LU86418A1 - NITROGENED POLYSACCHARIDES AND THEIR PROCESS FOR OBTAINING - Google Patents

Description

- * 'r "Nitrogenous polysaccharides and their production process"

The present invention relates, in general, to nitrogenous polysaccharides and to their process for obtaining. It refers in particular to a process for obtaining such nitrogenous polysaccharides, which consists of a fermentation process during which a nitrogenous polysaccharide compound is formed in the culture broth, this compound then being isolated from the culture broth in the form of an adsorption product, and this will be described more fully below.

The fermentation is carried out in an original manner, first using, as inoculum of the culture medium, a microorganism of the genus Zymomonas during the first days of the fermentation, and then carrying out another sowing in the same culture broth which is thus modified therefrom, using Pediococcus acidilactici, then continuing the process 15 until the desired compound is obtained in the culture broth and then separating it thanks to the formation of 'an adsorption product which contains almost all of this compound.

? Consequently, a fermentation which one could call "associated cultures" is used, which Dr. 3. Risler defines 20 in his work "Antibiotic adaptation complexes" (PACOMHY Editeurs, 7 rue Gustave-Nadaud, Paris (16th), or mixed culture ("fermentation mixed culture"), name due to CW Hesseltine (Ann. Rev. Microbiol. 37,575-601-1983), work in which we study obtaining different foods through a fermentation using 25 mixed cultures.

The fermen ation method according to the present invention differs from the technique which the last mentioned author calls "polyculture", in the sense that although more than one microorganism is used for fermentation and l When the desired final compound is obtained, these microorganisms are unknown, while in the present case the broth is inoculated with pure cultures of the two microorganisms - mentioned previously, providing for a phase shift between the additions of these microorganisms. The procedure is original and has various advantages, such as allowing a faster rate of growth of the two germs, a stable association of these, a much greater resistance to the action of phagocytes and a better yield of desired compound, as well as the possibility of employing mixed substrates in the preparation of the culture medium.

The following example sufficiently illustrates the invention.

In a metal container (preferably stainless steel), 90 kg of soybeans are placed and washed twice with water, the washing liquids are eliminated and water is then added in an amount sufficient to that the seeds are completely covered by this water. The container is closed and left to soak at a temperature of approximately 10 to ÔC (this temperature is not critical) for approximately 18 hours. This maceration time having elapsed, a coarse grinding, in the wet state, of the seeds 20 is carried out which will then form part of the culture broth. In the present case, the process represents an important modification compared to normal fermentation techniques, in which, in the preparation of the culture broth for its subsequent fermentation, with the aim of obtaining compounds having a therapeutic action. tick, finely ground ingredients are always used ("farinesi"), proceeding, as soon as the broth has been prepared, to its sterilization before the inoculation of the microorganism producing fermentation. In the case of the invention, when the coarse grinding process in the wet state is finished, the water is decanted, recovering from it the solid part constituted by the subdivided soybeans which, without sterilization, are placed in a fermenter of a useful working volume of approximately 14,000 to 15,000 liters, apparatus provided with a stirring system and to which approximately 3,000 liters of water have previously been added at a temperature of 39-40 ° C (this temperature 35 is not not critical). Maintaining moderate agitation, the following 3 components of the culture medium are added, these components having been previously finely sprayed. The addition is made in the following order: crystallized camphor: 65 kg; micronized sulfur: 65 kg; rosin-- ne: 40 kg; dry yeast extract: 75 kg; manganese dioxide: 5 2.5 kg; and manganese sulfate: 75 kg. When the addition of these components is complete, water is added to the fermenter, without stopping the stirring, at a temperature of approximately 39 ° C. + 2 ° C., until a final volume of 13,000 liters is reached. , the preparation of the culture broth having thus been completed.

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

Inoculation of the culture broth - Next, the culture broth is inoculated with a strain of Zymomonas movilis. The quantity of inoculum which is used is 10 cells per liter of culture broth, inoculum with which the fermentation process is initiated. On the third day, a new inoculation of the broth is carried out by a microorganism different from the previous one, this microorganism consisting of a strain called Pediococcus 20 acidilactici, the quantity of inoculum used in this case being 9 10 cells per liter of broth. The inoculated broth is maintained without stirring or aeration at a temperature between 37 and 40 ° C over a period of time which varies between 5 and 15 days. During this period, the process of fermentation and extraction of the components takes place, and the chemical modifications of these components, as well as a gradual decantation which causes the formation, within the liquid, of a gradient of solids. , particles and microorganisms.

Lysis-hydrolysis - The previous broth is subjected to a lysis-hydrolysis process by adding 120 kg of phosphori-30 acid only at 85% by weight, 120 kg of urea in pearls and 3.6 kg of pepsin powder d '' a title of 1 / 3.000.

All reagents are added with stirring until complete dissolution. Thanks to this process, combined with the action of the fermentative process described above, the following objectives are achieved: complete lysis of all microorganisms and solubilization of the nitrogen-containing polysaccha- 4 ride, as well as its chemical modification , so as to obtain the desired final compound, non-toxic and biologically active. The analyzes show that at this stage, the single macromolecule which remains as such is the nitrogen-containing polysaccharide (PN).

Precipitation and formation of the adsorption product: the broth which has thus undergone a lysis is filtered through Hyflo Super-Cel or a co-filter aid having similar properties. For this, a suspension of 5 kg of Hyflo Super-Cel is prepared in 100 10 liters of water, with which a layer is formed in a filter press of 10 frames of 40 x 40 cm, to which a pressure of 1 kg / cm2 inlet leaving the outlet free. Then and without interrupting the current, the lysate is filtered while maintaining the same pressure in the filter. Then 300 kg of calcium chloride slag (anhydrous) are added to the filtered solution, the mixture is stirred until complete dissolution is obtained and sodium hydroxide in a 10% solution is carefully incorporated. w / w, until the pH has a value of about 4.7. The approximate sodium hydroxide addition flow is approximately 20 liters / minute.

This operation being completed, the total of the filtered and partially neutralized liquid is quickly poured onto 4,800 liters of pure acetone. This change in polarity of the solvent determines the formation of an adsorption product (inorganic complex salt formed by calcium sulphate and phosphate, the analysis of which will be given in the chemical description of the compound) which adsorbs practically so selective, the macromolecule constituted by the polysaccharide containing nitrogen. There is also adsorption of small quantities of contaminating substances (reducing sugars, nitrogenous bases, amino acids) but these substances are eliminated almost completely by three successive washes of the adsorption product with 900 liters of acetone at 28% in volume / volume in water. These washing "acetone waters" are dried and the solid fraction suspended in these waters is separated by filtration using a rotary filter and is immediately dried in a hot air oven at a temperature of 40-60 ° C.

* * 5

Table I

Chemical composition of the polysaccharide containing nitrogen, as well as its inorganic support - Polysaccharide of 5 Active organic part manno-glucan 85.6 _ + 3.2 96 (2% w / w) Proteins 14.4 _ + 3, 2 96

Inorganic support Complex calcium phosphate salt (98 96 w / w) containing sulfur, in the form of sulphate 10 Characteristics of the adsorption product - White powder, odorless and tasteless (with a chalk flavor), 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 nitric solution gives hot, with the ammonium molybdate (reactive solution) a yellow precipitate of ammonium phosphomolybdate, which indicates the presence of phosphates.

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

The aqueous suspension of the adsorption product treated with a solution of the sodium salt of ethylenediamine-tetra-acetic acid gives a transparent and colorless liquid by forming a complex with this salt.

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

Table II

30 Chemical composition of the nitrogen-containing polysaccharide; polysaccharide fraction Composition in monosaccharides Glucose 23 + _ 4%

Mannose 7lf + _ 5% 35 Others 5 + _ 3% (*) 6 *

Table II (continued)

Structure using the degradation of SMITH-UNION 1-6 main payment.

5 (*) Mainly ribose, which probably comes from a little contamination by nucleic acids.

Table III

Chemical composition of the nitrogenous polysaccharide; polysaccharide fraction Acetylated derivatives (by CGL) (%) DE

10 Glycerol 90.5

Mannitol 4.1

Glucitol 2.9

Others 2.5

Table IV

15 Amino acid composition% in w / w

Phosphoserine 0.73 0.16

Aspartic acid 10.6 + 0.28

Threonine 9.2 _ + 0.83 20 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 25 Amino-butyric acid 3.5 _ + 0.25

Valine 2.5 + 0.16

Cysteine 3.2 _ + 0.23 Methionine 0.62 ^ + 0.31

Isoleucine 3.05 _ + 0.13 30 Leucine 5.4 _ + 0.17

Tyrosine 2.8 _ + 0.16

Phenylalanine 1.87 _ + 0.1

Ornitine 0.17 ^ 0.02

Lysine 9.2 _ + 0.52 35 Histidine 2.15 _ + 0.38

Arginine 6.65 _ + 0.36

P

7

P

The appended figure gives the infrared spectrum of two samples of the polysaccharide containing nitrogen, in the pure state, obtained from two different batches of adsorption product. As can be seen, the spectra obtained are equal. The position 5 of the most important bands (in cm L) is located at: 3.400, 2.940, 1.660, 1.135, 1.060, 980 and 815.

The polysaccharides according to the invention can be used in particular as therapeutic agents, in particular as imunostimulants, in particular for the stimulation of organic defenses, secondary immunodeficiency states and immunodeficiency problems in general.

Claims (9)

1. Nitrogenous polysaccharides, characterized in that they are obtained by fermentation of the culture broth, produced by the inoculation of two different classes of bacteria, belonging to the genera Zymomonas and Pediococcus, the polysaccharide being isolated in the form of a adsorption product. 2. Nitrogen polysaccharides according to claim 1, characterized in that the species of the genus Zymomonas is Zymomonas movilis and the species of the genus Pediococcus is Pediococcus acidilac-tici. 3. Method for obtaining a nitrogenous polysaccharide isolated in the form of an adsorption product, characterized in that the fermentation of the culture broth is carried out by the inoculation of two different classes of bacteria, the inoculated bacteria in the culture broth belonging to the genera Zymomonas and Pediococcus. 4. Method according to claim 3, characterized in that the inoculated species of the genus Zymomonas is Zymomonas movilis, and the species belonging to the genus Pediococcus is Pediococcus acidilactici. 5. Method according to either of claims 20 and 4, characterized in that the abovementioned strains are not inoculated at the same time in the culture medium, the Pediococcus acidilactici being inoculated on the third day after inoculation , in the culture broth, of the strain Zymomonas movilis. 6. Method according to any one of claims 4 to 5, characterized in that the culture broth is not heat sterilized and is maintained without stirring or aeration at a temperature of 37 to 40 ° C for 5 a 15 days. 7. Method according to claim 6, characterized in that, when the fermentation is complete, a process of lysis-hydrolysis of the culture broth is carried out, by addition to it of phosphoric acid, of urea and pepsin. 8. The method of claim 7, in that the broth having undergone lysis and having been filtered using a co-filtering aid, anhydrous calcium chloride and, V ** 9 κ "are added thereto. ft after dissolution, a solution of sodium hydroxide is added thereto until the pH reaches a value of approximately 4.7, then the solution obtained is poured onto a significant excess of acetone with which one obtains * · a complex precipitate in which the desired nitrogen polysaccharide 5 is retained in the form of an adsorption product. 9. Use of the nitrogenous polysaccharides according to claim 1 or 2 or as obtained according to any one of claims 3 to 8, as therapeutic agents, in particular as immunostimulants, in particular for stimulating the defenses. organic, secondary immunodeficiency states and immunodeficiency problems in general. Drawings: ......... / l ........ plate ^ w ™, i2 ... pages of which ....... y! ...... cover page .........- tL- pages of description ..........- claims pay ....... c brief description Luxembourg, ie -2 MA11986 The agent: Me Alain Rukavina