block preparation protocol describes the following steps: 1) the alginic acid is hydrolyzed in a heterogeneous medium at 100 ° C. for 3 to 5 hours with the consequence of the solubilization of the alternating zones and the insolubilization of the homopolymeric blocks;

2) the insoluble blocks are separated by filtration from the alternating soluble zones; 3) the homopolymeric blocks are solubilized by neutralization up to a pH of 7;

4) the blocks of α-L-guluronic acid (G) are precipitated specifically by adjusting the pH between 2.4 and 2.8;

5) the blocks of insoluble α-L-guluronic acid (G) are separated from the blocks of soluble β-D-mannuronic acid (M) by filtration;

6) the blocks of β-D-mannuronic acid (M) are precipitated to facilitate their recovery by lowering the pH to 1.3.

The process implemented limits the large dilutions necessary for the precipitation of the α-L-guluronic acid blocks (G) by directly dissolving the β-D-mannuronic acid blocks (M) after the hydrolysis step in adjusting the pH between 2.8 and 4. At these pHs, the blocks of α-L-guluronic acid (G) remain insoluble and are separated from the blocks of soluble β-D-mannuronic acid (M) by solid / liquid separation.

The documents EP-A-0 987 272 and EP-A-1 035 136 propose another manufacturing process. They concentrated on the production of α-L-guluronic block (G) with a degree of polymerization less than 20 having:

- calcium chelating properties for applications in the treatment of effluents and detergency;

- stabilization properties of colloidal systems for applications in the stabilization of printing inks;

- biological properties with regard to plants (improvement of root growth).

The methods described in these documents differ from that cited above by the choice of a first homogeneous hydrolysis step at a pH close to neutral. The blocks of α-L-guluronic acid (G) are precipitated at a more acidic pH of between 3 and 3.6. The blocks of α-L-guluronic acid (G) are separated from alternating zones and blocks of β-D-mannuronic acid (M) by solid / liquid separation. In addition, the process described in EP-A-1 035 136 intersperses an oxidation step to increase the chelating nature of the α-L-guluronic acid blocks (G) by increasing the number of carboxylic functions. However, numerous publications and patents describe the properties of a mixture of saturated and unsaturated oligo-alginates, with a degree of polymerization less than or equal to 20.

The work of Yonemoto (YONEMOTO Y., TANAKA H., YAMASHITA T., KITABATAKE N., ISHIDA Y., KIMURA A., and MURATA K., 1993 a. Promotion of germination and shoot elongation of some plants by alginate oligomers prepared with bacterial alginate lyase. Journal of fermentation and bioengineering, 75 (1): 68-70.) demonstrate the elicitor effect of unsaturated oligo-alginates with an average degree of polymerization of 9.5 obtained by enzymatic depolymerization of an alginate extracted from Eisenia bicyclis. The mixture obtained increases the elongation of the stem of Brassica grater, rice and tobacco calles.

More specifically, Natsume (NATSUME M, KAMO Y, HIRAYAMA M and ADACHI T., 1994. Isolation and characterization of alginate- derivated oligosaccharides with root growth-promoting activities. Carbohydrate Research, 258: 187-197.) Demonstrates that unsaturated trimers of homogeneous structure of the sodium salts of α-L-guluronic (ΔGG) or β-D- mannuronic (M) (ΔMM) acids stimulate the growth of barley roots. The trimers were isolated by chromatography from homopolymer blocks of degree of polymerization of 10 depolymerized by the enzymatic route.

In the nutrition sector, Akiyama (AKTYAMA H., ENDO T., NAKAKITA R., MURATA K., YONEMOTO Y. and OKAYOMA K., 1992. Effect of depolymerized alginates on the growth of bifidobacteria. Bioscience, biotechnology, and biochemistry, 56 (2): 355-356) has shown a positive effect of unsaturated oligo-alginates on the growth of bifidobacteria.

The oligo-alginates produced serve as an intermediary for the synthesis of new molecules.

American patent US Pat. No. 5,646,130 describes blocks rich in mannuronate units with a degree of polymerization of about 20, esterified with propanol, 2-propanol and methanol. The synthesized molecules are used to prevent thrombosis. The oligo-mannuronates could also serve as synthons for the synthesis of alkyl alkyl mannuronate and alkyl mannuronic acid used for their amphiphilic properties: foaming, emulsifying, dispersing power, ability to form supramolecular aggregates in dilute media, liquid crystals ...

Document WO-A-01/56404 relates to a technique for preparing alginate rich in mannuronates.

The process consists in adding an organic acid to the alginate, heating the solution, adjusting the pH to 2.5-3.5, and recovering the polymannuronate thus obtained.

The compounds produced have relatively high molecular weights.

The pH adjustment after hydrolysis is done using the same acid as that used for hydrolysis.

Document FR-A-1 426 213 relates to the extraction of alginate from algae. It is an extraction technique for conventional applications such as thickening and gelation, so that it is necessary to preserve the macromolecular nature of the alginate.

Thus, in Example 2, dried seaweed is extracted using sulfuric acid, then the liquid is filtered. After washing, water containing calcium carbonate is added. The mixture is left to stand and the undissolved algae particles are removed. Example 3 is focused on an alginate extraction method rich in guluronic acid.

These methods use a leaching step, carried out cold, which aims to demineralize the alginate to transform it into alginic acid.

A large part of the techniques described above shows the advantage of the applications of oligo-alginates, saturated or unsaturated. They are mainly produced from alginate by acid and / or enzymatic routes. They result in complex mixtures of alternating and homogeneous oligo-alginates. Homopolymer blocks of very low degree of polymerization (degree of polymerization of 10) were also used as raw material for the production of homogeneous and unsaturated oligo-alginate. However, the fact of having to depolymerize blocks produced from alginate, the cost of which is high, limits their industrial development. For many applications, it is indeed essential to implement homogeneous structures produced by simple processes. The object of the present invention is to overcome these drawbacks by proposing simple methods for obtaining oligo-mannuronates and oligoguluronates, which make it possible to dispense with the costly purchase of alginates and to obtain products. end products of homogeneous structure (and not mixtures of various products), these oligo-mannuronates and oligo-guluronates advantageously having relatively low degrees of polymerization which open them to diverse and varied applications.

The terms "oligo-mannuronates" and "oligo-guluronates" mean salts obtained from blocks of α-L-guluronic acid and β-D- mannuronic at least partially depolymerized. This definition is valid for the whole of the present application, including the claims.

Preferably, these products have a degree of polymerization less than or equal to 20, and even more preferably less than or equal to 4.

Thus, the process for obtaining oligo-mannuronates according to the invention essentially consists in implementing the following steps: a) dispersing brown algae in an acidic environment and under heat, at a pH less than or equal to 2; b) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from stage a), this insoluble fraction containing in particular the algal cellulose of the algae, and the α-L-guluronic blocks (G) and β-D- mannuronic (M); c) adjusting the pH of said insoluble fraction to around 2.8 by adding a base; d) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from step c), the soluble fraction containing blocks of β-D-mannuronic acid (M); e) subjecting said soluble fraction recovered in step d) to depolymerization by the acid route, or by the enzymatic route or by the mixed acid / enzymatic route, so as to collect saturated and / or unsaturated oligo-mannuronates.

As for the process for obtaining oligo-guluronates according to the invention, this consists in implementing the following steps: a) dispersing brown algae in an acidic environment and under hot conditions, at a pH less than or equal to 2; b) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from step a), this insoluble fraction containing in particular the algal cellulose from algae, and the α-L-guluronic (G) and β-D-mannuronic (M) blocks; c) adjusting the pH of said insoluble fraction to around 2.8 by adding a base; d) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from step c), the insoluble fraction in particular from the algal cellulose and the α-L-guluronic blocks; e ') adjust the pH of the insoluble fraction from d) to about 4, by adding a base; f) separating in particular by filtration, the insoluble fraction from the soluble fraction resulting from step e ¹ ) the soluble fraction containing blocks of α-L-guluronic acid (G); g ') subjecting the soluble fraction recovered in step f) to depolymerization by the acid route, or by the enzymatic route or by the acid / enzymatic route, so as to collect saturated and / or unsaturated oligo-guluronates.

Finally, the present applicant has realized that it was possible to obtain such alginate fractions directly from crude algae, by carrying out in a first step a particularly drastic acid hydrolysis, capable in particular of exchanging the cations linked to alginates by protons, to hydrolyze the alternate structures of the alginate and to dissolve the majority of algal molecules (mannitol, fucans, proteins, peptides, amino acids).

We can then recover the insoluble phase containing the blocks of α-L-guluronic acid (G) and β-D-mannuronic acid (M) to selectively treat them in subsequent steps.

According to other advantageous but non-limiting characteristics of these processes:

- in step a) dispersing fresh or dehydrated brown algae;

- in step a), an algae content (in dry equivalent relative to the total weight) of about 5% is used;

- using a strong mineral acid such as sulfuric acid;

- The reaction is continued for about 2 hours at 95 ° C;

in step b, a filter earth is used which is kept in the subsequent insoluble fractions; - In step c) and / or in step c '), sodium hydroxide is used; - When the soluble phase is depolymerized by the acid route, the latter is heated to approximately 95 ° C. and that saturated oligo-mannuronate is recovered, respectively saturated oligo-guluronates after neutralization;

- when the soluble phase is depolymerized by the enzymatic route, an alginate lyase is used, obtained by culturing the bacterial strain of

Pseudomonas alginovora deposited on March 6, 1998 with the CNCM (National Collection of Cultures of Microorganisms - Institut Pasteur) under the registration number 1.1989.

Finally, the present invention also relates to the uses of these oligo-mannuronates as eliciting agents for plants, prebiotics or immunological agents, and of these oligo-guluronates as prebiotics, immunological agents or chelating agents.

Other characteristics and advantages of the invention will appear on reading the detailed description which follows: The first step a) of the processes consists in dispersing the algae in hot acid medium in fresh or dehydrated form. The objective of this step is threefold. It makes it possible to exchange the cations linked to alginates by protons, to hydrolyze the alternating structures of the alginate and to dissolve the majority of the algal molecules: mannitol, fucans, proteins, peptides, amino acids. The algae content (dry equivalent) is around 5%. The pH is less than 2 obtained by acidifying the medium with an acid, in particular a strong mineral acid such as sulfuric acid. The final sulfuric acid content is 1.5%. The acid treatment lasts 2 hours at 95 ° C.

Step b) consists in separating the insoluble fraction containing the algal cellulose, the insoluble α-L-guluronic (G) and β-D-mannuronic (M) blocks from the soluble fraction containing the structural oligo-alginates alternating, cations and organic molecules such as fucans, laminaranes, proteins, peptides and amino acids, mannitol. In order to facilitate the solid / liquid separation, it is possible to proceed by filtration, in particular with filter earth added to the suspension.

Step c) consists in adjusting the pH to around 2.8 in order to specifically dissolve the mannuronate blocks.

Step d) consists in separating the insoluble fraction containing the cellulose, the blocks of α-L-guluronic acids (G) and the filtering earth, from the soluble fraction containing the blocks of β-D-mannuronic acid (M ). Step e) consists in specifically depolymerizing the blocks of β-D-mannuronic acid (M) by the acid route, by the enzymatic route or by the mixed acid and enzymatic route.

Acid hydrolysis in homogeneous phase can be done at the initial pH of 2.8, the block solution is heated to 95 ° C. The heating time will condition the composition of the mixture in terms of the size distribution of the oligo-alginates produced. Hydrolysis can be carried out at a pH greater than 2.8 or at a temperature below 95 ° C in order to better control the kinetics of hydrolysis. The ultimate hydrolysis leads to β-D-mannuronic acid (M) and / or to its lactone form.

The enzymatic depolymerization is carried out at a pH of 7.5 at 22 ° C., for example in the presence of an alginate lyase produced by the strain of Pseudomonas alginovora (described in WO-A-98/40 511). The level of enzyme will condition the average degree of polymerization of the final mixture. The final mixture has an average degree of polymerization of between 3 and 4. However, another lyase can be used, provided that it is a mannuronate-lyase.

The combination of these two depolymerization routes leads to saturated and unsaturated mixtures. The order of implementation of the two stages will influence the proportion of saturated and unsaturated units. Step e ') consists in dissolving the blocks of α-L-guluronic acid

(G) at a pH of 4. The pH is adjusted for example using sodium hydroxide.

Step f) consists in separating the cellulose and the insoluble filtering earth from the solution of α-L-guluronic acid (G). Step g ′) consists in depolymerizing the blocks of α-L-guluronic acid (G) by the acid route, by the enzymatic route or by the acid and enzymatic route.

Acid hydrolysis in homogeneous phase can be done at the initial pH of 4, the block solution is heated to 95 ° C. The heating time will condition the composition of the mixture in terms of the size distribution of the oligo-alginates produced. In order to produce saturated oligo-guluronates with the lowest possible degree of polymerization, acid hydrolysis is carried out in successive stages by adjusting the pH lower and lower. After the hydrolysis step at pH 4, a second step at pH 3 makes it possible to better hydrolyze the guluronate blocks. The enzymatic depolymerization takes place at a pH of 7.5 to 22 ° C in the presence of the alginate mentioned on the previous page. Here too, the enzyme level will condition the average degree of polymerization of the final mixture. The final mixture has a degree of polymerization between 3 and 4.

The combination of these two depolymerization routes leads to saturated and unsaturated mixtures. The order of implementation of the two stages will influence the proportion of saturated and unsaturated units.

Finally, the methods described made it possible to fractionate the algal matter into specific products such as saturated and / or unsaturated oligo-mannuronates, saturated and / or unsaturated oligo-guluronates and into products consisting of soluble fractions of the alga in the presence of oligo -alginates of alternating structure which can also be depolymerizable by the acid route, by the enzymatic route or by the mixed route, acid and enzymatic. The co-product of the process consists of algal cellulose, lipids and filter earth.

These processes can be represented as shown in the appended figures in which FIG. 1 is a diagram relating to the production of oligo-mannuronates, while FIG. 2 is a diagram relating to the production of oligo-guluronates.

A specific example of embodiment using micronized Laminaria digitata flour will be described below.

Hydrolysis step of the alternating zones (step a)

60 g of micronized flour of Laminaria digitata with 95.2% dry matter are dispersed in 1121.4 g of a 1.55% sulfuric acid solution in a flask fitted with a condenser for 3 hours with magnetic stirring (18.58 g d (95% sulfuric acid are diluted in 1102.8 g of ultra-pure water).

The flask is heated in an oil bath at 96 ° C for 2 hours. The temperature rise to reach 96 ° C is 30 minutes.

The reaction is stopped by cooling the flask in ice.

Separation of insoluble homopolymeric blocks and alternating soluble oligo-alginates (step b).

17.90 g of filter earth (Becoghur ref 1200) are dispersed in 1193.5 g of suspension for 2 hours with magnetic stirring (% of earth / suspension = 1.5%). The pH of the suspension is 1.2. The suspension is filtered on a bϋchner with a porcelain support and a paper filter. 1029.3 g of acid filtrate with 5.15% dry matter containing the alternating oligo-alginates are recovered as well as 173.7 g of filter cake.

Solubilization of homopolymeric blocks of β - D- mannuronic acid (partly in the form of sodium salt) (step c).

The filter cake is dispersed in 1026.3 g of ultra-pure water for one hour with magnetic stirring at room temperature. The pH of the suspension initially 1.8 is adjusted to 2.85 with 4.90 g of 30% sodium hydroxide and maintained for 1 hour with magnetic stirring.

Separation of the homopolymeric blocks of α -L-guluronic acid and of the homopolymeric blocks of β- D-mannuronic acid (partly in the form of sodium salt) (step d).

1198.85 g of suspension are filtered on a bϋchner with a porcelain support and a paper filter.

1045.7 g of solution containing the mannuronate blocks are recovered.

The M / G ratio of the blocks is 13, which corresponds to an M purity of 94%.

The analysis was carried out by proton NMR.

The dry matter of the filtrate is 0.74%. The block recovery yield is 13.5% relative to the dry mass of algae.

The mass of filter cake is 147.82 g.

Enzymatic depolymerization of β-D - mannuronate blocks (step e)

The pH of 480.24 g of β-D-mannuronic acid block solution is adjusted to 7.5 with 0.84 g of 30% sodium hydroxide. 0.746 g of enzymatic lyophilisate are added to the solution. The ratio of enzyme to block is

5%. The temperature is maintained at 22 ° C. in a thermostatically controlled enclosure and the solution mechanically stirred for 24 h. The pH is controlled and maintained at 7.5 if necessary. The distribution of the different unsaturated oligo-mannuronates is determined from the results obtained by low pressure liquid chromatography on a Bio-Gel P6. The eluent is a 0.05 mol / l sodium nitrate solution with 1 g / l sodium azide. The elution rate is 0.5 ml / min. The temperature is 30 ° C. Detection is done by refractometry.

Acid hydrolysis of β-D mannuronic acid blocks (step e)

The pH of 485.22 g of solution is adjusted to 3 with 1 g of sodium hydroxide M.

The solution is heated and stirred in a flask fitted with a condenser using an oil bath placed on a magnetic heating plate.

The temperature of 96 ° C is reached in 30 minutes and the solution maintained at this temperature for 8 h at reflux. The solution is cooled and neutralized to a pH of 7.5. The distribution of the various saturated oligo-mannuronates is determined from the results obtained by low pressure liquid chromatography on a Bio-Gel P6. The eluent is a 0.05 mol / l sodium nitrate solution with 1 g / l sodium azide. The elution rate is 0.5 ml / min. The temperature is 30 ° C. Detection is done by refractometry.

Solubilization of homopolymeric blocks of α— L- guluronic acid (partly in the form of sodium salt) (step e ').

147.82 g of filter cake are dispersed in 452.18 g of ultra-pure water for one hour with magnetic stirring at room temperature. The pH of the suspension initially 2.8 is adjusted to 4.0 with 26.3 g of sodium hydroxide at 1 mol / 1 and maintained for 1 hour with magnetic stirring.

Separation of the homopolymeric blocks of soluble α-L-guluronic acid from the insoluble fraction (filtering earth, cellulose and lipid fraction) (step f)

643.4 g of suspension are filtered on a bϋchner with a porcelain support and a paper filter. 467.0 g of solution containing the guluronate blocks are recovered.

The M / G ratio of the blocks is 0.1 which corresponds to a G content of the blocks of 91%.

The analysis was carried out by proton NMR.

The dry matter of the filtrate is 1.25%. The recovery yield of the guluronate blocks is 10.2% relative to the dry mass of algae. The filtrate is diluted to obtain 557.12 g of solution at a concentration of 1% of blocks.

The mass of filter cake is 167.33 g with a dry matter of 17.2%

Enzymatic depolymerization of α-L-guluronate blocks (step g ')

To 213.02 g of solution of α-L-guluronic acid blocks are added 0. 801 g of magnesium chloride hexahydrate. The pH is adjusted to 7.5 with 1.18 g of sodium hydroxide at 1 mole / 1. 0.1065 g of enzymatic lyophilisate are added to the solution. The ratio of enzyme to block is 5%.

The temperature is maintained at 22 ° C. in a thermostatically controlled enclosure and the solution mechanically stirred for 24 h. The pH is controlled and maintained at 7.5 if necessary. The distribution of the various unsaturated oligo-guluronates is determined from the results obtained by low pressure liquid chromatography on a Bio-Gel P6. The eluent is a 0.05 mol / l sodium nitrate solution with 1 g / l sodium azide. The elution rate is 0.5 ml / min. The temperature is 30 ° C. Detection is done by refractometry.

Acid hydrolysis of α-L- guluronic acid blocks (step g ')

The solution at pH 4 is heated and stirred in a flask fitted with a condenser using an oil bath placed on a magnetic heating plate.

The temperature of 96 ° C is reached in 30 minutes and the solution maintained at this temperature for 8 h at reflux.

After 8 h at pH 4, the solution is adjusted to pH 3 with 0.93 g of 30% hydrochloric acid and kept stirring at 96 ° C for 8 h. The solution is cooled and neutralized to pH 7.5 with 1.54 g of 30% sodium hydroxide.

The solution is cooled and neutralized to a pH of 7.5. The distribution of the various saturated oligo-guluronates is determined from the results obtained by low pressure liquid chromatography on a Bio-Gel P6.

The eluent is a 0.05 mol / l sodium nitrate solution with 1 g / l sodium azide. The elution rate is 0.5 ml / min. The temperature is 30 ° C. Detection is done by refractometry.

15

Claims

1. Process for obtaining saturated and / or unsaturated oligo-mannuronates, characterized in that it consists in implementing the following steps: a) dispersing brown algae in an acidic environment and at a warm temperature, at a lower pH or equal to 2; b) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from stage a), this insoluble fraction containing in particular the algal cellulose of the algae, and the α-L-guluronic blocks (G) and β-D- mannuronic (M); c) adjusting the pH of said insoluble fraction to around 2.8 by adding a base; d) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from step c), the soluble fraction containing blocks of β-D-mannuronic acid (M); e) subjecting said soluble fraction recovered in step d) to depolymerization by the acid route, or by the enzymatic route or by the mixed acid / enzymatic route, so as to collect saturated and / or unsaturated oligo-mannuronates. 2. Process for obtaining saturated and / or unsaturated oligo-guluronates, characterized in that it consists in implementing the following steps: a) dispersing brown algae in an acidic environment and at a warm temperature, at a lower pH or equal to 2; b) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from stage a), this insoluble fraction containing in particular the algal cellulose of the algae, and the α-L-guluronic blocks (G) and β-D- mannuronic (M); c) adjusting the pH of said insoluble fraction to around 2.8 by adding a base; d) separating, in particular by filtration, the insoluble fraction from the soluble fraction resulting from step c), the insoluble fraction in particular from the algal cellulose and the α-L-guluronic blocks; e ') adjust the pH of the insoluble fraction from d) to around 4, by adding a base f) separate, in particular by filtration, the insoluble fraction from soluble fraction from step e ') the soluble fraction containing blocks of α- L-guluronic acid (G); g ') subjecting the soluble fraction recovered in step f) to depolymerization by the acid route, or by the enzymatic route or by the acid / enzymatic route, so as to collect saturated and / or unsaturated oligo-guluronates. 3. Method according to one of claims 1 or 2, characterized in that in step a) dispersing fresh or dehydrated brown algae. 4. Method according to one of claims 1 to 3 characterized in that in step a), an algae content (in dry equivalent relative to the total weight) of about 5% is used. 5. Method according to one of claims 1 to 4, characterized in that a strong mineral acid such as sulfuric acid is used. 6. Method according to claim 5, characterized in that the reaction is continued for approximately 2 hours at 95 ° C. 7. Method according to claim 1 to 6, characterized in that in step b, a filter earth is used which is preserved in the subsequent insoluble fractions. 8. Method according to claim 1 to 7, characterized in that in step c) and / or in step c '), sodium hydroxide is used. 9. The method of claim 1 or 2 wherein the acid-soluble phase is depolymerized, characterized in that it is heated to about 95 ° C and that recovered saturated oligo-mannuronates, respectively saturated oligo-guluronates after neutralization. 10. The method of claim 1 or 2 wherein the soluble phase is enzymatically depolymerized, characterized in that an alginate lyase is obtained obtained by the cultivation of the bacterial strain of Pseudomonas alginovora deposited on March 6, 1998 of the CNCM under the registration number 1.1989. 11. Oligo-mannuronates obtained by implementing the method according to one of claims 1 and 3 to 10. 12. Oligo-guluronates obtained by implementing the method according to one of claims 2 to 10. 13. Use of the oligo-mannuronates according to claim li as plant elicitors, prebiotics or immunological agents. 14. Use of the oligo-guluronates according to claim 12 as prebiotics, immunological agents or chelating agents.