WO2008043892A1

WO2008043892A1 - Novel polysaccharide, process for the preparation thereof and uses thereof, in particular in the cosmetics field

Info

Publication number: WO2008043892A1
Application number: PCT/FR2007/001541
Authority: WO
Inventors: Asma Hassan; Wafa Achouak; Odile Berge; Thierry Heulin; Alain Heyraud; Michel Milas; Ludovic Deline; Ghislain Sanhaji; Anthony Bresin
Original assignee: Agro Industrie Recherches Et Developpements A.R.D.; Centre National De La Recherche Scientifique; Comissariat A L'energie Atomique C.E.A.
Priority date: 2006-10-09
Filing date: 2007-09-21
Publication date: 2008-04-17
Also published as: FR2906819A1; FR2906819B1

Abstract

The present invention relates to the use of the bacterial strain ALV104 of the family Rhizobiaceae, deposited with the CNCM on 21 March 2006 under number I-3591, for the preparation of a polysaccharide compound corresponding to the following formula (I): in which: - k ranges from 1 to n, - n is an integer from 5 to 300, in particular from 8 to 270, - Rk, Rk2, Rk3, Rk4, Rk5, Rk6, Rk7 and Rk8 are, independently of one another, H or an acyl group containing from 1 to 6 carbon atoms.

Description

NOVEL POLYSACCHARIDE, PROCESS FOR PREPARING THE SAME AND USES THEREOF ESPECIALLY IN THE COSMETIC FIELD

The subject of the present invention is a novel polysaccharide, as well as its method of preparation starting from a bacterial strain, and its uses in particular in the cosmetic field.

Soil is an inexhaustible reserve of microorganisms with innovative properties all related to a biological function. Within the part of the soil called the rhizosphere, there is a specific bacterial population having an ability to synthesize polysaccharides. The role of these polysaccharides at the soil level is to allow adhesion between the bacterium and the plant and to structure the soil in the near vicinity of the latter.

Many bacteria from the rhizosphere belong to the genus Rhizobium. These bacteria possess the ability to form symbiotic nodules with plants.

The present invention aims to provide a bacterial strain producing a polysaccharide compound having properties of interest for cosmetic applications.

The object of the present invention is to provide a polysaccharide having properties of interest for cosmetic applications.

The present invention relates to the use of the bacterial strain ALV1 04 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, or of a strain whose rrs gene has a higher percentage of identity. or equal to 99.9%, preferably equal to 100%, with the rrs gene of said strain ALVl 04, for the preparation of a polysaccharide compound, corresponding to the following formula (I):

in which: - k varies from 1 to n, n represents an integer from 5 to 300, in particular from 8 to 270,

- R ^k i, R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k ₈ represent, independently of one another, H or an acyl group comprising from 1, 2 , 3, 4, 5 or 6 carbon atoms, especially an acetyl group. The percent identity between two genes refers to the percentage of identical bases between these two genes.

The above-mentioned ALV 104 bacterium has been isolated for its particular properties in synthesizing a polysaccharide during a screening of microorganisms present in the soil of a common plant: Arabidopsis thaliana. This strain was classified through molecular genetic tools, as belonging to the family of

Rhizobiaceae and it was filed on 21 March 2006 with the CNCM under the Budapest Treaty under registration number 1-3591.

The above-mentioned polysaccharide compound of formula (I) consists of a repeating unit comprising 3 sugars, including 2 neutral sugars and 1 acidic sugar, namely mannuronic acid, glucose and galactose.

The aforementioned strain, filed on 21 March 2006 with the CNCM under the Budapest Treaty under registration number 1-3591, was identified after sequencing its rrs gene which codes for 16S ribosomal RNA.

The phylogenetic study of the rrs gene coding for the small ribosome subunit (16S rDNA) shows that the ALV 104 strain is a bacterium belonging to the genus

Rhizobium, from the family Rhizobiaceae, in the order Rhizobiales of the alpha subdivision of Proteobacteria.

The group of species most closely described phylogenetically of this strain are Rhizobium sullae (Squartini A, Struffi P, Deding H., Selenska-Pobell S., ToIa E., Giacomini A., Vendramin E., Velazquez E., Mateos PF, Martnez-Molina E, Dazzo FB,

Casella S. and Nuti MP (2002). Rhizobium sullae sp. Nov. (formly 'Rhizobium hedysarf), root-nodule microsymbiont of Hedysarum coronarium L. Int J Syst Evol Microbiol 52, 1267-1276), Rhizobium indigoferae (Wei GH, Wang AND ₅ Tan ZY, Zhu ME and Chen WX (2002) Rhizobium indigoferae, Nov., and Sinorhizobium kummerowiae, Nov., Indigofera spp., And Kummerowia stipulacea, Int J Syst Evol Microbiol, 52, 2231-2239), Rhizobium yanglingense (Tan ZY, Kan FL, Peng GX). , Wang ET, Reinhold-Hurek B and Chen WX (2001) Rhizobium yanglingense sp nov, isolated from arid and semi-arid regions in China, Int J Syst Evol Microbiol, 51, 909-914), Rhizobium gallicum (Amarger N, Macheret V and Laguerre G (1997) Rhizobium galicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules Int J Syst Bact, 47, 996-1006), and Rhizobium mongolense (van Berkum, P, D Beyene, Bao G ₅ Campbell TA and Eardly BD.1998. mongolense Rhizobium sp. November is one of three rhizobial genotypes identified which nodulate and form nitrogen-fixing symbioses with Medicago ruthenica [L. Ledebour] Int J Syst

Bact, 48, 13-22). However, the sequence of the rrs gene of the ALV 104 strain has a maximum identity percentage of 98.8% with that of the rrs gene of the standard strains of each of these species, suggesting that it belongs to another species.

The sequence of the rrs gene of the ALV 104 strain is also very close to those of three other strains, S 109, USDA1920 and YAS34, present in GenBank.

(Accession No. AY509211, U89823 and AF23924, respectively). Strain S109, erroneously named R. galicum, has an identity percentage of 99.7% with ALV 104 for its rrs sequence. For the strain Rhizobium sp. USDA 1920 this percentage is 99.6%. Van Berkum et al. (1998) showed that the USDA1920 strain belongs to a new genomic species that they have not formally described. Strain

YAS34 isolated from the rhizosphere of sunflower on French soil (Alami Y., Achouak W., Marol C. and Heulin T. (2000) Rhizosphere soil aggregation and plant growth promotion of sunflower by an EPS-producing Rhizobium sp. sunfiower roots, Appl., Environ Microbiol, 66 (8), 3393-3398), shares 99.5% of its rrs gene with ALV104 and belongs to a species that also remains to be described.

The genetic phylogenetic tree constructed from the one hundred GenBank sequences closest to that of the ALV104 rrs gene, makes it possible to visualize these four strains which form a monophyletic group and which probably represents one and the same new species. The DNA fingerprinting technique was used to show that the strain

ALV 104 is different from strain YAS34. The fingerprints are obtained by amplification of the total DNA of the strains in the presence of repeated rep type sequences. The REP primers were used with both strains and the electrophoretic profiles of amplified DNA fragments are clearly different for the two strains: the REP-PCR fingerprint of ALV104 comprises 9 major bands of sizes between 500 and 4000 pairs of bases (pb) including a very intense band of approximate size 2500 bp while in the same range, the REP-PCR footprint YAS34 strain comprises 6 major bands including a very intense size of approximately 1400 bp and none of size 2,500 bp. The strain deposited on March 21, 2006 with the CNCM under the Budapest Treaty under the registration number 1-3591 is a polymorphic, mobile, Gram-negative, strict aerobic, catalase positive and oxidase-negative bacillus.

Its ability to assimilate a wide variety of carbohydrate substrates allows the production of polymer by this strain from a wide variety of carbohydrate substrates derived from the valorization of co-products of agricultural origin.

This strain has the biochemical characteristics described in the table below:

The membership of this strain in the family Rhizobiaceae is determined by the results of the above tests, namely: Gram, fresh state, oxidase, catalase, respiratory type and blood agar.

The present invention also relates to the use as defined above, of the bacterial strain ALV 104 as defined above, for the preparation of a polysaccharide compound of formula (I) as defined above, in which at least 12.5% of all R \, ^k R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents an acetyl group. The present invention also relates to the use as defined above, of the bacterial strain ALV 104 as defined above, for the preparation of a polysaccharide compound of formula (I) as defined above, in which from about 40% to about 60%, especially from about 43% to about 56%, and preferably from about 43.75% to about 56.25% of all of the groups R ^k _ls R ^k ₂ , R ^k ₃ ,

^K R _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents an acetyl group.

The present invention also relates to the use as defined above, of the bacterial strain ALVl 04 as defined above, for the preparation of a polysaccharide compound of formula (I) as defined above, in which at least 12.5% of all groups

^K R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents a hydrogen atom.

The present invention also relates to the use as defined above, of the bacterial strain ALVl 04 as defined above, for the preparation of a polysaccharide compound of formula (I) as defined above, in which at least 60% of all R groups ^k i, ^k R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents a hydrogen atom.

A polysaccharide compound of formula (I) as defined above, in which at least 60% of all the groups R ^k i, R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k ₈ represents a hydrogen atom, corresponds to the deacetylated form of the polysaccharide compound of the invention.

The present invention also relates to the use as defined above, of the bacterial strain ALV1 04 of the family Rhizobiaceae, deposited at the CNCM March 21, 2006 under the number 1-3591, or a strain whose gene rrs has a percentage of identity greater than or equal to 99.9%, preferably equal to 100%, with the rrs gene of said strain ALVl 04, for the preparation of a compound of formula (I) as defined above. above, in a suitable culture medium, comprising in particular a nitrogen source, such as yeast extract or proteins of organic origin such as peptone plant peptones of wheat, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese. The present invention also relates to a polysaccharide compound having the following formula (I):

in which :

k varies from 1 to n, n represents an integer from 5 to 300, in particular from 8 to 270,

- R ^k _l3 R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k ₈ represent, independently of each other, H or an acyl group comprising from 1 to 6 atoms carbon, especially an acetyl group.

According to an advantageous embodiment, on the 8 hydroxyl groups of the repeating unit of the polymer of the invention, between 3.5 and 4.5 acetyl groups are present.

The number of n of the repeating unit is between 20 and 270, which corresponds to a molecular weight of between 250,000 and 3,000,000 g / mol during the synthesis of the polymer by the strain in the fermentation must. The present invention also relates to a compound as defined above, in which at least 12.5% of all the groups R ^k _b R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k ₈ represents an acetyl group, and in particular wherein at least 12.5% of all the groups

R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k ₈ , for a given value of k, represents an acetyl group. A preferred compound according to the present invention is a compound as defined above, wherein at least about 43% to about 56%, and preferably about 43.75% to about 56.25% of the total R \, R ^k _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents an acetyl group.

According to a particular embodiment, the compounds of the invention as defined above may be in the form of a double helix.

The present invention also relates to a compound as defined above, characterized in that at least 12.5% of all the groups R ^k _ls R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R ^k g represents a hydrogen atom. A preferred compound according to the present invention is a compound as defined above, wherein at least 60% of the total groups R _l5 ^k ^k R _2, R ₃ ^k, R ^k ₄ ^k R _5, R ₆ ^k , R ^k ₇ and R ^k g represents a hydrogen atom. This compound corresponds to the deacetylated form of the compound of the invention.

According to a preferred embodiment, the present invention also relates to a compound of formula (I) as defined above, wherein all groups R ^k _l3 R ^k ₂ R ^k _3, R ^k _4, R ^k _5, R ^k ₆ , R ^k ₇ and R ^k ₈ represent a hydrogen atom. Such a compound has the following formula (I-1):

The present invention also relates to a composition comprising a compound as defined above, in combination with a bacterial strain as defined above and / or the culture medium or a part of the culture medium of said strain, comprising in particular a a source of nitrogen, such as yeast extract or proteins of organic origin such as peptone, peptone, wheat, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese.

According to an advantageous embodiment, the composition of the invention is characterized in that it comprises from about 0.1% to about 8% by weight of a compound of formula (I) as defined above, and from about 0.05% to about 2% by weight of a bacterial strain as defined above.

The present invention also relates to a composition comprising a compound of formula (I) as defined above, in association with a bacterial strain as defined above, in particular strain ALVl 04.

The present invention also relates to a composition comprising a compound of formula (I) as defined above, in association with a culture medium comprising in particular a source of nitrogen, such as yeast extract or proteins of organic origin as plant peptones of the peptone type of wheat, alfalfa, potato, a source of carbon such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese. The present invention also relates to a composition comprising a compound of formula (I) as defined above, in association with a bacterial strain as defined above and the culture medium or a part of the culture medium of said strain, including a nitrogen source, such as yeast extract or proteins of organic origin such as plant peptones such as wheat peptones, alfalfa, potato, a carbon source such as glucose, maltose , mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese.

The present invention also relates to a pharmaceutical, agri-food or cosmetic composition, comprising a compound of formula (I) as defined above, in combination with a suitable vehicle.

The present invention also relates to a method for preparing a composition as defined above comprising a fermentation step in the presence of a bacterial strain ALV1 04 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, or of a strain whose rrs gene has a percentage of identity greater than or equal to 99.9%, preferably equal to 100%, with the rrs gene of said strain ALV104, and a medium of culture including a nitrogen source, such as yeast extract or proteins of organic origin such as plant peptones such as wheat peptones, alfalfa, potato, a carbon source such as glucose , maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese.

The present invention also relates to a process for preparing a compound of formula (I) as defined above, comprising the following steps:

a fermentation step in the presence of a bacterial strain ALV1 04 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, or of a strain whose rrs gene exhibits a percentage of identity greater than or equal to 99.9%, preferably equal to 100%, with the rrs gene of said strain ALV104, and a culture medium comprising in particular a source of nitrogen, such as yeast extract or proteins of organic origin such as plant peptones such as wheat peptones, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese, in order to obtain a composition comprising a compound of formula (I) as defined above, in association with a bacterial strain as defined above and the culture medium as defined above, and

a filtration step, in particular of tangential filtration, in order to get rid of said bacterial strain and of said culture medium, and to obtain the polysaccharide compound of the invention of formula (I).

The present invention also relates to a method of preparation as defined above, comprising, after the filtration step, a step of precipitating the polysaccharide compound of formula (I), in order to obtain said compound in powder form.

The present invention also relates to a process of preparation as defined above, comprising, below ^as the step of precipitating the polysaccharide compound, a step of dissolving the compound obtained in powder form, in order to conduct the deacetylation complete of said compound, and to obtain a deacetylated compound as defined above.

The present invention also relates to a method of preparation as defined above, characterized in that a complete deacetylation step of the compound of formula (I) as defined above is carried out after the fermentation step and before filtration step, in order to obtain a deacetylated compound as defined above. The present invention also relates to a process for preparing a deacetylated compound as defined above, comprising the following steps:

a fermentation step in the presence of a bacterial strain ALV1 04 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, or of a strain whose rrs gene exhibits a percentage of identity greater than or equal to 99.9%, preferably equal to 100%, with the rrs gene of said strain ALVl 04, and a culture medium comprising in particular a source of nitrogen, such as yeast extract or proteins of organic origin such as plant peptones such as wheat peptones, alfalfa, potatoes, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese, in order to obtain a composition comprising a compound of formula (I) as defined above , in combination with a bacterial strain as defined above and a culture medium as defined above, a complete deacetylation step of the compound of formula (I) as defined above, to obtain a composition comprising a deacetylated compound as defined above, in association with said bacterial strain and said culture medium, and

a filtration step, in particular of tangential filtration, in order to get rid of said bacterial strain and of said culture medium, and to obtain the deacetylated polysaccharide compound as defined above.

The present invention also relates to a process as defined above comprising a further step of hydrolysis of the polysaccharide of formula (I), this step making it possible to obtain a hydrolyzed polysaccharide of formula (I) in which n represents an integer inclusive from 8 to 120.

This hydrolysis step can be performed indifferently before or after the above-mentioned deacetylation step.

The present invention also relates to the use of the polysaccharide compound of formula (I) as defined above, for the preparation of a cosmetic composition.

The present invention also relates to the use of the polysaccharide compound of formula (I) as defined above, as a texturizer or touch, or as an emulsion stabilizer.

The term "texturing or touching agent" refers to a molecule or set of molecules that significantly modifies the sensory touch properties of a cosmetic preparation.

The term "emulsion stabilizing agent" refers to a molecule or a set of molecules, allowing in the presence of a surfactant molecule, to maintain in a single phase a naturally unstable fatty substance / water mixture. The present invention also relates to the use of the polysaccharide compound of formula (I) as defined above, in the field of microbiological diagnosis, in particular as a growth support for microorganisms. The present invention also relates to the use of the polysaccharide compound of formula (I) as defined above, in gel form, in total or partial substitution of the gelling bases used in the culture media, in particular to replace the gel of Agarose or Agar Agar. The present invention also relates to a method of preparation as defined above, comprising, after the step of fermentation of the bacterial strain, a step of pasteurization of the composition as defined above, comprising a compound of formula (I ) as defined above, in combination with a bacterial strain as defined above and / or the culture medium or part of the culture medium of said strain as defined above, in order to inactivate the bacteria constituting the bacterial strain, this pasteurization step corresponding to a step of maintaining said composition at a temperature of about 45 ° C. to about 90 ^° C. for about 5 minutes to about 60 minutes.

The present invention also relates to a composition comprising a compound of formula (I) as defined above, in association with a bacterial strain as defined above and / or the culture medium or part of said culture medium. strain as defined above, comprising in particular a nitrogen source, such as yeast extract or proteins of organic origin such as peptone plant peptones wheat, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese, characterized in that the bacteria constituting said bacterial strain are killed.

DESCRIPTION OF THE FIGURES

Figure 1 shows the evolution of the viscosity of a solution of 1 g / L of the polymer in 0.1 M NaCl at 25 ° C depending on the shear rate and the storage time. The ordinate axis represents the viscosity in mPa.s and the abscissa axis the shear rate in s ^"1. The curve with the black squares (m) corresponds to the initial time (no storage), the curve with the white diamonds (0) corresponds to a storage time of 4 days, the curve with the black crosses (x) corresponds to a storage time of 6.5 days, the curve with the black circles (•) corresponds to a time of storage time of 8.5 days, the curve with the white triangles (Δ) corresponds to a storage time of 11 days, the curve with the black diamonds (>) corresponds to a storage time of 39 days, the curve with the triangles black (A) corresponds to a storage time of 46 days, the curve with the circles (Θ) corresponds to a storage time of 46 days, the curve with the curves with the white circles (O) corresponds to a time storage time of 88 days and the curve with the white squares (D) corresponds to a storage time of 95 days. The absence of some of the symbols mentioned above stems from the perfect superposition of the different points.

Figure 2 shows the evolution of the viscosity of an emulsion as a function of pH. The abscissa axis represents the pH and the ordinate axis represents the viscosity in mPa.s (Brookfield DVIII Ultra Aig E-Vit 12 Helipath 2 min, 20 ^° C.). The solid curve with the black diamonds (4) corresponds to the product Sepigel 305; the dashed line curve with the black squares corresponds to the product Rheocare ATH (m) and the gray solid line curve with the black triangles corresponds to the product of the invention.

DETAILED DESCRIPTION OF THE INVENTION METHODS

The production of the polysaccharide of the invention at the industrial level follows two main stages, a first step of producing cells in small vials; this first culture called preculture is used to inoculate the fermenter which itself is carried out the production of the polymer over time ^(2nd step).

The preculture chain includes the revival of the strain preserved in a cryobank at -196 ° C (liquid nitrogen) by pouring the contents of a cryotube with a capacity of 2 ml (noted N ₂ ) into an Erlenmeyer flask of a capacity 500 ml containing a preculture medium whose composition is described below.

The preculture medium for the production of polymer has the following composition:

After incubation at 30 ^° C. +/- 3 ° C. for a period of between 15 and 30 hours, 10 ml of this culture are transferred to Erlenmeyer flasks with a capacity of 3 liters, labeled I for Inoculum, S for monitoring and Se. for help. Only the container marked I will be used to inoculate the fermenters.

The Erlenmeyer noted S is used for monitoring and that noted corresponds to the rescue in case of contamination of the Erlenmeyer noted I.

At each transfer a bacteriological control is carried out by a microscopic observation in phase contrast at a magnification of 100 times (Microscope Axioskop2 Cari Zeiss, Germany). The parameters of preculture as those of culture are the following ones:

- pH: 6.8 +/- 0.3

- Temperature: 30 ⁰ C +/- 3 ° C

- Duration 24 hours.

When the measurement of the optical density at 600 nm of the preculture reaches 2.00 or the total duration of the preculture is 24 hours, the medium is transferred sterilely into a 450 liter fermenter whose culture medium is as follows:

The inoculation rate of the fermenter is between 1 and 10% and preferably 5% volume of preculture / volume of the fermenter.

After sterilization of the medium, the glucose (carbon source) is added in order to reach a concentration of 25g / L at the time of inoculation of the fermenter.

The ratio between the carbon and the nitrogen present in the culture medium is an important parameter of the behavior of the strain in the face of a deficiency of carbon or nitrogen elements. To verify the influence of this ratio on the production of EPS (exopolysaccharide - polysaccharide compound of the invention) and on the production of biomass, the optical density at 600 nm and the final viscosity of the fermentation must are analyzed. The optical density at 600 nm accounts for the number of bacterial cells present in the culture medium while the final viscosity estimates the amount of polymer produced by the bacterium. The table below shows the influence of the C / N ratio on the OD 600 nm and on the viscosity:

The aforementioned tested media have the following compositions

In order to maximize the viscosity of the must and hence the production of the polymer, the C / N ratio for the production of the polymer should be between 10 and 50 and is preferably about 30.

The quality of the nitrogen present in the C / N ratio plays an important role in the growth of the bacteria and in the production of the polymer. Some amino acids are essential for the growth of the bacteria. Indeed, if the bacterial strain does not find a source of amino acids, it does not grow. Thus, the necessary amino acids are present in the form of peptides or proteins in a yeast extract or in peptones. Thus, the culture medium used in the method of the invention contains in particular peptones rich in amino acids essential for the bacterial strain.

An analysis of the qualitative needs of the strain provides the following results:

At the end of the industrial fermentation stage, the following results are obtained:

The latency phase is a phase during which nothing is observed, that is to say a phase during which there is no consumption of glucose and where no disorder is observed. The wort at the end of fermentation contains the culture medium as described above that has not been consumed by the bacteria, the bacterial cells (or bacteria) and the polysaccharide (or polymer) of the invention.

According to the purification method used, it is possible to obtain different forms of the molecule:

1) Raw must containing the polymer:

At the end of the fermentation, the bacterial strains are inactivated by the use of a chemical biocide. The culture must containing the polymer can thus be used for applications in the cosmetics, pharmacy or food industry for its particular rheological properties.

The final product obtained after this treatment contains the culture medium, the inactivated bacteria and the polymer. The polymer in this form has a number n of repeating units from 20 to 270.

2) Thermally treated fermentation must:

At the end of the fermentation, in order to "kill" the bacteria, the must (containing the culture medium, the bacteria and the polymer) is heated to a temperature ranging from about 35 ° C. to about 80 ^° C. and preferentially about 60 ^° C. for a period of about 25 minutes to about 2 hours and preferably about 40 minutes. This treatment allows both a sterilization of the fermentation must, but also gives the polymer new rheological properties that the native molecule does not present. The sterile fermentation must can be used in particular in the industrial sectors of cosmetics, pharmaceuticals or agri-food because of its particular rheological properties.

The final product obtained after this treatment contains the culture medium, the killed bacteria and the polymer. The polymer in this form has a number n of repeating units from 20 to 240.

3) Fermentation broth purified by filtration then precipitated:

At the end of the fermentation, the must having a viscosity between 550 and 1600 Centipoises (Cps) (RJiéomat 205 viscometer) is diluted with osmosis water following a dilution factor between 1/4 and 1/10 in order to obtain a viscosity of the wort diluted between 15 and 150 cps. A micro filtration step follows the dilution step.

The purification of this polymer can be carried out according to various techniques: centrifugation, frontal filtration as tangential or any other method allowing a separation of constituents.

Filtration allows the separation of bacterial cells from the culture medium containing the polymer. The thresholds of frontal filtration cutoff allowing an optimal separation of the product, lie in a range of 0.6 microns and 1.2 microns.

Thus, this first filtration makes it possible to eliminate the bacteria and to obtain a product containing only the culture medium and the polymer.

The table below describes the response of the product according to the cut-off threshold for a frontal filtration on a syringe filter at the laboratory stage.

Tangential filtration allows a separation compatible with the industrial imperatives of production. The membrane cut-off thresholds that can be used are between 0.1 μm and 1.2 μm. The wort dilution should be adjusted according to the viscosity parameters of the fermentation must in order to reduce the final must viscosity of 1600 cps to a viscosity of between 15 and 150 cps.

This diluted must is subsequently concentrated in order to obtain a concentration of polymer that meets the specifications of the cosmetics, pharmaceutical or agrifood industry, namely from about 0.1 to about 100 g / L and from preferably equal to about 10 g / l of polymer.

The techniques used for the concentration of the wort may be evaporation techniques such as evaporation under reduced pressure or thin film evaporator or preferably by ultrafiltration techniques. Ultrafiltration can be carried out by a tangential filtration system involving inorganic or organic membranes having a cutoff threshold of between about 10,000 Daltons and about 500,000 Daltons, with a cutoff threshold preferably of about 150,000 Daltons. Tangential filtration is preferably used for the sake of efficiency and cost of treatment. The material used for the microfiltration stage is a tangential filtration system with mineral membranes having a cutoff threshold of between 0.1 and 1.2 μm and preferably 0.22 μm.

At the end of the purification process, to the ultrafiltration retentate, 0.2 M NaCl is added in order to screen the molecule, that is to say to reduce the affinity of the polymer with respect to the water, then a first alcoholic solvent, for example, ethanol, is added to obtain a first precipitate. The precipitate is then filtered off and then brought into contact with a hydro-alcoholic mixture having an increasing alcoholic strength. The alcoholic solvent is used to precipitate the polymer and thus to eliminate the culture medium. The polymer in this form has a number n of repeating units of 20 to 220.

At the end of the precipitation step, the precipitate is dried under vacuum and conditioned. The pure polymer is obtained in the form of a powder exhibiting great stability over time. Thus monitoring the viscosity of a 1 g / l solution of the polymer of the invention stored at 25 ^° C. over a period of 95 days makes it possible to obtain the profiles represented in FIG. 1. The absence of a decrease in the viscosity at low shear stress (between

10 ^" and 10 ^" s ^" ) demonstrates that maintaining a solution at 1 g / L of polymer at 25 ⁰ C for 95 days has little effect on the polymer.

The polymer thus obtained has filmogenic properties.

4) Fermentation broth hydrolyzed then purified and precipitated:

After the fermentation, the must is adjusted to a pH of between 1.5 and 4.0 and preferably 2.0 by the addition of a strong acid such as, for example, sulfuric acid. The wort is then heated to a temperature of from about 60 ° C. to about 95 ^° C. and preferably about 92 ° C. over a period of about 3 to about 15 hours and preferably about 5 hours. At the end of these two stages, the polymer is hydrolyzed and the bacteria are killed.

After the fermentation, the must having a viscosity ranging from 550 to 1600 Centipoises (Cps) (Rheomat 205 Viscometer) is diluted with osmosis water at a dilution factor between 1/4 and 1/10 in order to obtain a viscosity of the must diluted between 15 and 150 cps. A microfiltration step to remove bacteria from the fermentation must follows the dilution step. Tangential filtration is preferred by a concern for efficiency and cost of treatment. The material used for the microfiltration stage is a tangential filtration system with mineral membranes having a cutoff threshold of between 0.1 and 1.2 μm and preferably 0.22 μm.

This hydrolysed and diluted must is subsequently concentrated in order to obtain a concentration of polymer that meets the specifications of the cosmetic, pharmaceutical or food industry, ie from about 0.1 to about 100 g / l and preferably about 10 g / l of polymer. The techniques used for concentration and precipitation are those as previously described in particular in paragraph 3.

At the end of the precipitation step, the precipitate is dried under vacuum and conditioned. The pure hydrolyzed polymer is obtained in powder form. The polymer in this form has a number n of repeating units from 8 to 120.

5) Deacetylated fermentation must then purified and precipitated:

After the fermentation, the must is cooled to room temperature (20 ° C) and the pH is adjusted to between 10 and 14, preferably to 12 by the addition of a base (sodium hydroxide for example) during a duration of 5 hours. Acetyl groups are removed through this treatment. This step corresponds to the deacetylation of the compound of the invention, which makes it possible to obtain a deacetylated compound of formula (1-1) as defined above.

At the end of the fermentation, the must having a viscosity between 550 and 1600 Centipoises (Cps) (Rheomat 205 Viscometer) is diluted with osmosis water at a dilution factor between 1/4 and 1/10 to obtain a viscosity of the wort diluted between 15 and 150 cps. A microfiltration step to remove the biomass follows the dilution step. Tangential filtration is preferred by a concern for efficiency and cost of treatment. The material used for the microfiltration stage is a tangential filtration system with mineral membranes having a cutoff threshold of between 0.1 and 1.2 μm and preferably 0.22 μm.

This deacetylated and diluted must is subsequently concentrated in order to obtain a concentration of polymer that meets the specifications of the cosmetic, pharmaceutical or food industry, ie from about 0.1 to about 100 g / L and from preferably equal to about 10 g / l of polymer. The techniques used for concentration and precipitation are those as previously described in particular in paragraph 3.

At the end of the precipitation step, the precipitate is dried under vacuum and conditioned. The pure deacetylated polymer is obtained in the form of a powder. This polymer has in particular filmogenic properties. The polymer in this form has a number n of repeating units of 20 to 220.

6) Deacetylated fermentation broth, hydrolyzed then purified and precipitated:

After the fermentation, the must is cooled to room temperature (20 ° C) and the pH is adjusted between 10 and 14, preferably to 12 by adding a base

(sodium hydroxide for example) for a period of 5 hours. Acetyl groups are removed through this treatment. This step corresponds to the deacetylation of the compound of the invention, which makes it possible to obtain a deacetylated compound of formula (1-1) as defined above. The deacetylated wort is adjusted to a pH of about 1.5 to about 4.0 and preferably about 2.0 by the addition of a strong acid such as, for example, sulfuric acid. The wort is then heated to a temperature of about 95 ° C. to about 60 ^° C. and preferably of about 92 ° C. for a duration of about 3 to about 15 hours and preferably of about 5 hours (step of hydrolysis) At the end of the fermentation, the must having a viscosity between 550 and 1600 Centipoises (Cps) (Rheomat 205 Viscometer) is diluted with osmosis water at a dilution factor between 1/4 and 1. In order to obtain a viscosity of the wort diluted between 15 and 150 cps. A microfiltration step follows the dilution step. Tangential filtration is preferably used for the sake of efficiency and cost of treatment. The material used for the microfiltration stage is a tangential filtration system with mineral membranes having a cutoff threshold of between 0.1 and 1.2 μm and preferably 0.22 μm.

This deacetylated, hydrolyzed and diluted must is subsequently concentrated in order to obtain a polymer concentration that meets the specifications of the cosmetic, pharmaceutical or food industry, ie from about 0.1 to about 100 g / L. and preferably equal to about 10 g / l of polymer.

The techniques used for concentration and precipitation are those as previously described in particular in paragraph 3. At the end of the precipitation step, the precipitate is dried under vacuum and conditioned. The deacetylated and hydrolyzed polymer is obtained in the form of a powder. The polymer in this form has a number n of repeating units from 8 to 120.

EXPERIMENTAL PART

The polymer of the invention contained in the thermally treated fermentation must, hereinafter referred to as "polymer of the invention" or Alcos 04, provides a significant improvement in the field of cosmetics.

Indeed, the viscoelastic properties of the product allow its use in the preparation of cosmetic emulsions in substitution of products commonly used in this sector of activity. The viscosity-providing molecules, such as polyacrylates or polyacrylamides, derive from the petroleum industry while the polysaccharide of the invention is produced naturally using renewable raw materials derived from agriculture.

A - Influence of pH on viscosity

The polymer may be used, for example, as a gelling agent in the manufacture of a stable cosmetic product. The chemical nature of the polymer makes it insensitive to pH variations compared to products derived from petrochemistry, thus promoting the stability of the formula.

Examples of cosmetic formulas:

The laboratory manufacturing process follows the following protocol: a. Phase A and B are weighed separately and heated to 75 ° C. b. Phase B is slowly added to the phase A under emulsifier at 3000 rpm (Polytron PT 300, Prolabo) for 5 minutes. vs. At 60 ^° C., phase C is added, still under an emulsifier. d. Phase D is then added if necessary. e. Cooled under a helix to room temperature.

The tested products are:

• Sepigel 305 (polyacrylamide / C13-14 isoparaffin / laureth-7) (SEPPIC)

• Rheocare ATH (sodium polyacrylate / ethylhexyl stearate / trideceth-6) (Cosmetics Rheologies)

• Alcos 04 Past the polymer of the invention.

The polymer of the invention is not very sensitive to pH variations when the latter varies from 2 to 12. In contrast to the non-natural products indicated above, the viscosity variation is small and does not alter the stability of the formed emulsions (see Figure 2).

The study of the viscosity and the stability of the emulsions by centrifugation of the latter makes it possible to check the stability of the tested formulas:

The polymer of the invention: stable emulsions throughout the pH range,

• Sepigel 305: loss of stability at pH 12,

• RHEocare ATH: unstable emulsions at pH 3 and 12.

The chemical nature of the polymer makes it otherwise insensitive to the variation of the ionic strength of the medium for adding salts in a cosmetic formula such as shampoos for example without stability variation thereof.

B - Influence of salt (0.2-0.5-1%) on viscosity

Formulations:

The laboratory manufacturing process follows the following protocol: a. Weigh phases A and B separately and heat to 75 ° C. b. Slowly add B on A under foaming agent at 3000 rpm (Polytron PT 300) for 5 min. vs. At 60 ^° C., add phase C, still under emulsifier. d. Cool under a fan until it reaches room temperature

Results:

Polymer of the invention: at 0.2% the emulsion is stable, between 0.2% and 0.5%, a slight stream of oil is observed on the surface of the tube; at 1% salt, a phase shift of 65% of water appears; the viscosity variation oscillates between 30 and 50%.

• Sepigel 305: at 0.2% the emulsion is stable; at 0.5% and 1% a water phase shift of 20 to 55% is observed, plus a re-release of oil; the variation in viscosity oscillates between 60 and 90%.

• Rheocare ATH: no emulsion is stable; phase shift of water of 20 to 35% and oil of the order of 19%; the variation in viscosity oscillates between 55 and 90%.

In addition, while under hydroalcoholic conditions, some molecules have their viscosity drop, the polymer of the invention is only slightly affected.

For example: 2 solutions in 20 and 36.2% ethanol 96 to 1.5% dry matter (DM) polymers are prepared for each of the 3 polymers (Sepigel 305, Rheocare ATH, Alcos 04 Past - polymer 'invention). Native pHs are between 6.8 and 7.2.

The study of the viscosity makes it possible to obtain the following results:

Polymer of the invention: viscosity of the order of 10 000 to 12 000 mPa.s, insensitive to the addition of ethanol 96;

• Sepigel 305: increase in viscosity to 20% ethanol and decrease to 36.2%;

• RHEocare ATH: little influence at 20% but total loss of viscosity at 36.2% of ethanol.

C - Influence of the nature of the oily base

The cosmetic industry uses a large number of different oily bases for the preparation of creams, lotions or milks. These raw materials of hydrophobic origin can modify the rheological behavior of the polymer-type molecules present in the formula.

The polymer of the invention is only slightly influenced by the chemical nature of the oily base, unlike other petrochemical molecules.

In the example below, 4 oils are tested: paraffin oil, sweet almond oil, isostearyl isostearate and dimethicone 200 350cps.

The formula is:

For the preparation of this formula it is advisable to proceed as follows: Phase A and B must be weighed separately and slowly add B to A under an emulsifier at 3000 rpm (Polytron PT 300, Prolabo) for 5 minutes.

Results

The study of the viscosity of the emulsions makes it possible to determine the impact of the oily base on the viscosity of the formula:

Polymer of the invention: viscosity of the order of 10 000 to 12 000 mPa.s, not very sensitive to the nature of the oil;

• Sepigel 305: viscosity in the range of 20,000 to 45,000 mPa.s;

• Rheocare ATH: viscosity in the range of 15,000 to 55,000 mPa.s. All of these properties make it possible to classify the polymer of the invention as an agent providing viscosity and stabilizing emulsions having low levels of emulsifier.

The use of the polymer of the invention makes it possible to obtain emulsion of the milk or serum type having a low viscosity, a light texture and a soft, silky feel.

Claims

1. Use of the ALV 104 bacterial strain of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under number 1-3591, or of a strain whose rrs gene has a percentage of identity greater than or equal to 99 , 9% with the rrs gene of said strain ALV 104, for the preparation of a polysaccharide compound, corresponding to the following formula (I):

in which: - k varies from 1 year,

N represents an integer from 5 to 300, in particular from 8 to 270,

- R ^k _l5 R ^k ₂ R ^k _3, R ^k _4, R ^k _5, R ^k _6, R ^k ₇ and R ^k ₈ independently from each other H or an acyl group comprising 1 to 6 carbon carbon, especially an acetyl group.

2. Use according to claim 1, of the bacterial strain ALV104 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, for the preparation of a polysaccharide compound of formula (I) as defined in claim 1, wherein from about 40% to about 60%, especially from about 43% to about 56%, and preferably from about 43.75% to about 56.25% of all R groups. \, ^k R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents an acetyl group.

3. Use according to claim 1, of the bacterial strain ALV1 04 of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, for the preparation of a polysaccharide compound of formula (I) such that defined in claim 1, wherein at least 60% of the total groups R ^k _l3 R ^k ₂ R ^k _3, R ^k _4, R ^k _5, R ^k _6, R ^k ₇ and R ^k ₈ represents an atom hydrogen.

4. A polysaccharide compound, corresponding to the following formula (I):

in which :

K varies from 1 to n,

N represents an integer from 5 to 300, in particular from 8 to 270,

- R. \, ^k R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ independently of one another, H or an acyl group comprising from 1 to 6 carbon atoms, especially an acetyl group.

The compound of claim 4, wherein at least from about 40% to about 60%, especially from about 43% to about 56%, and preferably from about 43.75% to about 56.25%. all the groups R ^k i, R ^k ₂ , R ^k ₃ , R ^k ₄ , R ^k ₅ , R ^k ₆ , R ^k ₇ and R s represents an acetyl group.

6. Compound according to claim 4, characterized in that at least 60% of the totality of groups R ^k _1; ^K R _2, R ₃ ^k, R ^k _4, R ₅ ^k, R ^k _6, ^k ₇ and R ^k R ₈ represents a hydrogen atom.

7. Composition comprising a compound according to any one of claims 4 to 6, in association with a bacterial strain as defined in claim 1 and / or the culture medium or a portion of the culture medium of said strain, comprising in particular a source of nitrogen, such as yeast extract or proteins of organic origin such as peptone plant peptones of wheat, alfalfa, potato, a carbon source such as glucose, maltose, mannose , arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese.

8. A pharmaceutical, food processing or cosmetic composition, comprising a compound according to any one of claims 4 to 6, in association with a suitable vehicle.

9. Process for the preparation of a composition according to claim 8 comprising a fermentation step in the presence of a bacterial strain ALV1 04 of the family Rhizobiaceae, deposited at the CNCM March 21, 2006 under the number I-3591, or d a strain whose rrs gene has a percentage of identity greater than or equal to 99.9% with the rrs gene of said strain ALVl 04, and a culture medium comprising in particular a nitrogen source, such as yeast extract or proteins of organic origin such as peptone plant peptones of wheat, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese.

Process for the preparation of a compound according to claim 4, comprising the following steps:

a fermentation step in the presence of an ALV 104 bacterial strain of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1-3591, or of a strain whose rrs gene exhibits a percentage of identity greater than or equal to 99.9% with the rrs gene of said strain ALV104, and a culture medium comprising in particular a nitrogen source, such as yeast extract or proteins of organic origin such as peptones vegetable types of wheat peptone, alfalfa, potato, a source of carbon such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron , sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese, to obtain a composition comprising a compound according to claim 4, in association with a bacterial strain as defined in claim 1 and the medium of culture as defined above essus, and

a filtration step, in particular of tangential filtration, in order to rid the aforementioned composition of said bacterial strain and of said culture medium, and to obtain the polysaccharide compound according to claim 4.

11. Preparation process according to claim 10, comprising, after the filtration step, a step of precipitating the polysaccharide compound according to claim 4, in order to obtain said compound in powder form, and comprising, if appropriate, after said step of precipitating, a step of dissolving the compound obtained in powder form, in order to carry out the complete deacetylation of said compound, and to obtain a compound according to claim 6.

12. Preparation process according to claim 10, characterized in that a complete deacetylation step of the compound according to claim 4 is carried out after the fermentation step and before the filtration step, in order to obtain a compound according to claim 6.

13. Process for the preparation of a compound according to claim 6, comprising the following steps: a fermentation step in the presence of an ALV104 bacterial strain of the Rhizobiaceae family, deposited at the CNCM on March 21, 2006 under the number 1 -3591, or a strain whose rrs gene has a percentage of identity greater than or equal to 99.9% with the rrs gene of said strain ALVl 04, and a culture medium comprising in particular a nitrogen source , such as yeast extract or proteins of organic origin such as plant peptones such as wheat peptones, alfalfa, potato, a carbon source such as glucose, maltose, mannose, arabinose, sucrose, ribose, xylose, lactose or mannitol, and a source of trace elements containing iron, sulfate, magnesium, calcium, chloride, zinc, boron, copper, cobalt or manganese, to obtain a composition comprising a compound according to claim 4, in association with a so bacterial agent as defined in claim 1 and a culture medium as defined above,

a step of complete deacetylation of the compound according to claim 4, to obtain a composition comprising a compound according to claim 6, in association with said bacterial strain and said culture medium, and a filtration step, in particular of tangential filtration, in order to to rid the aforementioned composition of said bacterial strain and of said culture medium, and to obtain the polysaccharide compound according to claim 6.

14. Use of the polysaccharide compound according to any one of claims 4 to 6, for the preparation of a cosmetic composition.

15. Use of the polysaccharide compound according to any one of claims 4 to 6, as a texturing agent or touch, or as an emulsion stabilizer, or in the field of microbiological diagnosis, especially as a as growth support of microorganisms.

16. Use of the polysaccharide compound according to any one of claims 4 to 6, in gel form, in total or partial substitution of the gelling bases in the culture media, in particular to replace the agarose gel or Agar Agar.

17. Preparation process according to claim 9, comprising, after the step of fermentation of the bacterial strain, a pasteurization step of the composition according to claim 7 in order to inactivate the bacteria constituting the bacterial strain, this corresponding pasteurization step at a step of maintaining said composition at a temperature of about 45 ° C to about 90 ° C for about 5 minutes to about 60 minutes.