NL8601154A - NITROGEN-CONTAINING POLYSACCHARIDES, THEIR PREPARATION METHOD AND USE AS THERAPEUTIC AGENTS. - Google Patents

Description

NL 33527 dJ / hf r

Nitrogen-containing polysaccharides, their method of preparation and their use as therapeutic agents.

The present invention relates, in general, to nitrogen-containing polysaccharides, to a process for their preparation, and to their use as therapeutic agents. The invention particularly relates to a preparation process of such nitrogen-containing polysaccharides, which consists of a fermentation process during which a nitrogen-containing polysaccharide compound forms in the culture broth, this compound then being isolated from the culture broth in the form of an adsorption product , which will be described more fully below.

The fermentation is carried out in an original manner, initially using a microorganism of the species Zymomonas as the inoculum of the culture medium during the first days of the fermentation, and subsequently inoculating another seed in the same culture broth, which is thus is modified using Pediococcus acidilactici. Thereafter, the process is continued to obtain the desired compound in the culture broth, and this compound is then isolated, owing to the formation of an adsorption product, which contains substantially the total amount of this compound.

Accordingly, one uses a fermentation, which could be called "united culture fermentation", which J. Risler describes in his work "Les complexes anti-biotiques d'adaption" (PACOMHY Publishers, 7 rue Gustave-Nadaud, Paris (16th)), or with a mixed culture ("fermentation with mixed culture"), so called by CW Hesseltine (Ann. Rev. Microbiol. 37,575-601-1983), in which work is being done to obtain different foods from a fermentation using mixed cultures.

The fermentation process of the present process differs from the technique which the latter author calls "polyculture" in that, although more than one microorganism is used for the fermentation and the v. ....

- 2 - preparation of the desired final compound, these microorganisms are unknown, while in the present case the broth is inoculated with pure cultures of the two aforementioned microorganisms, causing a phase shift between the additions of these microorganisms . This method is original and has several advantages, such as, for example, allowing a higher growth rate of the two germs, a stable association of the two, a much greater resistance against the action of phagocytes and a better yield of the desired compound, as well as the possibility to use mixed substrates in the preparation of the culture medium.

The following example adequately describes the invention.

90 kg of soybeans, which are washed twice with water, are then placed in a metal vessel (preferably made of stainless steel), the washing liquids are then removed and then enough water is added that the beans are completely covered by this water. The vessel is closed and the beans are soaked at a temperature of about 10-15 ° C (this temperature is not critical) for about 18 hours. After this soaking time has ended, proceed with a coarse grinding in the moist state of the seeds, which will then form part of the culture broth. In the present case, the method represents an important modification compared to normal fermentation techniques, in which finely ground ingredients are always used in the preparation of the culture broth with a view to its subsequent fermentation, in order to obtain compounds with a therapeutic effect. ("flour"), once the broth has been prepared, sterilization is continued prior to inoculation of the microorganism to produce the fermentation. In the case of the invention, the water is decanted after the coarse grinding process has been completed in the wet state, and the solid part, consisting of subdivided soybeans, which is transferred to a fermenter with a usable without recovery, is recovered therefrom. working volume of approximately - 3 - 14000 - 15000 liters, which device is equipped with a stirring system and in which approximately 3000 liters of water with a temperature of 39-40 ° C (this temperature is not critical) have been transferred in advance. While maintaining a moderate stirring, the following components of the culture medium are added, these components being finely powdered beforehand. The addition is made in the following order: 65 kg of crystallized camphor; 65 kg microfine sulfur; 40 kg of rosin; 75 kg of dry yeast extract; 2.5 kg of manganese dioxide; and 75 kg of manganese sulfate. After the addition of these components has ended, water is introduced into the fermentor, without stopping stirring, at a temperature of approximately 39 ° C + 2 ° C, until a final volume of 13,000 liters is obtained, after which the culture is prepared. -15 broth thus finished.

When the mixture has been prepared and homogenized, its temperature, which is to be maintained, is controlled during the fermentation process at preferably 39 ° C.

Inoculation of the culture stock - The culture stock is then inoculated with a strain of Zymomonas movilis. The amount of graft used is 10 cells inoculum per liter of culture broth, starting the fermentation process. On the third day proceed with a new inoculation of the broth with a microorganism different from the previous one, the microorganism being formed by a strain called Pediococcus acidilactici, while the amount of inoculum used in this case q is 10 cells per liter of broth. The inoculated broth is kept at a temperature between 37 - 40 ° C without stirring and without aeration for a period of time varying between 5 and 15 days. During this period, the fermentation process and the extraction of the components, and the chemical modifications thereof, as well as a gradual decantation which causes the formation of a gradient of solids, of particles and of micro-organisms in the liquid.

Lysis Hydrolysis - The previous broth is subjected to a lysis hydrolysis process by adding 120 kg of 85 wt% phosphoric acid, 120 kg of urea beads and 3.6 kg of pepsin powder with a titre of 1 / 3,000.

All reaction ingredients are added with stirring until completely dissolved. Thanks to this method, combined with the previously described action of the fermentation process, the following objectives are achieved: total lysis of all microorganisms and solubilization of the nitrogen-containing polysaccharide, as well as its chemical modification, in such a way that the desired, non-toxic and biologically active final compound is obtained. The analyzes show that at this stage the unique macromolecule that exists as such is the polysaccharide containing nitrogen (PN).

The precipitation and formation of the adsorption product: the broth, which has thus been lysed, is filtered over Hyflo Super-Cel or a filtration co-adjuvant with analogous properties. To this end, a suspension of 5 kg of Hyflo Super-Cel in 100 liters of water is prepared, with which a layer is formed in a pressure filter of 10 with a window of 40 x 40 20 cm, to which an inlet pressure of 1 kg / cm2 is applied, while the leave the exit free. Then, without interrupting flow, filtration of the lysate is continued, while maintaining the same pressure in the filter. Then 300 kg of calcium-25 chloride slag (anhydrous) is added to the filtered solution, followed by stirring until a total solution is obtained, followed by the careful addition of sodium hydroxide in a 10% w / w solution, until the pH reaches a value has about 4.7. The approximate addition amount of sodium hydroxide is about 20 liters / minute.

After this operation is finished, the total amount of filtered and partially neutralized liquid is quickly poured into 4,800 liters of pure acetone. This change in the polarity of the solvent causes the formation of an adsorption product (an inorganic complex salt formed by the sulfate and the phosphate of calcium, the analysis of which is given in the chemical description of the compound), which in a practically selective manner the nitrogenous polysaccharide macromolecule ad-

* * 4 U

3: - ^ - 5 - sorbs. Also adsorptions of small quantities of pollutants (reducing sugars, nitrogen bases, amino acids) are formed, but these substances are almost completely removed thanks to three successive washes of the adsorption product with 900 liters of acetone with 28% volume / volume in water. This "acetone water" from the washing is dried, and the solid fraction in suspension in this water is separated by filtration using a rotary filter, and the fraction is then immediately dried in a hot air oven at a temperature from 40-60 ° C.

Table A

Chemical composition of the nitrogen-containing polysaccharide, as well as its inorganic carrier Polysaccharide of

Active organic part manno-glucan 85.6 + 3.2% (2% w / w) Protein 14.4 _ + 3.2%

Inorganic carrier Complex salt of calcium phosphate (98% w / w) containing sulfur, in the form of sulfate

Characteristics of the adsorption product - White, odorless and tasteless (a chalk-flavored) powder, which is insoluble in water and soluble in dilute inorganic acids, nitric acid, hydrochloric acid, etc., and in strong organic acids, such as, for example, acetic acid.

Its solution in nitric acid, when heated, together with ammonium molybdate (reactive solution) gives a yellow precipitate of ammonium phospholyolybdate, indicating the presence of phosphates.

Its solution in dilute hydrochloric acid (2N), with a solution of barium chloride (reagent), gives a white precipitate, which indicates the presence of sulfates.

The aqueous suspension of the adsorption product, treated with a solution of the sodium salt of ethylene-diamine tetraacetic acid, yields a clear uncoloured solution by forming a complex with this salt.

The solution of the adsorption product in dilute 3-6 acetic acid, with ammonium oxalate, gives a white precipitate of calcium oxalate, which indicates the presence of calcium in the adsorption product.

Table B

5 Chemical composition of the nitrogen-containing polysaccharide; polysaccharide fraction Composition in monosaccharides Glucose 23 ± 4%

Mannose 74 ± 5% 10 Other 5 ± 3% (*)

Structure using the SMITH-UNION 1-6 principal analysis.

(*) Mainly ribose, which is likely to cause minor contamination by nucleic acids.

Table C

Chemical composition of the nitrogen-containing polysaccharide; polysaccharide fraction Acetyl derivatives (with GLC) (%) DE

Glycerol 90.5

Mannitol 4.1

Glucitol 2.9

Other 2.5

Table D

25 Amino acid composition% w / w.

Phosphoserine 0.73 ± 0.16

Aspartic acid 10.6 ± 0.28

Threonine 9.2 ± 0.83 30 Serine 8.01 ± 0.32

Glutamic acid 17.6 ± 1.22

Proline 6.1 ± 1.4

Glycine 18 ± 0.05

Alanine 3.9 ± 0.2 35 Aminobutanoic acid 3.5 ± 0.25

Valine 2.5 ± 0.16

Cysteine 3.2 ± 0.23 r - 7 -

Table D (continued)

Methionine 0.62 ± 0.31

Isoleucine 3.05 + 0.13

Leucine 5.4 ± 0.17 5 Tyrosine 2.8 ± 0.16

Phenylalanine 1.87 ± 0.1

Ornitine 0.17 ± 0.02

Lysine 9.2 ± 0.52

Histidine 2.15 ± 0.38 Arginine 6.65 ± 0.36

The attached figure shows the infrared spectrum of two samples of the nitrogenous polysaccharide, in pure state, obtained from two different batches of adsorption product. As can be seen from this, the spectra obtained correspond. The positions of the most important tires (and cm) are located at: 3,400, 2,940, 1,660, 1,135, 1,060, 980 and 815.

The polysaccharides of the invention are especially useful as therapeutic agents, especially as immunostimulants, especially for the stimulation of the organic defense mechanisms, of secondary immunodeficient states, as well as of immunodeficient problems in general.

Claims (9)

Nitrogen-containing polysaccharides, characterized in that it is obtained by the fermentation of the culture broth produced by inoculating two different classes of bacteria, belonging to the species Zymomonas and Pediococcus, the polysaccharide being isolated in the form of an adsorption product. Nitrogen-containing polysaccharides according to claim 1, characterized in that the specimen of the Zymomonas species is Zymomonas movilis and the specimen of the Pediococcus species is the Pediococcus acidilactici. 3. A process for the preparation of a nitrogen-containing polysaccharide, which is isolated in the form of an adsorption product, characterized in that the fermentation of the culture broth is effected by inoculating two different classes of bacteria, whereby the in the culture broth inoculated bacteria belong to the species Zymomonas and Pediococcus. 4. Method according to claim 3, characterized in that the inoculated specimen of the Zymomonas species is Zymomonas movilis and the specimen belonging to the Pediococcus species is used Pediococcus acidilactici. A method according to any one of claims 3 or 4, 25. characterized in that said strains are not inoculated simultaneously in the culture medium, wherein the Pediococcus acidilactici is inoculated on the third day after inoculating the strain Zymomonas movilis in the culture broth . A method according to any one of claims 4 or 5, characterized in that the culture broth is not sterilized by heat and is kept for 5-15 days without stirring and without aeration at a temperature of 37 - 40 ° C. Process according to claim 6, characterized in that, when the fermentation is finished, a lysis hydrolysis treatment of the culture broth is continued by adding thereto phosphoric acid, urea and pepsin-9. 8. Process according to claim 7, characterized in that the broth, after having undergone the lysis, is filtered using a filtration co-adjuvant, after which anhydrous calcium chloride is added and, after dissolving, a solution of sodium hydroxide. until the pH has reached about 4.7, then the resulting solution is poured into a large excess of acetone to give a complex precipitate in which the desired nitrogen-containing polysaccharide is retained in the form of an adsorption product. 9. Use of the nitrogen-containing polysaccharides according to claims 1 or 2, or obtainable according to any one of claims 3-8, as therapeutic agents, in particular as immunostimulating agents, in particular for the stimulation of organic defense mechanisms, of secondary immunodeficient states and of immuno-deficient problems in general. C "