METHOD FOR OBTAINING SURFACTANT COMPOSITIONS MADE FROM ALKYL-L-GULURONAMIDES

Abstract

Some embodiments relate to a novel method for obtaining surfactant compositions made from alkyl-L-guluronamides or mixtures of L-guluronamides and D-mannuronamides, the compositions obtained by the method and the uses thereof.

Claims

1. A process for preparing a composition, the process comprising: (i) alkyl L-guluronamides of formulae (Ia) and (Ib): ##STR00016## or (ii) a mixture of alkyl L-guluronamides of formulae (Ia) and (Ib) and of alkyl D-mannuronamides of formulae (IIa) and (IIb): ##STR00017## wherein: R.sub.1 is a linear or branched, saturated or unsaturated alkyl chain of 2 to 22 carbon atoms; and R.sub.2 is a hydrogen atom, a linear or branched, saturated or unsaturated alkyl chain of 2 to 22 carbon atoms which may comprise a terminal amine function; wherein the process further includes: a) performing a butanolysis and Fischer glycosylation reaction using poly(oligo)guluronates, oligoalginates, alginates and/or brown algae; and b) performing a aminolysis reaction on the reaction medium derived from the butanolysis and Fischer glycosylation reaction, in the presence of a linear or branched, saturated or unsaturated amine of formula RNH.sub.2 wherein R is composed of 2 to 22 carbon atoms.

2. The process as claimed in claim 1, further including performing of neutralizing the reaction medium derived from the butanolysis and Fischer glycosylation reaction before the aminolysis reaction.

3. The process as claimed in claim 1, wherein the butanolysis and Fischer glycosylation reaction is performed in the presence of: (i) water and/or of an ionic solvent and/or of a eutectic solvent, (ii) a linear or branched, saturated or unsaturated alcohol of the formula ROH, containing from 1 to 4 carbon atoms, and (iii) an acid catalyst.

4. The process as claimed in claim 3, wherein the acid catalyst is selected from the group consisting of consisting of: hydrochloric acid, sulfuric acid, an alkylsulfuric acid, a sulfonic acid, an alkylsulfonic acid or an alkyl sulfosuccinate, perhalohydric acids, metals, oxides thereof or salts thereof such as the halides thereof.

5. The process as claimed in claim 4, wherein the acid catalyst is methanesulfonic acid.

6. The process as claimed in claim 3, wherein the alcohol ROH is n-butanol.

7. The process as claimed in claim 1, wherein the aminolysis reaction is performed in the presence of a fatty amine selected from the group consisting of dodecylamine and oleylamine.

8. A composition obtained via the process as claimed in claim 1.

9. The composition as claimed in claim 8, wherein the composition is an oil-in-water or water-in-oil emulsion.

10. The use of the composition as claimed in claim 8 as a surfactant.

11. The use of a composition as claimed in claim 10, wherein the surfactant is selected from the group consisting of solubilizers, hydrotropes, wetting agents, foaming agents, emulsifying agents, emulsifiers and/or detergents.

12. The use of a composition as claimed in claim 8 as an antibacterial and/or antifungal agent.

13. A surfactant, comprising: the composition as claimed in claim 8.

14. An antibacterial and/or antifungal, comprising: the composition as claimed in claim 8.

15. The process as claimed in claim 2, wherein the butanolysis and Fischer glycosylation reaction is performed in the presence of: (i) water and/or of an ionic solvent and/or of a eutectic solvent, (ii) a linear or branched, saturated or unsaturated alcohol of formula ROH, containing from 1 to 4 carbon atoms, and (iii) an acid catalyst.

16. The process as claimed in claim 15, wherein the acid catalyst is selected from the group consisting of: hydrochloric acid, sulfuric acid, an alkylsulfuric acid, a sulfonic acid, an alkylsulfonic acid or an alkyl sulfosuccinate, perhalohydric acids, metals, oxides thereof or salts thereof such as the halides thereof.

17. The process as claimed in claim 16, wherein the acid catalyst is methanesulfonic acid.

18. The process as claimed in claim 15, wherein the alcohol ROH is n-butanol.

19. The process as claimed in claim 2, wherein the aminolysis reaction is performed in the presence of a fatty amine selected from the group consisting of dodecylamine and oleylamine.

20. The process as claimed in claim 1, wherein the aminolysis reaction is performed in the presence of a fatty amine selected from the group consisting of dodecylamine and oleylamine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0049] FIG. 1 represents the measurement of the interface tensions of compositions GN12, GN18, AlgN12, AlgN18 and crude Algn12.

[0050] FIG. 2 represents the measurement of the emulsifier power of compositions GN12, GN18, AlgN12, AlgN18 and crude Algn12 in comparison with the commercial references Xylance and Montanov.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Examples

Example 1: Process for Obtaining Alkyl L-Guluronamides from Poly(Oligo)Guluronates

[0051] ##STR00003##

[0052] Preparation of the starting materials: the poly(oligo)guluronates may be obtained, for example, according to the process of international patent application WO 03/099 870 [10].

1) Butanolysis and Fischer Glycosylation Reaction

[0053] 1 g of sodium L-poly(oligo)guluronate (7500 g/mol, degree of polymerization=44) whose M/G ratio is 0.25 (5.71 mmol, 1 eq) was mixed with 2 mL of distilled water. 80 mL (150 eq) of butanol were added to the alginate solution with stirring with 778 l of methanesulfonic acid (12 mmol, 2.1 eq). The medium was stirred at the reflux temperature of butanol (130-135 C.) for 12 hours. The waters present in the medium and those formed during the reaction were removed in Dean-Stark apparatus filled with butanol, via water-butanol azeotropic distillation. Given that it is denser than butanol, the water moves to the bottom of the Dean-Stark apparatus and a few ml of butanol pass into the flask to maintain the initial volume. After 12 hours, thin-layer chromatography (95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH) was performed on the reaction medium to ensure that the expected product had indeed been synthesized.

2) Aminolysis Reaction

[0054] The temperature of the medium was lowered to 60 C., followed by addition of 3 molar equivalents (17.14 mmol) of fatty amine (for n=11, 3.18 g and for n=17, 4.62 g) that may be required to increase the pH to 8.5. After stirring for 30 minutes at 65 C. and under a reduced pressure of 150 mbar, the butanol was evaporated off while reducing the pressure from 150 mbar to 6 mbar over a period of 1 hour. The medium was left under a reduced pressure of 6 mbar for 1 hour 30 minutes to ensure the evaporation of the traces of butanol that were formed.

[0055] The residue obtained was taken up in diethyl ether and then filtered through a sinter funnel and washed several times with diethyl ether to remove the salts and the unreacted starting sugar. The filtrate (containing our alkyl guluronamides) was concentrated under vacuum to remove the diethyl ether. A brown oil was thus obtained.

[0056] For n=11, after optional chromatography of the oil obtained on a column of silica gel (80 g, using as eluent 95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH), the presence of 0.95 g (2.27 mmol, 40% yield) of a mixture of four isomeric forms of N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (37%), N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (21%), N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (28%) and N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (14%) was determined. The pyranose/furanose ratio is 0.74. This surfactant composition was named GN12.

[0057] For n=17, after optional chromatography of the oil obtained on a column of silica gel (80 g, using as eluent 95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH), the presence of 1.08 g (2.16 mmol, 38% yield) of a mixture of four isomeric forms of N-(18-octadecyl)-n-butyl -L-gulurofuranosiduronamide (36%), N-(18-octadecyI)-n-butyl -L-gulurofuranosiduronamide (23%), N-(18-octadecyl)-n-butyl -L-guluropyranosiduronamide (25%) and N-(18-octadecyl)-n-butyl -L-guluropyranosiduronamide (16%) was determined. The pyranose/furanose ratio is 0.69. This surfactant composition was named GN18.

Example 2: Process for Obtaining Alkyl L-Guluronamides and D-Mannuronamides from Alginates

[0058] ##STR00004##

Preparation of the starting materials: Alginate extraction processes are conventionally used at the CEVA (Ren Perez La culture des algues marines dans le monde [Cultivation of marine algae throughout the world], Ifremer). They involve acidic leaching of fresh or dried algae (washing of the harvested algae with seawater, depigmentation in formaldehyde, grinding, extraction with 0.2N sulfuric acid at room temperature, draining and rinsing of the leached algae with distilled water), followed by dissolution of the sodium alginates by increasing the pH of the medium followed by solid/liquid separation so as to remove the algal residues (addition of a 1.5% Na.sub.2CO.sub.3 solution containing 50 g dry weight of leached algal material at a dry alga/1.5% Na.sub.2CO.sub.3 solution ratio of 0.025, stirring with an IKA reactor for 3 hours at 55 C., cooling in an ice-water bath to avoid excessive temperature differences, centrifugation for 5 minutes at 6000 rpm, and solid/liquid separation). At this stage, the liquid fraction may be frozen and freeze-dried and constitutes the semi-refined alginates in sodium alginate form. In order to obtain refined alginates, purification is performed in the preceding steps. After separation of the algal residues, this final purification step includes or consists of precipitation of the alginic acid by lowering the pH, followed by washing several times with acidic water so as to remove the co-products. Increasing the pH with Na.sub.2CO.sub.3 makes it possible once again to dissolve the sodium alginates while limiting the salts, relative to the use of sodium hydroxide. Lastly, a final step of freezing and then freeze-drying makes it possible to obtain the final product. In order to obtain saturated or unsaturated oligoalginates the alginate solution is treated enzymatically or with acid so as to lower the degree of polymerization of the alginates from 20 to 3.

A) Without Prior Neutralization of the Reaction Medium Before the Aminolysis Reaction

[0059] 1) Butanolysis and Fischer Glycosylation Reaction

[0060] 1 g of sodium alginate (110 200 g/mol, degree of polymerization=630, extracted from Sargassum vulgare) whose M/G ratio is 0.71 (5.71 mmol, 1 eq) was mixed with 3 mL of distilled water. 80 mL (150 eq) of butanol were added to the alginate solution with stirring with 927 l of methanesulfonic acid (14.27 mmol, 2.5 eq). The medium was stirred at the reflux temperature of butanol (130-135 C.) for 24 hours. The waters present in the medium and those formed during the reaction were removed in Dean-Stark apparatus filled with butanol, via water-butanol azeotropic distillation. Given that it is denser than butanol, the water moves to the bottom of the Dean-Stark apparatus and a few ml of butanol pass into the flask to maintain the initial volume. After 24 hours, thin-layer chromatography (95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH) was performed on the reaction medium to ensure that the expected product had indeed been synthesized.

[0061] 2) Aminolysis Reaction

[0062] The temperature of the medium was lowered to 60 C., followed by addition of 3 molar equivalents (17.14 mmol) of fatty amine (for n=11, 3.18 g and for n=17, 4.62 g) that may be required to increase the pH to 8.5. After stirring for 30 minutes at 65 C. and under a reduced pressure of 150 mbar, the butanol was evaporated off while reducing the pressure from 150 mbar to 6 mbar over a period of 1 hour. The medium was left under a reduced pressure of 6 mbar for 1 hour 30 minutes to ensure the evaporation of the traces of butanol that were formed.

[0063] The residue obtained was taken up in diethyl ether and then filtered through a sinter funnel and washed several times with diethyl ether to remove the salts and the unreacted starting sugar. The filtrate (containing the alkyl guluronamides and mannuronamides) was concentrated under vacuum to remove the diethyl ether. A dark brown oil is thus obtained.

[0064] For n=11, after optional chromatography of the oil obtained on a column of silica gel (80 g, using as eluent 95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH), the presence of 0.91 g (2.18 mmol, 39% yield) of a mixture of six isomeric forms (C.sub.22H.sub.43NO.sub.6, molar mass=417.59 g/mol) of N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (26%), N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (15%), N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (16%), N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (12%), N-(12-dodecyl)-n-butyl -D-mannofuranosiduronamide (18%) and N-(12-dodecyl)-n-butyl -D-mannopyranosiduronamide (14%) was determined. This surfactant composition was named AlgN12.

[0065] After 1D and 2D NMR spectral analysis of the crude reaction product (before purification), the following were calculated: the pyranose/furanose ratio of the guluronamide forms (0.72), the pyranose/furanose ratio of the mannuronamide forms (0.81) and the mannuronamide/guluronamide ratio (0.47).

[0066] In addition, the amount of dodecylammonium mesylate salts formed during the aminolysis reaction was able to be quantified. Starting with 1 g of alginate (1 eq), this amount formed was 1.8 molar equivalents (0.010 mol, 2.89 g).

[0067] Characterization of the associated amine salt: dodecylammonium mesylate

##STR00005##

[0068] For n=17, after optional chromatography of the oil obtained on a column of silica gel (80 g, using as eluent 95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH), the presence of 1.03 g (2.05 mmol, 36% yield) of a mixture of six isomeric forms (C.sub.28H.sub.55NO.sub.6, molar mass=501.75 g/mol) of N-(18-octadecyl)-n-butyl -L-gulurofuranosiduronamide (25%), N-(18-octadecyI)-n-butyl -L-gulurofuranosiduronamide (15%), N-(18-octadecyI)-n-butyl -L-guluropyranosiduronamide (16%), N-(18-octadecyI)-n-butyl -L-guluropyranosiduronamide (12%), N-(18-octadecyl)-n-butyl -D-mannofuranosiduronamide (18%) and N-(18-octadecyI)-n-butyl -D-mannopyranosiduronamide (14%) was determined. This surfactant composition was named AlgN18.

[0069] After 1D and 2D NMR spectral analysis of the crude reaction product (before purification), the following were calculated: the pyranose/furanose ratio of the guluronamide forms (0.71), the pyranose/furanose ratio of the mannuronamide forms (0.84) and the mannuronamide/guluronamide ratio (0.46). Similarly, the presence of the octadecylammonium mesylate salts formed during the aminolysis reaction was able to be quantified. Starting with 1 g of alginate (1 eq), this amount formed was 2 molar equivalents (0.011 mol, 4.16 g).

[0070] Characterization of the associated amine salt: octadecylammonium mesylate.

##STR00006##

B) With Prior Neutralization of the Reaction Medium Before the Aminolysis Reaction

[0071] For the purpose of developing a process not followed by a purification step, which is easier to perform on a pilot scale, while using a low amount of amine and avoiding the use of organic solvents (except for butanol), the protocol described below was performed:

[0072] 1) Butanolysis and Fischer Glycosylation Reaction

[0073] 1 g of sodium alginate (110 200 g/mol, degree of polymerization=630, extracted from Sargassum vulgare) whose M/G ratio is 0.71 (5.71 mmol, 1 eq) was mixed with 3 mL of distilled water. 80 mL (150 eq) of butanol were added to the alginate solution with stirring with 927 l of methanesulfonic acid (14.27 mmol, 2.5 eq). The medium was stirred at the reflux temperature of butanol (130-135 C.) for 24 hours. The waters present in the medium and those formed during the reaction were removed in Dean-Stark apparatus filled with butanol, via water-butanol azeotropic distillation. Given that it is denser than butanol, the water moves to the bottom of the Dean-Stark apparatus and a few ml of butanol pass into the flask to maintain the initial volume. After 24 hours, thin-layer chromatography (95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH) was performed on the reaction medium to ensure that the expected product had indeed been synthesized.

[0074] 2) Neutralization of the Reaction Medium

[0075] In order to reduce the amount of amines to be added during the second step of the process, the reaction medium was neutralized, after cooling, with 1M sodium hydroxide (8 mL) to a pH of 7. Next, the water was evaporated off on a rotary evaporator.

[0076] 3) Aminolysis Reaction

[0077] 1 molar equivalent of dodecylamine (5.71 mmol, 1.06 g) was added to the reaction medium. After stirring for 30 minutes at 65 C. and under a reduced pressure of 150 mbar, the butanol was evaporated off while reducing the pressure from 150 mbar to 6 mbar over a period of 1 hour. The medium was left under a reduced pressure of 6 mbar for 1 hour 30 minutes to ensure the evaporation of the traces of butanol that were formed.

[0078] A step to remove the salts included or consisted of adding 500 molar equivalents of water (60 mL) and the mixture was stirred at 70 C. for 15 minutes. After stopping the stirring, the amide organic products flocculated at the surface of the water. On leaving the medium to cool to room temperature, the organic phase solidified and it was then easy to remove the water containing salts, and the solid flocculates were recovered. These flocculates, which are an extremely intense brown color, are formed from the crude product of alkyl L-guluronamide and D-mannuronamide and of the dodecylammonium mesylate salts. The final mass of crude product obtained was 1.21 g (2.9 mmol). This surfactant composition was named crude AlgN12.

[0079] The presence of a mixture of six isomeric forms (C.sub.22H.sub.43NO.sub.6, molar mass=417.59 g/mol) of N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (24%), N-(12-dodecyl)-n-butyl -L-gulurofuranosiduronamide (14%), N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (18%), N-(12-dodecyl)-n-butyl -L-guluropyranosiduronamide (10%), N-(12-dodecyl)-n-butyl -D-mannofuranosiduronamide (19%) and N-(12-dodecyl)-n-butyl -D-mannopyranosiduronamide (15%) was determined from the analysis of the 1D and 2D NMR spectra of the crude reaction product (without purification).

[0080] These data made it possible to calculate the pyranose/furanose ratio of the guluronamide forms (0.73), the pyranose/furanose ratio of the mannuronamide forms (0.79) and also the mannuronamide/guluronamide ratio (0.51).

[0081] Furthermore, the presence of the dodecylammonium mesylate salts formed during the aminolysis reaction was able to be quantified. Starting with 1 g of alginate (1 eq), this amount formed was 0.4 molar equivalent (0.0023 mol, 0.64 g).

Example 3: Measurements of the Interface Tensions (in the Case of the Sunflower Oil-Water System) for Compositions GN12, GN18, AlgN12, Algn18 AND Crude Algn12

[0082] The interface properties of the surfactant compositions GN12, GN18, AlgN12, AlgN18 and crude Algn12 were evaluated by measuring the oil-water interface tensions. The surfactants were dissolved in sunflower oil at concentrations ranging from 0.12 to 2.28 g/L. In order to promote the solubility of the surfactants in the oil, the solutions were left in an ultrasonic bath for 10 minutes at 50 C.

[0083] The measurements of the interface tensions between the oil and water were taken at 25 C. with a ring tensiometer (Krss, model K 100 C). The ring used was made of calibrated iridium platinum.

[0084] The interface tension between sunflower oil (Carrefour brand) and water at 25 C. ranged between 24.71 and 25.04 mN/m.

[0085] For each surfactant composition, the machine initially measured the surface tension of sunflower oil containing the surfactant (low-density liquid) and then the surface tension of water (high-density liquid). Finally, the oil was added delicately to the water, while avoiding the formation of bubbles, and the machine began measuring the interface tension between the sunflower oil and the water (mean of 10 measurements).

[0086] FIG. 1 shows that the surfactant compositions were capable of similarly reducing the water/oil interface tensions to values low enough to give the compositions emulsifying power. It was found that the composition crude AlgN12 obtained via the direct process for obtaining alkyl L-guluronamides and D-mannuronamides from alginate, with prior neutralization of the medium before the aminolysis reaction, was more efficient than the surfactant composition AlgN12 corresponding to the mixture of alkyl L-guluronamide and D-mannuronamide surfactants purified by chromatography (direct process for obtaining alkyl L-guluronamides and D-mannuronamides from alginate, without prior neutralization of the medium before the aminolysis reaction). These results showed the possibility of using a crude reaction product without the need for additional purification. Similarly, the compositions derived from dodecylamine (GN12, AlgN12, crude AlgN12) were more efficient than those derived from octadecylamine (GN18, AlgN18).

Example 4: Measurement of the Emulsifying Power of Compositions GN12, GN18, AlgN12, Algn18 AND Crude Algn12

[0087] The stability of the oil-in-water (O/W) and water-in-oil (W/O) emulsions formed from the surfactant compositions GN12, GN18, AlgN12, AlgN18 and crude Algn12 was studied in comparison with that of commercial alkylpolyglycosides: Montanov 82 from SEPPIC and Xyliance from Soliance/ARD.

[0088] The stability of the two types of O/W and W/O emulsions was evaluated by considering the two water/oil ratios 75/25 and 25/75, respectively, in round-bottomed graduated tubes: 0.5% of the surfactant product is introduced (20 mg). The sunflower oil was introduced (1 or 3 mL) and the surfactants were then dissolved in an ultrasonic bath for 10 minutes at 50 C. After dissolution of the emulsifier, ultrapure water was added (1 or 3 mL).

[0089] The two phases were then emulsified using an IKA Ultra-Turrax T18 basic homogenizer for 10 minutes at 11 000 rpm. The emulsion was placed in a bath thermostatically maintained at 20 C.

[0090] The evolution of the emulsion and its gradual demixing was observed for a few hours to several weeks.

[0091] FIG. 2 shows the results of analysis of the emulsifying power of the compositions of some embodiments.

[0092] The surfactant compositions derived from dodecylamine (GN12, AlgN12 and crude AlgN12) formed very stable O/W emulsions, including in the case of the crude surfactant composition (crude AlgN12) and also stable W/O emulsions (GN12 and AlgN12). The surfactant compositions derived from octadecylamine (GN18 and AlgN18) formed very stable W/O emulsions; the other O/W emulsions underwent total demixing after 5 hours.

[0093] For the two types of emulsions (W/O and O/W), the novel compositions (GN12, AlgN12 and crude AlgN12) formed emulsions that were more stable than the commercial references (Xyliance and Montanov).

Example 5: Antibacterial Activity of Various Fractions of Modified Products

[0094] Two protocols were used. The first (protocol A) was applied to fractions enriched in N-(12-dodecyl)-n-butyl -L-guluronamide isomers by chromatography on silica gel. The second (protocol B) was followed to test the activity of the surfactant compositions derived from dodecylamine (GN12, AlgN12 and crude AlgN12) and of the surfactant composition derived from octadecylamine (AlgN18).

Protocol A

1) Preparation of the Culture Medium:

[0095] The culture medium used was a mixture of 21 g/L of Muller-Hinton broth and 10 g/L of agar in water. This mixture was stirred and then left to boil. Next, a step of autoclaving of this mixture, for 30 minutes, was desired in this embodiment so as to sterilize it before any manipulation. This culture medium was poured hot into Petri dishes and then left to cool.

2) Preparation of the Modified Test Products:

[0096] 5 mg of each modified sugar were dissolved in 1 mL of DMSO. Twofold serial dilution with DMSO was then performed using the stock solution, so as to obtain concentrations of 2.5 g/L, 1.25 g/L, 0.625 g/L and 0.3125 g/L.

3) Preparation of the Bacterial and Fungal Suspensions:

[0097] The bacterial strains used were Pseudomonas aeruginosa, Escherichia Coli, Enterococcus faecium and Staphylococcus aureus, and also the fungal strain Candida albicans. 10.sup.6 bacteria were taken and then transferred into 0.9% NaCl solution. Each Petri dish, containing Muller-Hinton medium, was flooded with a different bacterial suspension.

4) Protocol:

[0098] After allowing the bacterial suspensions to dry on the agar, 10 L of each test solution, and at various concentrations, were deposited on the surface of the agar flooded with the bacterial suspension. 10 L of DMSO were placed in each Petri dish as a negative control.

[0099] The positive controls used were disks soaked with ampicillin for Escherichia coli and Enterococcus faecium, ceftazidime disks for Pseudomonas aeruginosa and vancomycin disks for Staphylococcus aureus.

[0100] After drying, the Petri dishes were finally incubated at 37 C. in an oven, for 24 hours. The antibacterial activity was evaluated by measuring the clarification zone in millimeters around the point of deposition of the various test solutions.

[0101] By way of example, the activity of fractions I, II and III enriched in isomers of N-(12-dodecyl)-n-butyl -L-guluronamide (-L-furanoside form=Fraction I; -L-pyranoside form=Fraction II; -L-pyranoside=Fraction III):

TABLE-US-00002 Fractions P. E. E. C S. enriched in Concentration aeruginosa coli faecium albicans aureus N-(12- 5 0 0 9 mm 4.5 mm 10.5 mm dodecyl)-n- 2.5 9 mm 4 mm 5 mm butyl--L- 1.25 7 mm 4 mm 5 mm gulurofurano- 0.625 0 4 mm 0 siduronamide 0.3125 0 0 0 (75%) Fraction I N-(12- 5 0 0 20 mm 5 mm 18.5 mm dodecyl)-n- 2.5 20 mm 3 mm 17 mm butyl--L- 1.25 11 mm 3 mm 5 mm guluropyrano- 0.625 8 mm 0 0 siduronamide 0.3125 8 mm 0 0 (74%) Fraction II N-(12- 5 0 0 0 9 mm 20.5 mm dodecyl)-n- 2.5 7 mm 18 mm butyl--L- 1.25 5 mm 0 guluropyrano- 0.625 0 0 siduronamide 0.3125 0 0 (94%) Fraction III Positive Ceftazidime Ampicillin Ampicillin Vancomycin control 28 mm 22 mm 26 mm 19 mm [00007] [00008] [00009] [00010] Fractions P. E. E. C S. enriched in Concentration aeruginosa coli faecium albicans aureus N-(18- 5 0 0 6 mm 0 0 octadecyl)- 2.5 6 mm n-butyl--L- 1.25 6 mm gulurofurano- 0.625 4 mm siduronamide 0.3125 0 (47%) N-(18- 5 0 0 4.5 mm 0 10 mm octadecyl)- 2.5 4 mm 0 n-butyl--L- 1.25 4 mm 0 gulurofurano- 0.625 3 mm 0 siduronamide 0.3125 0 0 (63%) N-(18- 5 0 0 6 mm 0 0 octadecyl)- 2.5 4 mm n-butyl--L- 1.25 0 guluropyrano- 0.625 0 siduronamide 0.3125 0 (86%) Positive Ceftazidime Ampicillin Ampicillin Vancomycin control 28 mm 22 mm 26 mm 19 mm [00011] [00012] [00013] [00014] [00015]

[0102] For the studies of the antibacterial and antifungal activities, the fractions derived from the purification of crude N-(12-dodecyl)-n-butylL-guluronamide were tested. Each fraction is more enriched in one isomer than the others.

[0103] Against the Gram-positive bacteria Enterococcus faecium and Staphylococcus aureus, N-(12 dodecyl)-n-butyl L-guluronamide (fraction enriched in pyranose form) showed a substantial capacity for inhibiting the growth of these two bacteria, of the order of 20 mm and 18.5 mm, respectively. This inhibitory activity decreased as the concentration decreased. It appears that the pyranose forms, which were predominantly present (94%), had no inhibitory effect against Enterococcus faecium, but showed a greater effect on the growth of the yeast Candida albicans (9 mm at 5 g/L), in addition to the strongest inhibitory effect against Staphylococcus aureus (20.5 mm at 5 g/L).

[0104] On the other hand, N-(18 octadecyl)-n-butyl L-guluronamide showed no inhibitory effect either on the Gram-positive bacterium Staphylococcus aureus or on the yeast Candida albicans.

[0105] Against the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, neither the N-(12 dodecyl)-n-butyl L-guluronamide nor the N-(18 octadecyl)-n-butyl L-guluronamide (whether in furanose or pyranose form) showed any inhibitory activity on the growth of these two bacteria.

[0106] Protocol B

[0107] The advantageous results of the antibacterial and antifungal activity, obtained for the fractions enriched in N-(12-dodecyl)-n-butyl -L-guluronamide isomers, encouraged the applicants to test the antibacterial and antifungal activity of mixtures of surfactant products obtained from guluronates (GN12) and from alginates (AlgN12, crude AlgN12 and AlgN18). For these surfactant mixture products, a new method was tested based on the study of the capacity of the surfactants of some embodiments to kill the bacteria, followed by counting of the number of live bacteria on Muller-Hinton agar.

[0108] 1) Preparation of the Bacterial and Fungal Inoculum:

[0109] The inoculum was prepared at a turbidity equivalent to 0.5 MacFarland (Biomerieux France), and then diluted to 1/100 (10.sup.6 CFU/ml). From this inoculum, a series of dilutions to 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.6 and 10.sup.6 was prepared. 100 l of each dilution were spread (counting method) onto the surface of a Muller-Hinton agar (determination of the number of bacteria in CFU/ml in the inoculum N.

[0110] 2) Preparation of the Modified Test Products:

[0111] Stock solutions were prepared for each surfactant GN12 (430 mg/ml), AlgN12 (310 mg/ml), crude AlgN12 (216 mg/ml) and AlgN18 (219 mg/ml). Twofold serial dilution with DMSO was performed for each product in Muller-Hinton broth: the final dilution was 1/128.

[0112] 3) Protocol:

[0113] 1 ml of the bacterial inoculum was added to each tube of the surfactant dilutions.

[0114] After incubation for 24 hours at 36 C., 100 l of each clear tube were spread onto the surface of a Muller-Hinton agar, followed by incubation for 24 hours at 37 C.

[0115] The number of live bacteria was determined: N0=n10 CFU/ml (n=number of colonies).

[0116] The percentage of live bacteria was calculated: N0/N100:

TABLE-US-00003 Name of Concentration Name of the the prepared bacterium product (mg/ml) Results Escherichia AlgN.sub.12 310 mg/ml 155 mg/ml => Inhibition of 100% of the bacteria coli 77.5 mg/ml => Inhibition of 100% of the bacteria 38.75 mg/ml => Inhibition of 100% of the bacteria 19.375 mg/ml => Inhibition of 99.99% of the bacteria AlgN.sub.12 216 mg/ml 108 mg/ml => Inhibition of 100% of the bacteria (Crude) 54 mg/ml => Inhibition of 100% of the bacteria 27 mg/ml => Inhibition of 100% of the bacteria 13.5 mg/ml => Inhibition of 99.99% of the bacteria AlgN.sub.18 219 mg/ml 109.5 mg/ml => Inhibition of 100% of the bacteria 54.75 mg/ml => Inhibition of 100% of the bacteria 27.375 mg/ml => Inhibition of 99.99% of the bacteria GN.sub.12 430 mg/ml 215 mg/ml => Inhibition of 100% of the bacteria 107.5 mg/ml => Inhibition of 99.99% of the bacteria Pseudomonas AlgN.sub.12 310 mg/ml 155 mg/ml => Inhibition of 100% of the bacteria aeruginosa 77.5 mg/ml => Inhibition of 100% of the bacteria 38.75 mg/ml => Inhibition of 100% of the bacteria 19.375 mg/ml => Inhibition of 99.9% of the bacteria AlgN.sub.12 216 mg/ml 108 mg/ml => Inhibition of 100% of the bacteria (Crude) 54 mg/ml => Inhibition of 100% of the bacteria 27 mg/ml => Inhibition of 100% of the bacteria 13.5 mg/ml => Inhibition of 99.9% of the bacteria AlgN.sub.18 219 mg/ml 109.5 mg/ml => Inhibition of 100% of the bacteria 54.75 mg/ml => Inhibition of 100% of the bacteria 27.375 mg/ml => Inhibition of 100% of the bacteria GN.sub.12 430 mg/ml 215 mg/ml => Inhibition of 100% of the bacteria 107.5 mg/ml => Inhibition of 100% of the bacteria 53.75 mg/ml => Inhibition of 100% of the bacteria Enterococcus AlgN.sub.12 310 mg/ml 2.42 mg/ml => Inhibition of 100% of the bacteria faecium AlgN.sub.12 216 mg/ml 1.7 mg/ml => Inhibition of 100% of the bacteria (Crude) AlgN.sub.18 219 mg/ml 109.5 mg/ml => Inhibition of 100% of the bacteria 54.75 mg/ml => Inhibition of 100% of the bacteria 27.375 mg/ml => Inhibition of 100% of the bacteria GN.sub.12 430 mg/ml 3.36 mg/ml => Inhibition of 100% of the bacteria Candida AlgN.sub.12 310 mg/ml 2.42 mg/ml => Inhibition of 100% of the bacteria albicans AlgN.sub.12 216 mg/ml 1.7 mg/ml => Inhibition of 100% of the bacteria (Crude) AlgN.sub.18 219 mg/ml 109.5 mg/ml => Inhibition of 100% of the bacteria 54.75 mg/ml => Inhibition of 100% of the bacteria 27.375 mg/ml => Inhibition of 100% of the bacteria GN.sub.12 430 mg/ml 3.36 mg/ml => Inhibition of 100% of the bacteria Staphylococcus AlgN.sub.18 219 mg/ml 109.5 mg/ml => Inhibition of 100% of the bacteria aureus

[0117] Against the Gram-positive bacterium Enterococcus faecium, the mixture of crude AlgN12 (with neutralization prior to the aminolysis reaction) appears to be more efficient than the mixture AlgN12 (process without neutralization prior to the aminolysis reaction) since a concentration of 1.7 mg/mL of AlgN12 (crude) is sufficient to inhibit 100% of Enterococcus faecium, whereas this concentration is higher in the case of AlgN12 (2.42 mg/ml). Furthermore, the mixture of surfactants derived from the chemical modification of alginate (mixture of mannuronamide and guluronamide) has stronger power against Enterococcus faecium than the surfactants based on guluronamides alone (higher concentration of GN12 of the order of 3.36 mg/mL to inhibit 100% of Enterococcus faecium). This observation is the same for the inhibition of Candida albicans.

[0118] Against the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, very high concentrations of surfactants may be required to inhibit these two bacteria to 100%; showing that AlgN12 (38.75 mg/ml), crude AlgN12 (13.5 mg/ml) and GN12 (53.75 mg/ml) had low antibacterial power against these two types of bacteria.

[0119] By comparing AlgN12 and AlgN18, the alginate amide surfactant product bearing an octadecyl chain has lower antibacterial power against the Gram-positive and Gram-negative bacteria tested. A high concentration of AlgN18 may be necessary to inhibit 100% of the bacteria Enterococcus faecium (27.375 mg/ml), Pseudomonas aeruginosa (27.375 mg/ml), Escherichia coli (54.75 mg/ml) and Candida albicans (27.375 mg/ml).

LIST OF REFERENCES

[0120] 1Hill and Lehen-Ferrenbach, In Sugar-based surfactants fundamentals and Applications, C. C. Ruiz (Ed.), 1-20, CRC Press, ISBN 978-1-4200-5166-7, 2009 [0121] 2Laurent et al., J. Surfact. Deterg., 14: 51-63, 2011 [0122] 3International patent application WO 92/06984 [0123] 4International patent application WO 93/03004 [0124] 5U.S. Pat. No. 7,655,611 [0125] 6U.S. Pat. No. 5,872,111 [0126] 7U.S. Pat. No. 2,670,345 [0127] 8Benvegnu and Sassi, Topics in Current Chemistry, 294: 143-164, 2010 [0128] 9International patent application WO 03/104 248 [0129] 10International patent application WO 03/099 870 [0130] 11International patent application WO 2009/134 368 [0131] 12International patent application WO 98/40511