Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-06-25
2003-01-21
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S104000, C435S072000, C435S252200, C536S123000, C536S123100, C536S126000, C536S127000
Reexamination Certificate
active
06509176
ABSTRACT:
The present invention relates to a heteropolysaccharide (HP) characterized in that it can be obtained by fermentation of a medium including at least one
Agrobacterium radiobacter I
-2001 (or DSM 12095) strain, a recombinant thereof or mutant thereof, and a source of carbon which can be assimilated by said strain, a recombinant thereof or a mutant thereof.
In many industrial domains, there is a constant search for novel compounds with:
improved rheological properties, and which are capable of forming gels,
increased compatibility with the media into which they are incorporated,
great stability over a wide temperature and pH range.
In the case of compounds obtained at the end of a bacterial fermentation, it is also important for the compound to have good productivity.
The ability to gel is very advantageous since they are systems which are particularly attractive by virtue of the diversity of the domains in which they have applications: some applications require the use of a gel.
Thus, for example, the agrofoods industry provides a wide range of gelled products (creams, yoghurts, diverse jellies, ice creams, etc.), and the pharmaceutical industry uses gels as active principle or thickening agent supports.
In entirely another domain, some paints do not drip since they have gel characteristics when standing, whereas they spread easily with a paintbrush (rheofluidifying profile).
Aqueous gels are also used as chromatographic supports or for developing contact lenses.
Heteropolysaccharides of bacterial origin, such as for example xanthan gum, have already been described and used for their effective rheological properties under extreme temperature and pH conditions. However, these heteropolysaccharides, which are suitable in applications in solution, do not always produce gels.
It is known that the gelling of a medium takes place when a three-dimensional network is formed subsequent to the crosslinking of the components of said medium.
Conventionally, this gelling is brought about by adding additional cations in particular of alkali metal or alkaline earth metal type (for example calcium and/or magnesium) to the medium, by switching the pH toward acid or basic pHs, by adding another compound, in particular another polysaccharide (for example the combination of xanthan and carob), or by modifying the temperature.
Whatever the application envisaged, the abovementioned gelling conditions may:
harm the stability and the compatibility of the final gel due to the interactions between the additional cations or the coadditive, which must be introduced in order to obtain the gel, and the other ingredients present in said compositions, or
denature the heteropolysaccharide and/or the other ingredients present in said compositions due to the high temperatures and/or the pH changes.
In the context of the present invention, the term “gel” refers to a pseudosolid (behavior close to a solid) resulting from the association, at least partial, of heteropolysaccharide chains dispersed in a liquid. In a stressing frequency range &ohgr;, the pseudo-solid gels are in general characterized, with regard to their solid component, by an elastic modulus G′ (&ohgr;) also called storage modulus and, with regard to their liquid or viscous component, by a viscous modulus G″ (&ohgr;) also called loss modulus.
The mechanical values G′ (&ohgr;) and G″ (&ohgr;) can be measured using a controlled strain rheometer and operating in oscillatory mode. By way of nonlimiting indication, mention may be made, for example, of a Rheo-Fluid Spectrometer® rheometer.
G′ and G″ can also be measured on a controlled stress rheometer and operating in oscillatory mode. By way of indication, mention may be made, for example, of a CARRIMED® rheometer.
The principle of the measurement consists in determining, firstly, the range of reversible mechanical strain in which the response of the gel to the mechanical stressing is linear as a function of said strain. Secondly, the gel is subjected to a set value of mechanical strain included in the linear range determined above. The rheometer then carries out a frequency sweep &ohgr;.
The stress response of the gel which is in phase with the strain gives the elastic modulus G′ (&ohgr;). G′ (&ohgr;) corresponds to the energy stored by the gel in elastic form and can be recovered.
The stress response of the gel which is out of phase by an angle of 90° with the strain gives the viscous modulus G″ (&ohgr;). G″ (&ohgr;) corresponds to the energy dissipated by the viscous flow and can not be recovered.
A gel is termed strong or true when, throughout the stressing frequency range (&ohgr;) swept, the G′/G″ ratio is greater than or equal to 10, i.e. when the elasticity of the gel remains high and when the value of G′ (&ohgr;) is greater than or equal to 10 Pa.
The aim of the present invention is precisely to provide heteropolysaccharides which have very good rheological properties, in particular in terms of thickening and pseudoplastic (rheofluidifying) properties, and also the ability to produce true gels without adding additional cations to the medium and without switching the pH, this being at temperatures lower than or equal to 40° C.
The aim of the present invention is also to provide a heteropolysaccharide with very good rheological properties at low concentrations.
The present invention therefore relates to a heteropolysaccharide (HP) characterized in that it can be obtained by fermentation of a medium including at least one
Agrobacterium radiobacter I
-2001 (or DSM 12095) strain, a recombinant thereof or a mutant thereof, and a source of carbon which can be assimilated by said strain, a recombinant thereof or a mutant thereof.
The
Agrobacterium radiobacter
strain was deposited in accordance with the Treaty of Budapest, with the Collection Nationale de Culture des Micro-organismes (CNCM) [National Collection of Microorganism Cultures], on Apr. 3, 1998, where it can be accessed publicly under the number I-2001. It was also deposited with the Deutsche Sammlung von Mikro-organismen und Zellculturen GmbH (DSMZ), on Apr. 21, 1998, where it can be accessed publicly under the number DSM 12095.
Pure culturing of
Agrobacterium radiobacter I
-2001 (or DSM 12095) can be carried out in Petri dishes incubated at a temperature of between 25° C. and 30° C., and more particularly of between 25° C. and 28° C., for approximately 24 hours.
The sources of carbon and nitrogen which can be assimilated by
Agrobacterium radiobacter I
-2001 (or DSM 12095) can be chosen from glucose, fructose, galactose, trehalose, mannose, melobiose, sucrose, raffinose, maltotriose, maltose, lactose, lactulose, methyl-&bgr;-galactopyranoside, methyl-&agr;-galactopyranoside, cellobiose, gentobiose, methyl-&bgr;-D-glucopyranoside, methyl-&agr;-D-glucopyranoside, esculin, ribose, arabinose, xylose, palatinose, rhamnose, fucose, melezitose, D(+) arabitol, L(−) arabitol, xylitol, dulcitol, tagatose, glycerol, myo-innositol, mannitol, maltitol, turanose, sorbitol, adonitol, lyxose, erythritol, D(−) tartrate, D(+) malate, L(−) malate, cis-aconitate, trans-aconitate, 2-keto-D-gluconate, N-acetylglucosamine, guinate, betaine, succinate, fumarate, glycerate and glucosamine.
Among the possible maintenance media for the strain, the maintenance medium of the type Difco MY agar (reference 0712-01-8) is considered to be particularly advantageous. Said Difco MY agar medium has the following composition:
bacto-yeast extract
3 g
malt extract
3 g
bacto-peptone
5 g
bacto-dextrose
10 g
bacto-agar
20 g
For conserving the strain, it is preferable to anticipate at least one preculturing step. The term “preculturing step” is intended to mean a step which consists in developing and multiplying the bacterial strain, without polysaccharide production.
It has been possible to demonstrate that, in general, the heteropolysaccharide (HP) includes glucose motifs and/or derivatives thereof, galactose motifs and/or derivatives thereof, glucuron
Cantiani Robert
Chevallereau Paule
Senechal Alain
Simon Jean-Luc
Vaslin Sophie
Prats Francisco
Rhodia Chimie
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