DE3121548A1 - Process for the preparation of a polysaccharide - Google Patents

Abstract

A polysaccharide with a molecular weight of more than 200,000 is obtained by cultivation of Bacillus polymyxa No. 271 (FERM-P 1824) in an aqueous culture medium which contains assimilable saccharides. The polysaccharide and its salts with alkali metals, alkaline earth metals, transition metals, manganese, aluminium, zinc, copper and with ammonium ions have excellent activity in reducing the serum and liver cholesterol levels and the atherogenic index and are therefore suitable as component to pharmaceutical compositions and dietetic foods.

Description

DESCRIPTION

the invention relates to a method for producing a polysaccharide.

A method for producing a polysaccharide using of Bacillus polymyxa No. 271 is known from Japanese Patent Application 67-7600. Bacillus polymJa No. 271 has been registered with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry and has been given the depository number FERM-P 1824.

In Japanese Patent Application 67-7600 there is a method of manufacture of a highly viscous polysaccharide by cultivating Bacillus polymyxa No. 271 in a culture medium containing an assimilable carbon source such as glucose, sucrose and containing lactose. That obtained from the viscous fermentation broth Polysaccharide consists of glucose, mannose, galactose and glucuronic acid. It was also known that the mixture of the above-described acidic polysaccharide with the neutral polysaccharide was obtained from a medium containing sucrose contained as a carbon source.

The molecular weight; that by Bacillus polyiuyxa No. 271 in the Glucose-containing medium formed polysaccnarias was determined using the Staudinger formula determined, a value of about 1,300,000 was found. This is in journal of Agricultural Chemical Society of Japan, Volume 42, No. 7, pages 431 to 434 (1968) described.

The invention is based on the object of a method for production a polysaccharide with a molecular weight of more than 200,000 are available to provide that activity to suppress an increase in serum and liver cholesterol levels and the atherogenic index.

It is also an object of the invention to provide a method of production a salt of this polysaccharide with a cation having a degree of substitution with cations greater than 0.2 to provide the activity to lower an increase in serum and liver cholesterol levels and the atherogenic index Ha.

According to the invention, these objects can be achieved with the aid of a method for Production of a poly saccharide, according to Bacillus polymyxa No. 271 (FERM-P 1824) in a culture medium containing an assimilable carbon source contains, is grown until highly viscous polysaccharide is accumulated in the medium is, and this polysaccharide is obtained from the medium. Bacillus poylmyxa No. 271 is preferably in the medium for a period of more than 20 hours bred. The enriched polysaccharide can be obtained by ultrafiltration and / or precipitation be cleaned with an alcohol.

Another object of the invention can be achieved, typically a method for the preparation of a salt of a polysaccharide with a cation in which Bacillus polymyxa No. 271 is grown in a culture medium that is an assimilable source of carbon and a sufficient amount of the Contains cation to a salt of a polysaccharide with a degree of substitution of more than 0.2 to form a highly viscous salt of the polysaccharide in the medium is enriched, and this salt of the polysaccharide is obtained from the medium.

The culture medium preferably contains a total number of equivalents of Cations of at least 0.005 per titer of medium and in the medium a pH of more than 4.5 is maintained.

If the growing conditions are not suitable for the production of the desired salt of the polysaccharide or the enriched salt of the polysaccharide undergoes decationization, then can to obtain the desired salt of the polysaccharide to prepare a salt of the desired cation to the fermentation broth or the free polysaccharide are added and stirred for a sufficient time, so that a salt of the polysaccharide is formed with the cation, its degree of substitution (Degree of cationization) is more than 0.2.

The salt of the polysaccharide enriched in the medium can directly or after treatment with the salt of the cation by ultrafiltration and / or Precipitation can be cleaned with alcohol.

These polysaccharides with a molecular weight of more than 200,000 and the salt of the polysaccharide or polysaccharides with a cation with a Substitution degrees of more than 0.2 have a remarkable degradation effect of serum and liver cholesterol levels and the atherogenic index and are therefore for the therapeutic or prophylactic treatment of arteriosclerosis or hypercholesterolemia suitable.

The invention is explained below on the basis of preferred embodiments described.

The invention relates to a method for producing polysaccharides and their salts by cultivating Bacillus polyrnyxa No. 271 (F3.f {M P 1824) in a culture medium.

These polysaccharides and their salts have a remarkable effectiveness Suppression of increases in serum and liver cholesterol and atherogenic levels Index.

It is well known that blood cholesterol levels rise sharply the amount and type of food is affected. The chlosteremia is called considered to be one of the dangerous factors that prevent the occurrence of arteriosclerosis, like from myocardial infarction.

Polysaccharide, a component of food, was previously used by From a dietary standpoint, neglected, RUS-taken the digestible and absorbable Polysaccharides, such as starch.

According to the invention, animal experiments were carried out with rats to obtain a Means for lowering the cholesterol level in the serum and the liver from the polysaccharide produced by Bacillus polymyxa No. 271. (That by Bacillus polymyxa No. 271 is hereinafter referred to as B.p. abbreviated.) It B.P., which has an average molecular weight in excess of 200,000 possesses the effect of remarkably lowering an increase in the level of cholesterol in serum and liver and activity to surprisingly increase cholesterol levels of high-density lipoprotein (hereinafter abbreviated as HDL) having as a factor for the prevention of arteriosclerosis we are known.

The effect of increasing the cholesterol level of MDL shows the Activity for lowering the atherogenic index indicated by the following formula becomes: Serum Cholesterol HDL Cholesterol HDL Cholesterol This is an unpredictable one Effect of B.p.

The mean or average molecular weight (hereinafter referred to only as the molecular weight) of F.pv was determined by the method neCh Nlnomiya et al in J. Agr. Chem.

Soc. Japan 42 (7), p. 431 (1968).

The polysaccharide of the present invention can be produced by Bacillus polymyxa No. 271 (FERM-P No. 1824) aerobically in an aqueous culture medium is grown that contains 3 to 5% of a carbon source such as glucose, sucrose, Contains lactose, molasses and other saccharides, and which is a source of nitrogen, such as peptone, corn steep liquor, yeast extracts and urea, and salts such as magnesium sulfate, are added.

The enriched viscous polysaccharide is precipitated with Alcohol and ultrafiltration purified. It is essential that the molecular weight by B.p. more than 200,000 is to suppress the increase in activity To show serum and liver cholesterol levels.

The conditions for the cultivation of the microbes and for the purification of B.p. were fully investigated as the fermentation broth was very viscous and their treatment, such as by filtration, concentration and drying in the purification process was very difficult.

The cultivation time is usually more than 20 hours to get the Polysaccharide with an average molecular weight greater than 200,000 to obtain.

A polysaccharide with a molecular weight of 190,000 is made by Cultivation of the microorganism formed for 16 hours.

The polysaccharide with an average molecular weight of more than 200,000 can also be obtained by considering the polysaccharide, which is a Has molecular weight less than 200,000, with alcohols in lower concentration treated. The higher molecular weight polysaccharide is obtained from alcohols precipitated in lower concentration, while that of lower molecular weight is precipitated with alcohols of higher concentration. Salts, such as sodium chloride or Potassium chloride, can lower the precipitation of the polysaccharide with alcohol Facilitate concentration.

The lower molecular weight polysaccharide falls by Adding salts easier from without adding salts when alcohol of each same concentration is used.

The high molecular weight polysaccharide can be mixed with acids or Alkalis are hydrolyzed, the polysaccharide being of lower molecular weight is obtained.

It is preferred to remove the polysaccharide before removing the cells hydrolyze from the culture broth by filtration, because this reduces the resistance to filtration is decreased.

The polysaccharide produced with the aid of the method according to the invention shows activity to lower serum and liver cholesterol levels as tested on rats treated with a 5% B.p. containing diet raised became.

The B.p. can be used as a powder or as an aqueous solution or used as an aqueous suspension. It is given either on its own or added to the diet or different types of food (health promotional foods).

It has also been found that the salts of Bacillus polymyxa No. 271 produced polysaccharide cholesterol lowering activity show in serum and liver. The investigation was carried out by acidic Polysaccharide has been combined with different cations to establish the relationship between the activity to lower the cholesterol level, the type of B.p. to be bound Determine cations and the degree of substitution with these cations.

The salts of the polysaccharide with cations, of which at least one member from the group of alkali metals, alkali metal metals, transition metals, manganese, aluminum, Zinc, copper and ammonium ions are selected that have a degree of substitution of more than 0.2 have remarkable cholesterol lowering effectiveness in serum and liver.

The salts of B.p. with cations can be made, others Bacillus polymyxa No. 271 is grown aerobically in a culture medium containing 3 to 5, o a carbon source1 such as glucose, galactose, lactose, molasses and others Contains saccharides and a nitrogen source, such as peptone, corn steep liquor, Yeast extracts and urea as well as salts are ugesetzt with suitable cations are combined, such as phosophate, magnesium sulfate.

The polysaccharide accumulated in the medium is highly viscous and consists of glucose, D-mannose, D-galactose and -D-glucuronic acid, the ratio of these components being 3: 3: 1: 2. From the ratio of the sugars present as constituents and the combination of two cations with a molecular weight of 1,500, the degree of substitution with cations is calculated as follows: Mi: content of cations in g / kg Ni: gram equivalent of cations Suitable alkali metals include sodium and potassium, and alkaline earth metals calcium and magnesium.

The amount of the cations contained in the culture medium is preferably at least 0.005 per liter, expressed as total cation equivalent value, and the The pH of the medium must necessarily be greater than 4.5, preferably in the range between 6 and 7.

If the culture medium is not suitable for growing the desired Salt from B.p. it may happen that the enriched salt of B.p. does not have the predetermined degree of substitution. The B.p. or that Polysaccharide that undergoes a decationization treatment such as ion exchange and has been subjected to electrolysis can with the desired Salts treated and stirred for sufficient time to achieve the desired Salt of b: p. with a degree of substitution greater than 0.2 to form before the end product is purified by ultrafiltration or precipitation with alcohol.

Not always all cations are necessary for the microorganism to breed is preferred to B.p. to treat with the gem desired salts after the cation from the salt of B.p. removed with the help of clay exchange or electrolysis was used to buy only the salt from B.p. with the special cation.

The polysaccharide salt produced by the method according to the invention with a degree of substitution of more than 0.2 has degradation activity the cholenterin level in the serum and in the liver according to the tests on rats, who were fed a diet containing 5% of the salt of B.p. contained.

The salt of the invention from B.p. can be used as a powder or as an aqueous solution or as an aqueous suspension and is used as an ingredient in various Foods, so-called health foods or dietetic foods, incorporated.

The methods of making B.p. and its salts and their Cholesterol lowering activities are in the examples below and experiments described.

Example 1 600 ml of the culture broth from the 24 hours carried out Preculture with Bacillus poylmyxa No. 271 (FERM-P 1824) were 12 ml (pH 7.0) of one Culture medium, the 4% crystalline glucose, 0.1% peptone, 2% corn steep liquor, Contained 0.2% magnesium sulfate and 6 ppm magnesium sulfate. Breeding was carried out at a Temperature of 28 ° C under ventilation with 0.8 VVM for 72 hours. the The viscosity of the culture broth had one high value of 9.5 Pa.s. (9,500 cP). The broth was diluted with five times the amount of hot water and heated to 70 ° to 80 ° C. The cells of the microorganism were covered with a diatomaceous earth Pre-coated paper filter filtered off and removed from the broth. 40 1 of the filtered Broth were filtered with an ultrafilter (UF-SW4-85V-1, membrane module Abcor SWM-85M of the Bioengineering Co., LTD), with constant volume filtration up to one Value W / WO = 5 (ratio of the amount of water added to the original broth) purified, concentrated and freeze-dried, taking 50 g of powdered polysaccharide (Sample 1) were obtained.

Example 2 to 4. Comparative Example 1 4 samples of 40 1 of those in Example 1 filtered broth was adjusted to pH with hydrochloric acid set of 1.5. and for 5 minutes (experiment 2) 15 minutes (experiment 3), 45 Minutes (experiment 4) and 2 hours (experiment 5) heated to 80 ° C. and then with sodium hydroxide neutralized. Then each sample was treated as in Example 1 by ultrafiltration and dried, 48 g of powdered polysaccharide (sample 2), 46 g (sample 3), 45 g (sample 4) and 40 g (sample 5) of the powdery polysaccharide in each case Examples 2 to 4 and Comparative Example 1 were obtained.

The molecular weight and analytical data of Examples 1 to 4 and Comparative Example 1 are shown in Table 1.

Table 1 Sample No. Molecular weight of crude protein (%) Ash content (c; o ') 1 93 x 104 1.16 7.6 2 54 x 104 1.02 7.5 3 56 x 104 0.87 7.2 4 25 x 104 0.89 8.1 5 16 x 104 0.78 8.2 Example 5 A filtered broth was passed through Cultivation of the microorganism as in Example 1, using a culture medium (pH 7.0) was used, the 4 Vo Hydrol as a dry by-product from the production of crystalline glucose, 0.1% peptone, 2% corn steep liquor, 0.2% magnesium sulfate and contained 6 ppm manganese sulfate. 40 l of the filtered broth were twofold Volume of ethanol added.

The fibrous nipple formed thereby was by centrifugation won. The precipitate was dewatered, dried and again in 15 l of water solved. After heating the aqueous solution to 70 to 80 ° C. and filtering 25 liters of ethanol were added to the filtrate. The precipitate formed was recovered, dried under vacuum at 50 ° C. and crushed, with 50 g of powdered polysaccharide (Sample 6) were obtained.

Example 6 40 l of the filtered broth obtained in Example 5 were used with an ultrafilter (UF-SWM-35V-1, Membrane-Module-Abcor SWM-85V from Bioengineering Co., LTD), with constant volume filtration up to a W / Wo of 5 (ratio of the amount of added water to the original broth) purified, concentrated and freeze-dried, leaving 50 g powdered polysaccharide (sample 7) were obtained.

Example 7 600 ml of the preculture of Bacillus polymyxa for 24 hours No. 271 obtained preculture broth was added to 12 l of a culture medium (pH 7.0), the 5% crystalline glucose, 0.3% powdered yeast extract, 0.05% urea, 0.2, o 'contained magnesium sulfate and 6 ppm manganese sulfate. The direction was at carried out at a temperature of 28 ° C. with ventilation at 0.8 VVM for 120 hours. 40 l of the filtered broth were obtained in the same way as in Example 1 and the Purification was also carried out as in Example 1, with the modification that methanol was used as a precipitant. Thus, there was obtained 40 g of powdery polysaccharide (Sample 8).

The molecular weight and analytical data of samples 6 to 8 from Examples 5 to 7 are shown in Table 2.

Table 2 Sample No. Molecular Weight Crude Protein (%) Ash (%) 6 104 x 104 2.0 8.1 .7 100 x 104 1.4 6.2 8 136 x 104 1.9 7.5 Example 8 600 ml of the through 24-hour preculture of Bacillus polymyxa No. 271 obtained preculture broth added to 12 l of a culture medium (pH 7.0) containing 4% crystalline glucose, 0.5 °, S Peptone, 0.5% potassium primary phosphate, 0.1% magnesium sulfate (.7H20), 6 ppm manganese sulfate (4 to 6 H20) and 0.1% silicone as a foam brake. The breeding took place at a temperature of 28 0C with aeration with 0.8 VVt for 96 hours. the The viscosity of the culture broth was 14 Pa.s (14,000 cP). The broth was five times Volume of hot water earned and treated in the same way as in Example 5, whereby 53 g of powdery polysaccharide (Sample 9) was obtained.

Example 9 40 ml of a saturated aqueous solution of NaCl was added added as precipitant to 40 l of the filtered broth obtained in Example 7, 80 l of ethanol were also added to the broth and this was then as in Example 5 to obtain 55 g of powdery polysaccharide (Sample 10).

Comparative experiment 2 40 1 of the filtered ones obtained in Example 8 Broth was diluted with the same amount of water and 300 ml of the ion exchange resin Amberlite 200 (H-Form) and 6o0 ml of the ion exchange resin Amberlite IRA-411 (OH-Form) were added to the diluted filtered broth. The resulting mixture became Stirred for 3 hours at a temperature of 400C. The liquid thus treated was with the help of an ultrafilter (UF-SW1-85V-1, Membrane-SIodul-Abcor SWM-85V from Bioengineering Co., LTD.) To a value of W / WO = 5 (ratio of the amount of Water to that of the original broth) purified, concentrated and freeze-dried, whereby 50 g of powdery polysaccharide (Sample 11) was obtained.

Example 10 The filtered broth obtained in Example 8 became like treated in comparative experiment 2 with the ion exchange resin and 50 ml of an aqueous one Solution containing 5 30 potassium chloride was added to the broth. That The resulting mixture was left to stand for one night and as in the comparative experiment 2 to obtain 51 g of powdery polysaccharide (Sample 12).

Example 11 The same treatment of the broth as in Example 10 was made carried out using 20 ml of an aqueous solution containing 5 7 'NaCl in place of the potassium chloride used in Example 10 were used. In this way 51 g of powdery polysaccharide (Sample 13) was obtained.

The molecular weight, the analytical data, the cation content and the degree of substitution with cations for samples 9 to 13 are given in Table 3.

Table 3 Sample Molecular Raw Ash Cation Content (ppm) Total Substi- weight protein equivalent of the equiva- Cation / kg lentgrad Na K Mg Ca count X 104%% 9 114 1.46 7.4 5400 18000 3400 <50 - 0.23 0.46 0.28 0.002 0.97 0.73 10 98 1.23 7.2 15000 9700 2700 <50 - 0.65 0.25 0.22 0.002 1.12 0.84 11 118 0.74 3.0 590 120 270 <50 - 0.025 0.003 0.002 0.002 0.052 0.039 12 118 0.73 4.5 550 22 300 260 <50 - 0.024 0.57 0.021 0.002 0.62 0.47 13 116 0.73 4.9 6900 130 270 <50 - 0.30 0.003 0.002 0.002 0.33 0.25 Example 12 600 ml of the preculture broth obtained over 24 hours with Bacillus polymyxa No. 271 were added to 12 l of a culture medium (pH 7.0) containing 5% crystalline glucose, 0.625% peptone, 0.5% primary potassium phosphate, 0.2 Va contained magnesium sulphate (.7H20), 0.5% calcium carbonate, 6 ppm Ingansulphate (4-GH20) and 0.1 °, 4 soybean oil. The cultivation was carried out at a temperature of 2800 with aeration of 0.8 VYM for 96 hours. The viscosity of the culture bridge was 16 Pa.s (16,000 cP). 40 liters of the filtered broth were treated as in Example 5 to obtain 58 g of powdered polysaccharide (sample 14).

Example 13 40 l of the filtered broth obtained in Example 12 were used subjected to ultrafiltration under the same conditions as in Comparative Example 2, whereby 62 g of powdery polysaccharide (Sample 15) was obtained.

Example 14 40 l of the filtered broth obtained in Example 12 were used diluted with the same amount of water and demineralized by adding the broth in flow rate (SV3) through a tower (diameter 5 cm) was made with a mixture of 300 ml Amberlite 200 (H-form) and 600 ml Amberlite IRA-411 (OH shape) was filled.

The mixture of salts (35 g primary potassium phosphate and 15 g magnesium sulfate (.7H20), the amount of which is calculated from the salts contained in the original culture medium was dissolved in a small amount of water. It then became the demineralized one Given broth. The resulting mixture was further neutralized with sodium hydroxide and left to stand for one night, after which it was carried out according to the comparative example 2 described method of ultrafiltration was subjected. 60 g powdered polysaccharide (Sample 16) obtained.

Comparative Example The one obtained by ion exchange in Example 14 Broth was subjected to ultrafiltration without the addition of salts, yielding 56 g of powdery Polysaccharide (sample 17) were obtained.

The molecular weight, the analytical data for the cation content and the degree of substitution with cations for Examples 12 to 14 and the comparative example 3 are shown in Table 4.

Table 4 Sample Molecular Raw Ash Cation Content (ppm) Total Substi- weight protein equivalent of the equiva- Cation / kg lentgrad Na K Mg Ca count %% 14 104 X 104 2.0 8.1 5470 18000 7850 4250 - 0.24 0.48 0.65 0.21 1.58 1.18 15 100 1.4 8.9 6670 15600 4420 4120 - 0.29 0.40 0.36 0.21 1.26 0.95 16 89 0.9 6.1 5610 16350 6700 920 - 0.24 0.42 0.55 0.046 1.26 0.95 17 91 0.9 1.2 670 70 310 3190 - 0.029 0.002 0.025 0.16 0.216 0.16 Example 15 600 ml of the preculture broth obtained by culturing Bacillus polymyxa No. 271 for 24 hours was added to 12 liters of a culture medium (pH 7.0) containing 4% crystalline glucose, 0.5% peptone, 0.5% primary potassium phosphate , 0.5 5'o calcium carbonate, 0.1% magnesium sulfate (.7H20), 6 ppm manganese sulfate and 0.1% silicone as a foam brake. The cultivation took place at a temperature of 28 ° C. with a load of 0.8 VVM for 72 hours. The viscosity of the culture broth was 10 Pa.s (10,000 cP). The broth was diluted with five times the volume of hot water and heated to 70-80C. The cells of the microorganism were filtered off from the broth with the aid of a paper filter precoated with diatomaceous earth and removed. 40 l of the filtered broth were treated with an ultrafilter (UF-SWM-85V-1, Membrane-Module-Abcor SWT45V from Bioengineering Co., Ltd.) to a value W / WO = 5 (ratio of the amount of added Water to that of the original broth), concentrated and freeze-dried to give 50 g of powdered polysaccharide (sample 18).

Example 16 to 20 6 samples of 40 1 of the filtered broth were made obtained by dividing the broth formed in Example 15. Every sample was diluted with the same amount of water and demineralized by adding the broth allowed to flow down through a tower (5 cm diameter) at a flow rate (SV = 3) with a mixture of 900 ml Amberlite 200 (H-form) and 600 ml Amberlite IRA-411 (OH shape) was filled. One of the 6 samples was designated Sample 19. Each of the other 5 samples was treated with 1 liter of a 10% aqueous solution of NaCl, KCl, MgC12, CaCl2 or NH4Cl added. Each sample was then taken for one night left to stand and according to the ultrafiltration method described in Example 1 subjected to 40 to 45 g of the special salts of the polysaccharide obtained (samples 19 to 24).

Example 21 1 1 of a 10 percent strength aqueous solution of iron (III) chloride became the broth after treatment with an ion exchange resin as in Example 16 given. The resulting mixture became overnight with occasional Stirring left to stand. The precipitate formed was recovered by centrifugation, washed twice with demineralized water and dried under vacuum, whereby the iron TII salt of the polysaccharide (sample 25) was obtained.

Example 22 1 1 of a 10 percent aqueous solution of aluminum chloride became that after treatment with an ion exchange resin as in Example 16 Given broth. The pH was adjusted to 4.5. The resulting mixture became left to stand for one night with occasional stirring. The precipitate obtained was treated as in Example 21 to obtain the aluminum salt of the polysaccharide was (sample 26).

The molecular weight, the cation content, the total cation equivalents and the degree of substitution of Examples 18-26 are shown in Table 5.

Table 5 Sample Treat- Molecular Cation Content, ppm Total Substitute No. ment weight (equivalents / kg) equivalent degree Na K Mg Ca other 18 Ultra- 89 x 104 5860 13000 3720 2850 filtra- 0.25 0.33 0.31 0.14 1.03 0.78 tion 19 demining 660 85 250 1750 rali- 59 0.03 0.002 0.02 0.09 0.14 0.10 20 salt formation 19500 210 70 380 dung with 63 0.85 0.005 0.005 0.02 0.88 0.66 Na 21 salt formation 54 1150 23400 70 280 dung 0.05 0.60 0.005 0.01 0.67 0.50 with K 22 salt formation 68 78 130 13 700 200 dung 0.003 0.003 1.13 0.01 1.15 0.86 with Mg 23 salt formation - 250 110 130 19500 dung 0.01 0.003 0.01 0.98 1.00 0.75 with Ca 24 salt formation 70 1090 75 32 280 NH4: 18600 0.05 0.002 0.002 0.01 1.03 1.09 0.82 with NH4 25 salt bil- - 93 120 50 75 Fe 24 300 dung 0.004 0.003 0.004 0.004 1.30 1.32 0.99 with fur 26 salt bil- - 25 70 3 25 Al 11600 dung 0.001 0.002 - 0.001 1.29 1.29 0.97 with Al Attempt 1 Seven groups of rats that had been reared for four weeks were ranked each Group consisted of five rats were fed a test diet for four days, to consider the influence of the molecular weight of B.p. on the activity of humiliation to examine cholesterol levels.

Seven test groups were formed. It was the standard diet group (without cholesterol), the control diet group (with cholesterol), and test diet groups (with cholesterol and 5% B.p. from each of samples 1 to 5 according to Table 1).

The composition of the diet is shown in Table 6.

The rats were fed the diet and water free.

After the test, the rats' abdomen was anesthetized with ether opened and blood drawn from the abdominal artery. The liver was removed and weighed. The serum was centrifuged and collected from the blood. The liver and the serum were frozen and preserved. The serum cholesterol level was up directly with the aid of the "Determiner TC" device from KYOWA HAKKO Co., LTD. measured. The liver was saponified and the unsaponifiable material was separated. The cholesterol level the unsaponifiable material was determined using the same device TC measured.

The level of HDL cholesterol was determined with the help of HDL Sterozyme of FUJI ZOKI SEIYAKU CO. LTD. measured.

The results obtained are shown in Table 7.

Table 6 Composition of the diet (% by weight) Composition standard control test diet diet diet - Casein 22 22 22 Salt mixture 4 4 4 4 Vitamin mixture2) (water soluble) 0.85 0.85 0.85 Soybean oil3) (mixed with oil 1 1 1 soluble vitamin Choline 0.15 0.15 0.15 Lard 10 10 10 Cholesterol 0 0.5 0.5 1 sodium salt of Cholic acid 0 0.25 0.25 Bp O 0 5 (3) Sucrose 62 61.25 56.25 (58.25) total 100 100 100 1) Harper's salt mixture 2) Harper's vitamin mixture 3) mixed with vitamin A (3000 IU), vitamin D (300IE) and vitamin E (100 mg / kg) of the diet) 4) the expression in brackets indicates the addition of 3% Bp the diet.

Harper's Salt Mix: CaCO3, 29.29; CaHPO4, 2H2O, 0.43; KH2PO4, 34.31; NaCl, 25.06; MgSO4, 7H2O, 9.98 and others Table 7 Diet Cholesterol Level (mg / dl) atherogenic portion 1) group serum HDL liver index of the enriched (mg / g) Cholesterol in the liver standard 91 # 16 59.6 # 16.0 3.7 # 1.2 0.6 # 0.4 diet Control 291 # 53 31.6 # 7.0 22.2 # 0.6 8.2 # 2.0 50.4 # 4.7 diet test diet 5% B.p. 116 # 16 39.5 # 4.8 7.6 # 1.1 1.9 # 0.3 14.2 # 1.7 Sample 1 5% B.p.

Sample 2 129 # 40 38.9 # 8.4 10.2 # 1.9 2.4 # 1.0 16.6 # 6.4 5% B.p. 165 # 41 34.5 # 4.7 9.6 # 2.5 3.8 # 1.2 15.9 # 4.7 Sample 3 5% B.p. 190 # 37 40 # 9.7 12.9 # 0.9 4.2 # 2.6 20.6 # 6.0 Sample 4 5% B.p. 243 # 35 30.5 # 4.8 20.6 # 1.7 7.0 # 2.0 35.4 # 5.3 Sample 5 1) Liver cholesterol content of liver cholesterol content Test or control diet group - the standard diet group X 100 cholesterol intake As as clearly shown in Table 7, the B.p.

with a molecular weight of more than 200,000 (samples 1 to 4) die Effect of a remarkable reduction in serum and cholesterol levels Liver.

The appearance of the liver of flatten raised with standard diet The livers of rats that were on the control diet were reddish-brown and the liver had been raised was yellowish-brown. This was clearly the most characteristic Color of a fatty liver. On the other hand, the liver of rats showed that with the addition by B.p. according to samples 1 to 4 had been grown, a healthy reddish-brown Coloring. The livers of rats given Sample 5 were yellowish brown such as those from rats fed the control diet. This was evident the characteristic color of fatty liver.

Experiment 2 The test was carried out on rats given the samples 6 to 8 according to Table 2 had been administered in order to reduce the activity detect the cholesterol level. 8 diet groups were set up in this experiment.

The composition of the diet and the experimental method were up the same as in experiment 1.

The results are shown in Table 8.

Table 8 Diet cholesterol level (mg / dl) atherogenic proportion of the group ~~~~~~~~~~~~~~~~~~~~~~~~~ Index enriched-serum HDL liver ten Choleste- (mg / g) liver Standard 96 + 13 61.8 + 4.5 2.9 + 0.2 0.6 + 0.2 -diet control 323 + 19 36.3 + 1.2 22.4 + 1.0 8.3 # 0.4 52.4 + 3.8 diet test diet 3% B.p. 213 # 11 32.1 # 1.5 11.9 # 0.6 5.7 # 0.5 19.8 # 2.5 Sample 6 5% B.p. 150 # 11 39.7 # 2.1 9.5 # 0.5 2.9 # 0.5 14.6 # 1.4 sample 6 3% B.p. 138 # 11 34.9 # 1.2 7.9 # 0.5 3.0 # 0.3 9.9 # 1.1 Sample 7 5% B.p. 90+ 3 45.0j2.4 5.4 + 0.6 1.0 + 0.1 3.8 + 1.3 sample 7 3 O, o B.p. 201+ 8 35.8 + 2.2 11.3 + 0.8 4.5 + 0.3 18.8 + 2.0 sample 8 5% B.p. 155 # 14 37.1 # 1.9 9.6 # 0.3 3.0 # 0.5 14.7 # 1.5 sample 8 As can be clearly seen from Table 8, the B.p. the activity a noticeable decrease in serum and liver cholesterol increases and a lowering of the atherogenic index when the diet is 3% B.p.

or 5% B.p. were added. This activity was particularly pronounced at the B.p. (Sample 7) treated by ultrafiltration. Sample 8, which had the highest molecular weight of 1,360,000 showed almost the same Activity as sample 6 with a molecular weight of 1,040,000.

It appears that the activity of B.p.

not above a molecular weight of about 1,000,000 more continues to rise.

The livers of rats raised on a diet which contained the samples according to the invention had a reddish-brown color like that fed with the standard diet.

Experiment 3 The animal experiment was carried out with the samples given in Table 3 carried out to determine the relationship between the degree of substitution and the activity Detect a decrease in cholesterol levels. 5% of the samples were diet added. The minorities of the Frügmedthode were the same as in experiment 1. The results are shown in Table 9.

Table g Diet cholesterol level (mg / dl) atherogenic proportion of the group Serum HDL liver index enriched (mg / g) cholesterol in d.

Liver standard 88T 5 50, 9 # 4.2 3.2 + 0.3 0.7 + 0.1 diet control 251 # 26 28.9 # 2.7 20.6 # 0.5 8.2 # 1.8 49.8 # 2.2 diet test diet 5% B.p. 106 # 6 49.9 # 6.0 7.7 # 1.1 1.3 # 0.3 8.6 # 2.9 Sample 9 5% B.p. 116 # 16 41.0 # 6.1 7.4 # 1.4 2.2 # 0.7 7.8 # 4.2 sample 10 5% B.p. 291 # 147 31.5 # 4.2 23.4 # 0.5 8.4 # 1.3 51.4 # 1.8 Sample 11 5% B.p. 105 # 11 32.0 # 4.2 8.3 # 1.6 2.3 # 0.7 7.5 # 3.1 Sample 12 5% B.p. 154 # 21 28.1 # 2.7 9.8 # 1.6 4.8 # 1.1 12.9 # 4.0 sample 13 the clearly shown in Table 9, Samples 9, 10, 12 and 13 possessed, respectively a degree of substitution of nehr than 0.2 and in particular one Degrees of 0.73, 0.84, 0, and 0.25, respectively, had a noticeable reduction in activity the cholesterol level in serum and liver.

The B.p. (Sample 11) with a degree of substitution of 0.0n9 had one effect similar to that shown in the control diet group.

Samples 12 and 13, to which salts were added after demineralization showed fairly good activity, as did samples 9 and 10.

The presence of the B.p. combined Katien is essential Factor for the activity shown for lowering the cholesterol level. That simple admixture of cations to B.p. did not show such activity as shown in Table 6 as in the 4% Harper's salt mixture of the diet were added to the test diet group. Sample 11 did not show any activity.

In observing the liver of the experimental animals, the liver was revealed of rats fed with the standard diet and the test diet with the inventive B.p. were fed as reddish-brown and those of the animals on the control diet received was yellowish-brown. The appearance of the liver of rats using the invention B.p. was far better than that of the control diet rats, as in FIG Experiments 1 and 2 are described.

Experiment 4 The animal experiment was carried out on rats to which the Samples 14 to 17 according to Table 4 were administered in Examples 12 to 14 and Comparative Example 3 had been prepared. 10 diet groups, i.e. the standard diet group, the control diet group and 8 test diet groups (with cholesterol and samples 14 to 17 according to Table 4 in concentrations of 3% and 5%) were established. The composition the diet and the test method were the same as in Experiment 1.

The results of the test are shown in Table 10.

Table 10 Diet cholesterol level (mg / dl) atherogenic proportion of the group Index serum HDL liver enriched (mg / g) cholesterol in d.

Liver standard 96 # 13 61.8 # 4.5 2.9 # 0.2 0.6 # 0.2 diet control 323 # 19 36.3 # 1.2 22.4 # 1.0 8.3 # 0.4 52.4 # 3.8 diet test diet 3% B.p. 213 # 11 32.1 # 1.5 11.9 # 0.6 5.7 # 0.5 19.8 # 2.5 Sample 14 5% B.p. 150 # 2.1 39.7 # 2.1 9.5 # 0.5 3.0 # 0.3 14.6 # 1.4 Sample 14 3% B.p. 138 # 11 34.9 # 1.2 7.9 # 0.5 3.0 # 0.3 9.9 # 1.1 Sample 15 5% B.p. 90 # 3 45.0 # 2.4 5.4 # 0.6 1.0 # 0.1 3.8 # 1.3 sample 15 3% B.p. 127 # 16 42.2 # 1.0 6.6 # 0.4 2.0 # 0.4 6.5 # 1.1 Sample 16 5% B.p. 91 # 8 45.1 # 4.3 5.3 # 0.4 1.1 # 0.3 2.5 # 1.2 Sample 16 3% B.p. 251 # 17 33.9 # 1.2 15.6 # 1.5 6.5 # 0.4 31 # 3.4 sample 17 5% B.p. 227 # 12 32.4 # 1.6 15.5 # 1.3 6.1 # 0.4 29.1 # 3.2 Sample 17 As clearly shown in Table 10, Samples 14, 15 and 16 were superior to the comparative sample and Sample 17 was superior to one Degree of substitution of 0.16 showed somewhat reduced activity. Sample 16, its degree of substitution Was 0.95 and that obtained by adding salts to the demineralized B.p. and through Ultrafiltration had been produced showed almost the same Effectiveness as sample 15 with a degree of substitution of 0.95 and manufactured by Ultrafiltration without demineralization. The activity of lowering cholesterol was completely restored by the addition of salts.

The color and appearance of the liver of rats given the B.p. had been administered were far better in comparison with the control group, as described in Experiments 1 to 3 above.

Experiment 5 The animal experiment was carried out using samples 18 to 25 (Table 5) carried out, which had been obtained in Examples 15 to 22, to determine the activity of the salts of B.p.

with different cations.

The amount of salt of B.p. added to the diet was 4%.

The experiment was carried out as in Experiment 1. The results of the Experiments are shown in Table 11.

Table 11 Diet cholestetin value (mg / dl) atherogenic proportion of the group -------------------------- Index angerei serum HDL liver cherten (mg / g) cholesterol in d.

Liver standard 92 # 14 43.3 # 7.3 2.5 # 0.3 1.2 # 0.5 diet control 284 # 31 31.8 # 5.4 17.7 # 0.6 8.1 # 1.8 44.1 # 3.1 diet test diet ultrafil- 114 # 16 39.1 # 13.9 7.3 # 1.1 1.9 # 0.3 12.2 # 2.4 trated B.p.

Sample 18 Sample 19 106 # 13 34.6 # 6.5 7.8 # 1.0 1.8 # 0.6 12.8 # 3.7 Na salt sample 20, 92 # 25 40.7 # 12.7 6.5 # 2.2 1.6 # 1.4 8.5 # 5.3 K salt sample 21, 88 # 35 50.3 # 13.2 6.2 # 1.8 1.1 # 1.7 7.7 # 5.0 Mg Salt Sample 22, 85 # 24 47.8 # 11.7 5.8 # 1.6 0.9 # 0.9 6.1 # 3.9 Ca Salt Sample 23, 72 # 17 46.9 # 4.9 4.6 # 1.8 0.5 # 0.2 3.9 # 5.0 NH4 salt sample 24, 102 # 17 37.0 # 6.1 7.7 # 1.3 1.8 # 0.7 12.1 # 3.3 Fe salt sample 25, 110 # 17 42.5 # 17.0 6.5 # 1.9 1.9 # 1.1 8.6 # 5.6 Al Salt As shown in Table 11, the diet with the salts of B.p. with various cations a remarkable effect on lowering the serum cholesterol level, Liver cholesterol levels and the atherogenic index on and the proportion of in the Lebel fortified cholesterol was comparable to the control diet. This shows, that this bound cation in the salt of B.p. strong activity to lower cholesterol.

The ammonium, calcine and magnesium salts of B.p. had special high activities to lower the atherogenic index, which is closely related with the appearance of atherosclerosis. The index showed the same or one lower value than obtained with the standard diet.

When observing the livers of rats exposed to the invention Salts of B.p./terad were found to have the color and appearance the liver were far better than the rats on the control diet, which were in the vorstenender Attempts have been described.

Claims (11)

Process for the preparation of a polysaccharide PATENT CLAIMS 1. Process for producing a polysaccharide having a molecular weight greater than than 200,000 and activity to suppress increases in serum and hepatic choleterin levels and the atherogenic index, by the fact that one is Bacillus polymyxa No. 271 (RERM-P 1824) grows in an aqueous culture medium, the assimilable Contained saccharides until the polysaccharide has accumulated in the medium, and recovering the enriched polysaccharide from the medium. 2. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that the cultivation of bacillus polymyxa No. 271 in the culture medium is more than 20 hours. 3. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that the enriched polysaccharide is subjected to ultrafiltration in the medium, before recovering the enriched polysaccharide. 4. Process for the preparation of a cationic salt of a polysaccharide with a degree of cation substitution greater than 0.2 and with activity for suppression an increase in serum and liver cholesterol levels and the atherogenic index, by noting that Bacillus polymyxa No. 271 (FERE-P 1824) cultured in an aqueous culture medium, the assimilable saccharides and cations until the cationic salt of the polysaccharide accumulates in the medium and the cationic salt of the polysaccharide is recovered from the medium. 5. The method according to claim 4, characterized in that g e k e n n -z e i c h n e t that at least one cation from the group of alkali metals, alkaline earth metals, Transition metals, manganese, aluminum, zinc, copper and ammonium ions are used. 6. The method according to claim 4 or 5, characterized in that g e -k e n n z e i c It does not mean that a culture medium is used whose total cation equivalent is at least Is 0.005 per liter of the medium, and the cultivation of Bacillus polymyxa No. 271 in this culture medium at a pH of more than 4.5. 7. The method according to any one of claims 4 to 6, characterized g e k e n n notices that salt is added to the fermentation broth before the salt of the Polysaccharide is obtained. 8. The method according to any one of claims 4 to 7, characterized g e k e n n notices that the enriched salt of the polysaccharide of ultrafiltration is used subjects before extracting the salt of the polysaccharide. 9. The method according to any one of claims 4 to 8, characterized g e k e n n notices that the enriched salt of the polysaccharide can be added by adding wins an alcohol as a precipitant to the fermentation medium from the medium. 10. Medicines with activity to reduce serum and liver cholesterol levels, containing an active ingredient and optionally a pharmaceutically suitable carrier and, if appropriate, pharmaceutically suitable additives, thereby g e k e n n z e i c h n e t that there is one obtained according to one of claims 1 to 9 as active ingredient Contains polysaccharide in free form or in the form of a salt. 11. Dietetic food, not indicated a content of a polysaccharide or polysaccharide salt prepared according to a the i% ns1reche 1 to 9.