PROCESS FOR THE PREPARATION OF THE POLYSACCHARIDES K4 AND K5 FROM ESCHERICHIA COLI Field of the Invention The present invention refers to the preparation of the polysaccharides K4 and K5 from Escherichia coli carried out by a fermentation process.

Prior Art The polysaccharides K4 and K5 are known which may be obtained from strains of Escherichia coli responsible for extra-intestinal infections.

The polysaccharide K4 is composed of equimolecular quantities of glucuronic acid and N-acetylgalactosamine in a linear chain bonded with ß-1,3 bonds and fructose bonded in a lateral chain with carbon 3 of glucuronic acid.

The polysaccharide K5 is composed of equimolecular quantities of glucuronic acid and N-acetylglucosamine, which make up the alternate linear repetitive unit 4-P- glucuronil-1,4 a-N-acetylglucosamine. This repetitive unit presents a structural analogy with completely desulphated and N-acetylated heparin, hence the use of K5 may be hypothesised in processes of semi-synthesis to obtain heparin-like sulphated polysaccharides. The possibility of producing the polysaccharides K4 and K5 by means of a fermentative process therefore opens up new prospects for obtaining biologically active molecules by semi-synthesis as an alternative to extraction from animal organs.

The polysaccharide K5 was initial obtained in capsular form from broth culture in toto with yields of about 40-50 mg/l of broth culture [Eur. J. Biochem. 116,359- 369,1981].

Subsequently the production of the polysaccharide K5 in extracellular form was described, which contemplates the isolation of K5 from the culture filtrate with yields varying from 200 to 700 mg/l of broth culture [J. Bioact. Comp. Polym. 11, 301-311,1996; European Patent EP 0489 647 A2,1991].

Also the polysaccharide K4 was initial obtained in capsular form from broth culture in toto with yields of about 80-90 mg/l of broth culture [Eur. J. Biochem.

117,112-124,1988].

Subsequently the production of the polysaccharide K4 in extracellular form was described, which contemplates the isolation of K4 from the culture filtrate alone

with yields of about 200 mg/I of broth culture [Biotechnol. Letters 18,383-386, 1996].

The yields given above do not allow economically advantageous results.

Summary We have now found a new process which allows the polysaccharides K4 and K5 to be obtained by fermentation, isolating them from broth cultures of Escherichia colt'.

The process for the production, isolation and purification of the polysaccharides K4 and K5 according to this invention allows these polysaccharides to be obtained with high purity and with higher yields than when using the known processes.

The invention process comprises the following stages: a) fermentation in a submerged culture of a strain of Escherichia coli which produces the polysaccharide K4 or of a strain of Escherichia coli which produces the polysaccharide K5; b) centrifugation of the broth culture, concentration of the culture filtrate by ultrafiltration and precipitation of the polysaccharide by treatment with an organic solvent; c) dissolving of the precipitate in a suitable buffer solution and treatment with protease; d) passage through an ion exchange column followed by one or more stages of dialysis and re-precipitation with ethanol.

The innovation of the process lies in the fact that the culture medium for said fermentation is an aqueous medium comprising: defatted soya flour, mineral salts and glucose, or the dialyse portion of yeast autolysate, mineral salts and glucose.

Detailed description of the invention The characteristics and the advantages of the process for the preparation of the polysaccharides K4 and K5 by fermentation, isolating them from broth cultures of Escherichia co//"according to this invention, will be illustrated at greater length during the following description. The strains of Escherichia coli suitable for the production of K4 or of K5 may be obtained from public collections such as the International Escherichia Centre [Denmark], ATCC [American Type Culture Collection-USA], DSM [Deutsche Sammlung von Mikroorganismen, Federal

Republic of Germany] and others. Alternatively, strains of E. coli which produce K4 or K5 may be obtained by isolation from clinical findings and subsequent characterisation as described in the literature [Eur. J. Biochem. 117,112-124, 1988].

In the experimentation for this invention, the following strains were used: E. coli 05: K4: H4 available from ATCC number 23502, for the preparation of K4; E. coli 010: K5: H4 available from ATCC number 23506, for the preparation of K5.

These strains present the following characteristics (+ = positive and-= negative).

Reaction/Enzyme Substrate E. coli E. coli 010: K5: H4 05: K4: H4 ß-galactosidase O-nitrophenyl-+ + galactoside arginine dehydrolase Arginine lysine decarboxylase Lysine + + ornithine decarboxylase Ornithine-- use of citrate Sodium citrate production of H2S Sodium thiosulphate-- urease Urea tryptophan deaminase Tryptophan production of indol Tryptophan + + production of acetoine Sodium pyruvate gelatinase Gelatine fermentation of glucose Glucose + + fermentation-oxidation (other Mannitol + + S) fermentation-oxidation I nositol fermentation-oxidation Sorbitol + + fermentation-oxidation Rhamnose + + fermentation-oxidation Saccharose fermentation-oxidation Melibiose + + fermentation-oxidation Amygdalin fermentation-oxidation Arabinose + + cytochrome oxidase Tetramethylpara enyldiamine production of N02 Glucose + + reduction from N02 to N2 Glucose + +

The fermentation conditions in submerged culture may vary according to the characteristics of the strain used. Proceeding according to this invention, the fermentation temperature is between 30 and 40°C, and preferably 37°C, while the time is between 2 and 24 hours. As regards the culture medium we found that the fermentation yield of K4 and of K5 in the extracellular form is particularly high when using as a culture medium an aqueous medium with the ingredients defatted soya flour (0.1-5 g/I) or the dialyse portion of yeast autolysate (from 5 to 30 g/I) (10 g are dissolved in 50 ml and dialyse against 500 ml of water), mineral salts and glucose.

The content of said mineral salts and glucose is as follows: from 5 to 15 9/l of K2HPO4, from 0.5 to 5 g/l of KH2PO4, from 0.01 to 1 gel of MgCI2, from 0.05 to 2 gui of sodium citrate, from 0.1 to 3 gui of ammonium sulphate and from 0.5 to 4 g/l of glucose.

At the end of fermentation the cells are separated from the culture mixture, preferably by centrifugation. The culture filtrate is then concentrated to 1/4-1/5 of the initial volume, for example by ultrafiltration with a tangential flow or using flat or spiralled membranes with a different molecular cut-off (8,000-300,000 D).

The precipitation of the polysaccharide K4 or K5 is carried out at 4°C overnight, using 4-5 volumes of solvent (96% ethanol or isopropanol or acetone) per volume of concentrated culture filtrate. The precipitate collecte for example by centrifugation, dissolved in a suitable buffer, is subjected to enzymatic deproteinization, using a fungal protease (for example Protease Type XXIII: fungal crude from Aspergillus oryzae).

The subsequent purification of the polysaccharide is carried out by passing it through an ion exchange column followed by one or more stages of dialysis, for example for ultrafiltration against distille water, and subsequent re-precipitations

with ethanol. Normally two cycles of dialysis and precipitations are sufficient to obtain the polysaccharide in a sufficiently pure form. The sample may later by dehydrated using the traditional techniques such lyophilization, treatment with solvents and drying.

For the purpose of illustration, the following examples are given which describe in detail the process of the invention for the production of the polysaccharides K4 and K5.

Example 1 Preparation of the polysaccharide K5 in a flask with soya flour.

For the production of the polysaccharide K5 in a flask, 750 ml flasks with a deflector were used, each containing 100 ml of culture medium.

Defatted soya flour was used (PROVABIS Prodotti Gianni, Milan) (2 g/2) and the culture medium SD having the following composition (g/I): K2HPO4: 9.7 KH2PO4: 2.0 MgC'2:0. 1 sodium citrate: 0.5 ammonium sulphate: 1.0 glucose: 2.0 well water: 1,000 ml, pH 7.3, sterilisation at 118°C for 20 min.

The glucose solution was prepared separately, sterilised at 115°C for 30 min. and added in sterile condition to the culture medium.

Each flask was inoculated starting from a cellular suspension from a slant of TSA (Tryptic Soya Agar) incubated at 37°C for 24 h. The flasks were placed to incubate with alternative agitator (6 cm of movement, 160 cpm) at 37°C for 24 h and samples were taken at different times. The microbe development was assessed by means of a direct count in the optical microscope using the Burker chamber.

At the end of fermentation, heat treatment was carried out (80°C for 10 min) in order to inactivate any enzymatic components present.

Isolation of the polysaccharide K5 The broth culture obtained by means of the described fermentation was

centrifuged at 10,000 rpm in order to separate the cells from the culture fiitrate.

The following operations were performed to isolate the polysaccharide from the culture filtrate: Concentration The culture filtrate (1,000 ml) was subjected to concentration by ultrafiltration using membranes with cut-off 8,000-10,000 D down to a final volume of about 1/5 of the initial volume (200 ml). During this phase various types of filtering modules may be used; in particular, in this preparation a Minitan cell (Millipore) was used with flat membranes of polysulfone (PTCG).

Precipitation The polysaccharide K5 was precipitated by adding 4 volumes (800 ml) of 96% ethanol at 4°C overnight. The polysaccharide K5 has a natural tendency to sediment, thus making it possible to separate most of the supernatant liquor by siphoning; the residual precipitate was then separated by centrifugation at 10,000 rpm for 20 min. These conditions were adopted in all the subsequent precipitation stages.

Deproteinization The precipitate was subjected to enzymatic deproteinization, using a fungal protease (Protease Type XXIII: fungal crude from Aspergillus oryzae 3.2 U/mg, code 4755, Sigma) adopting the following conditions: -the precipitate was dissolved in 45 ml of a solution of NaCI 0.1 M-EDTA 0.15 M at pH 8, containing sodium dodecylsulphate (SDS) at 0.5% (p/v); -addition of 4 U of protease per litre of initial culture filtrate and thermostat control at 37°C for 90 min: -dialysis by ultrafiltration (2 cycles) using the Minitan cell, with 50 mi of distille water; -precipitation with ethanol.

Purification Purification was carried out by means of two extraction cycles with 30 ml of NaCI 1 M, followed by dialysis by ultrafiltration with 50 mi of distille water and precipitation with ethanol.

The precipitate obtained from the second precipitation was dissolved in H20 and

subjected to chromatography with ion exchange on a DEAE column, washing the column with 1 volume of NaCI 0.4 M and separating the polysaccharide with 1 volume of NaCI 0.6 M.

The fermentation yield in purified K5 is about 850 mg/l.

Characteristics of K5 The samples obtained at the end of purification were analysed according to the following procedures and with the following results.

Content of uronic acids about 40% determined using the carbazole method (Bitter et a/., Anal. Biochem. 4,330-334,1962).

Spectrometry 13C NMR: -Varian Gemini 200 operating at 50.3 MHz at 22°C using TSP as standard; -sample 50 mg/ml in D2O.

The spectrums of the polysaccharide produced were similar to the spectrums for the polysaccharide K5 reported in the literature.

The principal signals related to N-acetylglucosamine and to D-glucuronic acid bonded in sequence were particularly shown.

Assessment of the molecular weight by HPLC chromatography with molecular exclusion: column: (1 x 30 cm) Superdex 75 HR (Pharmacia) eluent: phosphate buffer 0.1 M, pH 7 with addition of NaCI 0.15 M flow: 1 ml/min detector: UV at 210 nm sample: 20 1 of a solution 2 mg/ml.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70%) and the other with PM 5,000 D (30%).

Example 2 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 gui of defatted soya flour (Cargill Foods S. R. L., Milan, 200/20 S).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 800 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 3 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 gui of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 900 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the

carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 4 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 g/l of defatted soya flour (Cargill Foods S. R. L., Milan, 200/20 S).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 780 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 5 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 g/I of defatted soya flour (ABS Food-Defatted Soya Flour-India).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration

The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 820 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 6 Preparation of the polysaccharide K5 in a flask with a proteinaceous concentrate extracted from soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 g/l of proteinaceous concentrate obtained from defatted soya flour (ABS Food-ABSPS60-India).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 720 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 7 Preparation of the polysaccharide K5 in a flask with a proteinaceous concentrate extracted from soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 2 g/l of defatted soya flour (Cereol).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 710 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 8 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 1 g/l of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 780 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 9 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared as in example 1 using the culture medium SD and 1 g/I of defatted soya flour (ABS Food-Defatted Soya Flour-India).

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 815 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 10 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 gui of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 1% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 750 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 11 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 g/t of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared

in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 830 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 12 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 g/t of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 1% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 800 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 13 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 g/I of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 930 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 14 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 g/l of defatted soya flour (ABS Food-Defatted Soya Flour-India).

Each flask was inoculated with 1% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 790 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 15 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 g/I of defatted soya flour (ABS Food-Defatted Soya Flour-India).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 810 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 16 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 gui of defatted soya flour (ABS Food-Defatted Soya Flour-India).

Each flask was inoculated with 1% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 810 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 17 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 g/t of defatted soya flour (ABS Food-Defatted Soya Flour-India).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 820 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 18 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 g/I of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium and prolonging the fermentation time to 30 h.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 850 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 19 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 1 g/l of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium and prolonging the fermentation time to 48 h.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as

example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 710 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 20 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 g/l of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium and prolonging the fermentation time to 30 h.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 830 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of

about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 21 Preparation of the polysaccharide K5 in a flask with soya flour.

The polysaccharide K5 was prepared using the culture medium SD as in example 1 and 2 g/l of defatted soya flour (Cargill Foods S. R. L., Milan, 100/20 S).

Each flask was inoculated with 10% (v/v) using a culture in liquid of 16 h prepared in the same culture medium and prolonging the fermentation time to 48 h.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 720 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 22 Preparation of the polysaccharide K5 in a flask with dialyse yeast autolysate.

For the production of the polysaccharide K5 in a flask, 750 ml flasks with a deflector were used, each containing 100 mi of culture medium.

The culture medium AL was used having the following composition (g/l): yeast autolysate: 10.0 (10.0 g are dissolved in 50 ml and dialyse against 50 ml of water) K2HPO4: 9.7

KH2PO4: 2.0 MgCI2:0. 1 sodium citrate: 0.5 ammonium sulphate: 1.0 glucose: 2.0 well water: 1,000 ml, pH 7.3, sterilisation at 118°C for 20 min.

The glucose solution was prepared separately, sterilised at 115°C for 30 min. and added in sterile condition to the culture medium.

Each flask was inoculated starting from a cellular suspension from a slant of TSA (Tryptic Soya Agar) incubated at 37°C for 24 h. The flasks were placed to incubate with alternative agitator (6 cm of movement, 160 cpm) at 37°C for 24 h and samples were taken at different times. The microbe development was assessed by means of a direct count in the optical microscope using the Burker chamber.

At the end of fermentation, heat treatment was performed as described in example 1.

The polysaccharide was separated with the same procedure used in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out in the same conditions as example 1.

The fermentation yield in purified K5 is about 780 mg/l.

The samples of K5 obtained presented a content of uronic acids, dosed with the carbazole method, of about 40%.

The polysaccharide was made up of two components: one with an apparent PM of about 16,000 D (70-80%) and the other with PM 5,000 D (20-30%).

Example 23

Preparation of the polysaccharide K5 in a fermenter.

The preparation was carried out in an automated 14 I fermenter with a working volume of 10 1 (Chemap-Braun, Melsungen, Germany). The culture medium SD was used having the same composition as in example 1.

The fermentation conditions adopted were the same as in example 1, with inoculation of 10% (v/v) from a submerged culture of 24 h prepared in a flask using the same culture medium, with aeration 1 vvm (volume per volume per minute), agitation 400 rpm, temperature 37°C, fermentation time up to 24 h.

During fermentation, the following parameters were assessed: trend of the ph trend of the dissolved oxygen residual glucose polysaccharide produced microbe development At the end of fermentation, heat treatment was carried out (80°C for 10 min) in order to inactivate any enzymatic components present.

The polysaccharide was separated with the same procedure used in example 1.

The culture filtrate (10 I) was subjected to concentration by ultrafiltration using membranes with cut-off 8,000 and 10,000 D down to a final volume of about 2 1 using a SS 316 module (MST, Gallarate, Varese) with spiralled polymer membranes (PES).

The polysaccharide was precipitated by adding 4 volumes of ethanol at 4°C overnight, then separated from the supernatant liquor by centrifugation at 10,000 rpm for 20 min.

For deproteinization the same fungal protease was used as in example 1, dissolving the precipitate in 450 ml of reaction buffer and proceeding in the same conditions as example 1, using the SS 316 module and 500 ml of distille water for ultrafiltration.

The samples of K5 obtained at 24 h presented a content of about 70-80% of the component at 16,000 D and about 20-30% of the one at 5,000 D.

Yield: 820 mg/l.

Example 24

Preparation of the polysaccharide K4 in a flask.

The polysaccharide K4 was prepared using the medium SD in the same conditions as example 1, checking the same parameters during fermentation.

At the end of fermentation, heat treatment was carried out (80°C for 10 min) in order to inactivate any enzymatic components present.

The polysaccharide was isolated as in example 1.

Concentration The concentration of the culture filtrate was performed as in example 1 using a 300,000 D membrane for filtration.

Precipitation The precipitation of the polysaccharide was carried out in the same conditions as example 1.

Deproteinization Deproteinization was carried out in the same conditions as example 1.

Purification Purification was carried out by means of two extraction cycles with 30 ml of NaCI 1 M, followed by dialysis by ultrafiltration with 50 ml of distille water and precipitation with ethanol.

The precipitate obtained from the second precipitation was dissolved in H2O and subjected to chromatography with ion exchange on a DEAE column, washing the column with 1 volume of NaCI 0.4 M and separating the polysaccharide with 1 volume of NaCI 0.6 M.

The fermentation yield in purified K4 is about 400 mg/l.

Characterization of K4 The samples obtained at the end of purification were analyse according to the following procedures and with the following results.

Content of uronic acids about 25% determined using the carbazole method (Bitter et a/., Anal. Biochem. 4,330-334,1962).

Fructose content: about 14.1 % determined using the enzymatic test for fructose (hexokinase/phosphoglucoisomerase/glucose-6-phosphate dehydrogenase/NAPD Kit, Boehringer, Mannheim, Germany), after hydrolysis of the K4 (triphluoroacetic acid 0.1 M at 100°C for 30 min).

Galactosamine content: about 23% determined using the Elson-Morgan reactant after hydrolysis of the K4 (HCI 4M at 100°C for 18 h).

Spectrometry 13C NMR Varian Gemini 200 operating at 50.3 MHz at 22°C using TSP as standard.

Sample 50 mg/ml in NaCI 1 M.

The spectrums of the polysaccharide produced were similar to the spectrums for the polysaccharide K4 reported in the literature.

The principal signals related to N-acetylgalactosamine and to D-glucuronic acid bonded in sequence and to fructose bonded in a lateral chain to glucuronic acid were particularly shown.

Assessment of the molecular weight by HPLC chromatography with molecular exclusion: column: (1 x 30 cm) Superdex 75 HR (Pharmacia) eluent: phosphate buffer 0.1 M, pH 7 with addition of NaCI 0.15 M flow: 1 ml/min detector: UV at 210 nm sample: 20 1 of a solution 2 mg/ml.

The polysaccharide was made up of a single component with an apparent PM of about 300,000 D.

Example 25 Preparation of the polysaccharide K4 in a fermenter.

The preparation was carried out in an automated 14 I fermenter with a working volume of 10 1 (Chemap-Braun, Melsungen, Germany).

The culture medium SD was used having the same composition as in example 1.

The following fermentation conditions were adopted: inoculation of 10% (v/v) from a submerged culture of 24 h prepared in a flask using the same culture medium as example 1, aeration 1 vvm (volume per volume per minute), agitation 400 rpm, temperature 37°C, fermentation time up to 24 h.

Isolation and purification of the polysaccharide were carried out as in example 3, using 300,000 D membranes for concentration.

Yield: 420 mg/l.