GB2296249A

GB2296249A - Recovering polysaccharides from fermentation musts

Info

Publication number: GB2296249A
Application number: GB9525951A
Authority: GB
Inventors: Francis X Quinn; Frederic Monot
Original assignee: IFP Energies Nouvelles IFPEN
Current assignee: IFP Energies Nouvelles IFPEN
Priority date: 1994-12-20
Filing date: 1995-12-19
Publication date: 1996-06-26
Also published as: DE19547748A1; FR2728269A1; GB9525951D0; FR2728269B1

Description

1 A METHOD FOR TREATING A FERMENTATION MUST CONTAINING POLYSACCHARIDE

2296249 The present invention relates to a method of processing an aqueous solution of a fermentation must containing exopolysaccharides. This method allows the solution to be purified, in particular, in a very simple manner by concentrating the polysaccharide in one of the liquid phases that separate out during the process.

Polysaccharide musts are obtained by means of a well- known fermentation method: a polysaccharide-producing microorganism (fungus, bacterium) is cultured in a nutritive aqueous medium containing a carbon substrate, more specifically glucose.

is Xanthomonas campestris, for example, can be used to produce xanthan, Sclerotium rolfaii or Sclerotium glucanicum can be used to produce scleroglucan and Schizophyllum commune fungus can be used to produce schizophyllan.

Documents US-4326052, US-4326053, US-4377636 and US- 4385123 disclose polysaccharide S-60, known as gellan, which can be used in an aqueous solution as a thickening, gelling and stabilising agent and is well known in the food, paints or adhesives industries.

Gellan is an exocellular polysaccharide produced by the bacterium Pseudomonas elodea (ATCC 31461), sometimes classified as Auromonas elodea. This bacterium was isolated from plant tissue (elodea) in Pennsylvania. The bacterium has been characterised by Kang and Veeder and was described in patent US-4326053. The gellan is also produced by 2 aerobic fermentation.

The method conventionally used to extra the gellan from the fermentation must consists in particular of a process of pasteurisation, followed by removal of the cells by filtration or centrifugation, after which the polysaccharide is precipitated with a solvent such as isopropanol.

However, precipitation is a cumbersome method to implement because it requires large quantities of solvent, heating and dilution of the must prior to adding the solvent. In addition, it can lead to an aggregation of the polymer in solution.

The present invention, therefore, relates to a method of treating an aqueous solution of a fermentation must containing polysaccharides of microbial origin. During the process, the phases in the solution are separated by adding a specified quantity of a surfactant.

The surfactant may be added drop by drop, without stirring.

Between 0.1 and 10 g/1 of surfactant may be used for 1 g/1 of polysaccharide contained in the solution and preferably between 1 and 5 g/1 of surfactant.

A first phase containing virtually purified polysaccharide can be recovered.

The first phase can be treated by adding trifluorotrichloroethane, putting it through a process of homogenisation and then centrifugation essentially to remove the remaining low concentration of proteins completely.

A second phase may contain mainly residues and the surfactant.

3 before The surfactant may be anionic or non-ionic.

The surfactant may be sodium dodecylsulfate.

The polysaccharides may be selected from the group comprising gellan, welan, rhamsan, xanthan, scleroglucan, schizophyllan, curdlan, pullulan, the dextrans, alginate and succinoglucan.

The invention also relates to a compound consisting of a fermentation must containing polysaccharides of microbial origin and a useful quantity of a surfactant.

The product obtained by the method of the invention can be used in all the currently known applications of polysaccharides of microbial origin. By preference, the applications may be use as a thickening, stabilising or viscosity-improving agent in the food, cosmetics, paints and is paper industries or with oil well fluids.

In addition, in the case of gellan, the product obtained by the present invention has the particular advantage of being easy to obtain in esterified form.

In the known method, commercial gellan is produced by means of three steps:

- the native gellan is obtained directly from the fermentation must after heating thereof to 950C for 5 minutes and precipitation with isopropyl alcohol, - the second step, known as esterification of the native gellan, consists in adjusting the pH of the solution to 10 using soda or potash when the fermentation must is at 800C. The pH is maintained at this value for 10 minutes, - the solution is then neutralised with sulphuric acid the product is precipitated in alcohol. The non - 4 clarified gellan is made up of about 50% of insoluble material mainly consisting of whole cells and cell fragments, in which case these impurities are removed from the de-esterified product by a process of centrifugation or 5 filtration at 700C.

This process causes the ester bonds between the lateral groups in the native polysaccharide, which contain an acetate and a glycerate, to rupture. De-esterified polysaccharide is marketed under the name of GELRITE or KELCOGEL by MERK & Co (USA). The description of the product and a method of producing it are disclosed in documents US4326052, US-4326053, US- 4377636 and US-4385123.

It is known that gels prepared from gellan become increasingly fragile, depending on the degree to which the polysaccharide is de-esterified. When producing gels from de-esterified gellan, for application in the food industry for example, large quantities of cations have to be added in order to stabilise the polymorphic lattice. In order to improve the performance of their de- esterified gellan, MERK & Co. (USA) generally adds the following concentrations of cations: Na (2 mg/g), K+ (20 mg/g), Ca+ (5 mg/g), Mg' (1. 5 mg/9) - Compared with the gels produced using commercial deesterified gellan, the gels produced from esterified gellan in accordance with the invention are less fragile and their stability may even be increased at extreme temperatures and high salinities. In practice, the gellan structure is such that the esterified parts clearly impart to it a very good stability.

The invention will be more readily understood from the following examples.

Test No. 1:

- An aqueous solution of gellan is prepared from a fermentation must containing approximately 9.2 g/1 of gellan and 3.3 g/1 of cells, half of which are proteins. The solution is diluted at a concentration of 1 to 2 g/1 of gellan.

- The solution is stirred at about 300-750 revolutions/minute at a temperature of 300C.

- An equal volume of an aqueous solution of SDS (sodium dodecylsulfate) is added, drop by drop, to the gellan solution, at a rate of approximately one drop per second, without stirring and at 300C.

is - A phase separation occurs as the surfactant is added.

- The solution obtained in the above manner is placed in a cooling means at about 40C for 1 to 30 days, for example, in order to accelerate precipitation of the free surfactant in solution.

- The upper phase mainly containing the virtually purified gellan is removed with a syringe. Approximately 75% of the initial gellan is contained in this phase.

Clearly, this operation can be repeated with the remaining solution.

- The upper phase is put through a process of centrifugation at 12,000 revolutions /minute at 40C for 30 minutes.

- The gellan solution is left to decant, after which the remaining proteins in the gellan solution are separated 6 by adding a quantity of trifluorotrichloroethane substantially equal to that of the solution. They are mixed to bring about homogenisation and then put through a process of centrifugation.

The supernatant obtained is rich in esterif ied gellan and contains very few proteins. The measured protein concentration is below the limit of detection by the biuret method, i.e. less than 0.2 g/1. The biuret method is described by D. Herbert, P. J. Phipps and R. E. Strange in "Methods in microbiology, Vol. 5-BI1, Eds. J.R. Norris and D.W. Ribbons, Academic Press, London, 209-344 (1971).

The remaining proteins can also be removed by dialysis, using a membrane with a separation threshold of 104 to 105 daltons.

Test No. 2:

The operation of the invention for treating a polysaccharide must was repeated using a xanthan must.

The xanthan used for this test has a molecular mass of 5. 106, a pyruvate proportion of 70% and an acetate proportion of 100%.

The xanthan concentration in the must is in the order of 30 g/1.

An aqueous solution of the must is prepared with 10 grams of must and 180 ml of pure water. The xanthan concentration in the solution is approximately 1 g/1.

An equal volume of an aqueous solution of 5/gl of surfactant SDS (sodium dodecylsulfate) is added, drop by drop, at ambient temperature and without stirring.

A phase separation occurs whilst the surfactant is 7 The protein concentration in the xanthan-rich phase is below the limit of detection of the biuret method.

The two tests were repeated with surfactants other than SDS, using the following anionic surfactants, for example:

- Sodium a-olefin sulfonate - Ethoxylated alcohol phosphates Sodium lignosulfonate - Sodium alkyl lauryl sulfonate - Sodium di-octyl sulfosuccinat - Acidic form of sophorolipid - Raw form of sophorolipid pH=6 - Raw form of sophorolipid pH=3.85 For the non-ionic surfactants, ethoxylated alkyl phenols were used.

The results obtained are very similar to those obtained with SDS, which shows that anionic and non-ionic surfactants can be used in the processing method of the invention.

It should be noted that the surfactant used is concentrated in the phase containing less polysaccharide and can easily be recovered for other applications, after a stage of separating the residues, proteins and cells, using known methods.

8

Claims

1. A method of processing an aqueous solution of a fermentation must containing exopolysaccharides of microbial origin with a view to the extraction thereof, wherein a separation of the phases in the solution is brought about by adding a specified quantity of surfactant, which is incorporated drop by drop and without stirring, and in that a supernatant phase containing mainly practically purified polysaccharide is recovered.

2. A method as claimed in one of the previous claims, wherein between 0.1 and 10 g/l of surfactant are added to 1 g/l of polysaccharide contained in the solution, and preferably between 1 and 5 g/l of surfactant.

3. A method as claimed in one of the previous claims, wherein the said supernatant phase is treated by adding trifluorotrichloroethane, followed by homogenisation and then centrifugation essentially to remove the remaining weak concentration of proteins completely.

4. A method as claimed in one of the previous claims, wherein the surfactant is recovered from a phase lower than the said supernatant phase.

5. A method as claimed in one of the previous claims, wherein the said surfactant is anionic or non-ionic.

6. A method as claimed in one of the previous claims, wherein the said surfactant is sodium dodecylsulfate.

7. A method as claimed in one of the previous claims, wherein the said polysaccharides are selected from the group comprising gellan, welan, rhamsan, xanthan, scleroglucan, schizophyllan, curdlan, pullulan, the dextrans, alginate and 9 succinoglucan.

8. A method substantially as hereinbefore described.