FR2728269A1 - PROCESS FOR TREATING A FERMENTATION MUST CONTAINING POLYSACCHARIDE - Google Patents

Abstract

The present invention relates to a process for treating a fermentation must intended for the production of exopolysaccharide of microbial origin. - The process comprises the addition of a surfactant to an aqueous solution of the fermentation must which contains the polysaccharide. - In the process, the polysaccharide can be, for example, gellan, welan, rhamsane, scleroglucan or xanthan. The invention also relates to a composition comprising a fermentation must containing polysaccharides of microbial origin and a useful amount of a surfactant.

Description

The present invention relates to a method for treating a solution

  aqueous solution of a fermentation must containing exopolysaccharides. This process makes it possible in particular to purify the solution very simply by concentrating the polysaccharide in a

  liquid phases which separate during the process.

  The polysaccharide musts are obtained by a well-known fermentation process: a polysaccharide-producing microorganism (fungus, bacteria) is cultured in a nutrient aqueous medium containing a carbon substrate, plus

especially glucose.

  For the production of xanthan, for example Xanthomonas campestris can be used to produce scleroglucan, Sclerotium rofsii or Scerotiumon can be used.

  glucanicumwn, and for schizophyllan the common Schizophyllum fungus.

  Document US-4326052, US-4326053, US-4377636 and US4385123, the polysaccharide S-60 called gellane whose use in aqueous solution as thickening agent, gelling and stabilizer is known in the fields

  agri-food, paints or adhesives.

  Gellan is an exocellular polysaccharide produced by the bacterium Pseudomona elodea (ATCC 31461) sometimes classified as Auromonas elodea. This bacterium has been isolated from plant tissues (elodea) collected in Pennsylvania. The bacterium has been characterized by Kang and Veeder and described in US-4326053. Gellan is also produced by

aerobic fermentation.

  The method for extracting gellan from the fermentation broth conventionally used comprises, in particular, pasteurization followed by elimination of the cells by filtration or centrifugation, and then precipitation of the polysaccharide with a solvent such as isopropanol. But precipitation is a heavy method to implement because it requires large amounts of solvent, heating and dilution of the must before adding

  solvent. In addition, it can cause aggregation of the polymer in solution.

  Thus the present invention relates to a method of treating an aqueous solution of a fermentation must containing exopolysaccharides of microbial origin. In the process, phase separation of the solution is caused by

  the addition of a specific amount of a surfactant.

  The surfactant can be added dropwise without stirring. It is possible to incorporate between 0.1 and 10 g / l of surfactant per 1 g / l of polysaccharide

  contained in the solution, and preferably between 1 and 5 g / l of surfactant.

  We can recover a first phase which contains mainly

  substantially purified polysaccharide.

  The first phase can be treated by the addition of trifluorotrichlorothane, homogenization and centrifugation can be carried out to substantially remove

  totally the low remaining concentration of protein.

  A second phase may comprise mainly residues and the surfactant.

  The surfactant may be anionic or nonionic.

  The surfactant may be sodium dodecyl sulphate.

  The polysaccharides may be chosen from the group consisting of gellan, welane, rhamsane, xanthan, scleroglucan, schizophyllane, curdlane, pullulant,

  dextrans, alginate and succinoglucan.

  The invention also relates to a composition comprising a fermentation must containing polysaccharides of microbial origin and a useful amount of a surfactant. All currently known applications of polysaccharides of microbial origin can be carried out using the product obtained by the method according to the present invention. The applications can be preferably as thickener, stabilizer or viscosifier, in the food, cosmetic, size,

  the paper industry or oil well fluids.

  Moreover, in the case of gellan, the product obtained by the process according to the present invention has the advantage of being easily obtained in a form

esterified.

  In the known process, obtaining the commercial gellan is the result of three steps: the native gellan is obtained directly from the fermentation must after heating it to 95 ° C. for 5 minutes and precipitation with alcohol isopropyliqlue - the second step, called de-esterification of native gellan, consists in bringing the pH of the solution to 10 with sodium hydroxide or potassium hydroxide when the fermentation must is at C The pH is maintained at this value for 10 minutes, - the solution is then neutralized with sulfuric acid, then the product is precipitated with alcohol The unclarified gellan is composed of about 50% of insoluble material consisting mainly of whole cells and cellular debris, these impurities are in this case removed from the de-esterified product by centrifugation or filtration at 700 ° C. This process causes the ester bonds of the side groups of the native polysaccharide to rupture. contains an acetate and a glycerate The de-esterified polysaccharide is marketed under the name GELRITE or KELCOGEL by MERK & Co

  (USA). The description of the product and a process for obtaining it are disclosed in

  US-4326052, US4326053, US-4377636 and US-4385123.

  It is known that gels prepared from gellan become increasingly brittle depending on the degree of de-esterification of the polysaccharide. When preparing gels from de-esterified gellan, for example for agro-food applications, large quantities of cations must be added in order to stabilize the polymorphic network. In order to improve the performance of their de-esterified gellan, MERK & Co (USA) generally adds the following cation concentrations: Na + (2 mg / g), K + (20 mg / g),

Ca ++ (5 mg / g), Mg ++ (1.5 mg / g).

  Compared to the gels obtained with commercial de-esterified gellan, the gels obtained with the esterified gellan obtained according to the present invention are less fragile and can be increased in stability at extreme temperatures or at high salinity. Indeed, the structure of the gellan is such that it is clear that the esterified parts give it a

very good stability.

  The invention will be better understood and its advantages will appear more clearly in the

read the following examples.

  Experiment 1: An aqueous gellan solution is prepared from a fermentation broth containing about 9.2 g / l of gellan and 3.3 g / l of cells, half of which is composed of

  proteins. The solution is diluted to the concentration of 1 to 2 g / l of gellan.

  The solution is stirred at about 300-750 rpm at a temperature of 30.degree.

  An equal volume of an aqueous solution of SDS (sodium dodecyl sulphate) is added dropwise to the gellan solution at a rate of approximately one drop per second, without stirring and at 30 ° C.

  - Phase separation takes place as the surfactant is added.

  The solution thus obtained is placed in a cooling medium at approximately 4 ° C., for example for 1 to 30 days in order to accelerate the precipitation of the free surfactant in solution. - With a syringe, remove the upper phase which contains mainly

  virtually purified gellan. About 75% of the initial gellan is contained in this phase.

  Of course, it is possible to repeat the operation on the remaining solution.

  - The upper phase is centrifuged at 12000 rpm at 4 C for 30 minutes.

  The gellan solution is allowed to settle, then the remaining proteins are separated from the gellan solution by adding to the solution a substantially equal amount of trifluorotrichloroethane. We mix for homogenization then we

performs a centrifugation.

  The resulting supernatant is rich in esterified gellan and contains very little protein.

  The measured protein concentration is below the limit of detection of the biuret method, ie less than 0.2 g / l. The biuret method is described by D. Herbert, PJ. Phipps and R. E. Strange in "Methods in Microbiology, Vol 5-B", Eds J. R. Nonis and

  D. W. Ribbons, Academic Press, London, 209-344 (1971).

  Remaining proteins can also be removed by dialysis, using a

  membrane having a cut-off of 104 to 105 daltons.

  Experiment 2: The operation of treating a polysaccharide must according to the invention was

  repeated with a must of xanthan.

  The xanthan used in this test has a molecular weight of 5.

  70% pyruvate and 100% acetate.

  The concentration of xanthan in the must is of the order of 30 g / l.

  An aqueous solution of the must is prepared with 10 grams of must and 180 ml

  pure water. The concentration of xanthan in the solution is about 1 g / l.

  An equal volume of an aqueous solution of surfactant SDS (sodium dodecyl sulphate)

  sodium) at 5 g / l is added dropwise at room temperature and without stirring.

  Phase separation takes place during the addition of surfactant. The sentence

  superior is rich in xanthan, with a yield greater than 75%.

  The protein concentration in the xanthan-rich phase is below the

  limit of detection of the biuret method.

  Both experiments were repeated with other surfactants than SDS: for example for anionic surfactants: - sodium α-olefin sulfonate - Ethoxylated alcohol phosphates - Sodium lignosulfonate - Sodium alkyl lauryl sulphonate - Sodium octyl sulfosuccinate - Sophorolipid acid form - Sophorolipid crude form pH = 6 Sophorolipid crude form pH = 3.85

  For nonionics: ethoxylated alkyl phenols.

  The results obtained are very similar to those obtained with SDS, which shows that anionic or nonionic surfactants can be used in the process of

treatment according to the invention.

  It should be noted that the surfactant used is concentrated in the phase which is less rich in polysaccharide and can be easily recovered for other uses, after a step of

  separation of residues, proteins and cells, according to known methods.

Claims (8)

  1) Process for the treatment of an aqueous solution of a fermentation must containing exopolysaccharides of microbial origin, characterized in that a phase separation of the solution is induced by the addition of a determined quantity a surfactant.   2) Method according to claim 1, characterized in that one incorporates said   surfactant drop by drop, without stirring.   3) Method according to one of the preceding claims, characterized in that one   incorporates between 0.1 and 10 g / 1 of surfactant for 1 g / l of polysaccharide contained in the   solution, and preferably between 1 and 5 g / l of surfactant.   4) Method according to one of the preceding claims, characterized in that one   recover a first phase which contains mainly polysaccharide purified.   Process according to claim 4, characterized in that said first phase is treated by addition of trifluorotrichloroethane, homogenization is carried out and then centrifugation to substantially completely remove the remaining low concentration of proteins.   6) Method according to one of the preceding claims, characterized in that a   second phase mainly comprises residues and said surfactant.   7) Method according to one of the preceding claims, characterized in that said   surfactant is anionic or nonionic.   8) Method according to one of the preceding claims, characterized in that said   surfactant is sodium dodecyl sulfate.   9) Method according to one of the preceding claims, characterized in that said   polysaccharides are selected from the group consisting of gellan, welan, rhamsane, xanthan, scleroglucan, schizophyllan, curdlane, pullulan, dextrans, alginate and succinoglucan. ) Composition comprising a fermentation must containing polysaccharides of microbial origin, characterized in that it comprises a useful amount a surfactant.