NL8102628A - PROCESS FOR PREPARING A POLYSACCHARIDE FROM BACTERIA. - Google Patents

Description

. £; - 1 -

Process for the preparation of a polysaccharide from bacteria.

This invention relates to a method of preparing a polysaccharide or a salt thereof which has the property of lowering elevated cholesterol levels in blood serum or liver and also reducing the atherogenic index.

5 It is known that the cholesterol level in the blood is strongly influenced by the nature and amount of the food. Cholesteremia is believed to be one of the most dangerous factors leading to arteriosclerosis (including heart attack). Polysaccharide, one of the main constituents of the food, has been somewhat neglected by the researchers, with the exception of the digestible polysaccharides such as starch.

Using rat tests, it has been found that the polysaccharide secreted by Bacillus polymyxa No. 271 has the property of lowering blood serum and liver cholesterol levels and also reducing the atherogenic index so that it is useful for curing and preventing arteriosclerosis or hypercholesterolaemia.

It was found that the polysaccharide from Bacillus polymyxa with an average molecular weight above 20,000,000 (hereinafter abbreviated to "pmps") has the surprising property of lowering elevated blood serum and liver cholesterol levels and also the cholesterol content in high density lipoprotein (hereinafter referred to as abbreviated to "HDL"), which is known as a factor in preventing arteriosclerosis.

25 Increasing the level of HDL cholesterol means a decrease in the atherogenic index which is defined as: serum cholesterol - HDL cholesterol HDL cholesterol 30

This was an unforeseeable effect of pmps.

The average molecular weight in the study which led to this invention was determined by the method of Ninomiya et al., Described in the Journal of Agricultural Chemical Society of Japan, 42, no. 7 (1968) 431-434.

The polysaccharide of the invention can be obtained by Bacillus polymyxa No. 271 (FERM-P No.

5 1824) to be grown aerobically in an aqueous medium containing 3-5% of a suitable carbon source (such as glucose, sucrose, lactose, molasses or other sugars) and a nitrogen source such as peptone, corn soak water, yeast extract or urea) and salts such as magnesium sulfate. The accumulated polysaccharide (which makes the solution viscous 10) is separated and purified by alcohol precipitation and ultrafiltration. In addition, one usually has to incubate longer than 20 hours in order to obtain a polysaccharide with a sufficiently high average molecular weight. If one incubates for only 16 hours, one obtains a polysaccharide with a molecular weight of 190,000 or less. Suitable conditions in this culture are further specified in the examples below. The bacterial strain used, Bacillus polymyxa No. 271, was deposited with the Fermentation Research Institute, Department of Industrial Science and Technology of the Ministry of International Trade and Industry, and was given the number FEEM-P No. 1824; this strain is available to the public.

For the effect of lowering blood serum cholesterol and liver levels, it is essential that the average molecular weight be above 200,000. The desired higher molecular fraction can be separated from a preparation with an average molecular weight below 200,000 by adding alcohol to an aqueous solution to a low alcohol concentration. The presence of salts such as NaCl and KCl can support this precipitation. Polysaccharides with too high an average molecular weight can be hydrolyzed with acid to polysaccharides with a lower and somewhat more convenient molecular weight. When hydrolysing, it is desirable to do this before removing the bacterial cells from the broth, since filtration is easier.

35 The polysaccharide that accumulates in the culture liquid and makes it highly viscous, appears to have been built up ................———1 0 2 6 2 8 ..... ...... * 4 - 3 - from D-glucose, D-mannose, D-galactose and D-glucuronic acid in a molar ratio of 3: 3: 1: 2. It is thus a polymeric acid and is usually obtained in the form of some salt. It can be deduced from this composition that two cations can be bound at a weight of 1500 units, so that the degree of salt formation = Σ (where Mi is the content of cation in g / kg and Ni is the equivalent weight of the cation.

In most preparations, the degree of salt formation is above 0.2.

These salts of the pmps are as effective as the polysaccharide as such. They originate from Bacillus polymyxa No. 271 in a medium that contains salts in addition to sufficient carbon source and nitrogen source. The amount of cation in the culture medium is preferably at least 0.005 M, and the cations are preferably those of the alkali and alkaline earth metals. The pH should then be above 4.5, preferably between 6 and 7.

If the obtained preparation does not contain the desired cations, it can be freed from that cation in an appropriate manner, for example by means of an ion exchanger or by electrolysis, and then the desired cations are introduced into it by adding suitable salts. This preferably takes place before the polysaccharide is separated and purified by alcohol precipitation and / or ultrafiltration.

The poly saccharide and the salts thereof obtained according to the invention have the remarkable property of lowering the cholesterol content of blood serum and liver; this was found in experiments on rats fed a diet containing 5% pmps.

The pmps according to the invention or any salt thereof can be taken as a powder or as an aqueous solution or suspension. It can be taken as such, but also after ingestion or processed in some food (for example in a diet). If it is in the form of a salt, the cations therein are those of the alkali metals, alkaline earth metals, transition metals, manganese, aluminum, zinc, copper and / or ammonium, and generally 8102628 '= ·· - 4 - ψ * · with a degree of salt formation above 0.2.

The invention is now further illustrated by the following non-limiting examples.

Example I

5 From a liquid containing Bacillus polymyxa

No. 271 (FERM-P No. 1824) was bone and incubated for 24 hours, 600 ml was added to 12 liters of culture broth containing 4% glucose,

0.1% peptone, 2% corn soak water, 0.2% magnesium sulfate and 6 ppm manganese sulfate and had a pH of 7.0. Incubation was done at 28 ° C under aeration with 0.8 liters of air per liter of liquid per minute. After 72 hours, the viscosity of the liquid was 9500 cP. The whole was diluted with 5 parts by volume of hot water and heated to 70-80 ° C. The bacterial cells were removed by passing the diluted liquid through filter paper 15 on which a layer of diatomaceous earth was first deposited. 40 liters of the filtrate were drawn through an ultra-filter (UF-SWM-85V-1, membrane module Abcor SWM-85M from Bioengineering Co. Ltd.) with continuous replenishment of the liquid to constant volume and a filtrate of 200 liters. This filtrate was concentrated and lyophilized to give 50 g of powdered polysaccharide. Example II

From the filtrate obtained in Example I, four 40 liter samples were acidified with hydrochloric acid to pH = 1.5, after which they were kept at 80 ° C for several times, namely preparation no. 2 5 minutes, preparation no. 3 15 minutes, preparation no. 4, 45 minutes and preparation no. 5 for 2 hours. The liquids were then neutralized with NaOH, and then each sample was ultrafiltrated as in Example I.

This gave 48 g of preparation no. 2, 46 g of preparation no. 3, 45 g of preparation no. 4 and 40 g preparation no. 5.

The molecular weights, protein and ash contents of the obtained preparations are shown in Table A.

35 8102628 - 5 "

F F

Table A

Preparation Average. mole Protein content Ash content no. molecular weight (%) (%) 1 93 x 104 1.16 7.6 5 2 54 x 104 1.02 7.5 3 36 x 104 0.87 7.2 4 25 x 104 0, 89 8.1 5 16 x 104 0.78 8.2

Example III

In accordance with Example I, Bacillus polymyxa was grown on a medium containing 4% "residue" (obtained as a by-product of the preparation of crystalline glucose), 0.1% peptone, 2% corn soak water, 0.2% magnesium sulfate and 6 ppm man-sulfate contained. 40 liters of the filtered liquid were diluted with 2 parts by volume of ethanol. The fibrous precipitate thus formed was collected by centrifugation, dehydrated and dried, and then taken up again in 15 liters of water. After this solution had warmed to 70-80 ° C and filtered, 25 liters of ethanol was added. The precipitate thus obtained was collected again, dried under vacuum at 50 ° C and ground to powder; yield 50 g (preparation no. 6).

Example IV

40 liters of the filtered liquid of example was ultra-filtered (UF-SWM-85V-1, membrane module 25 Abcor SWM-85V from Bioengineering Go., Ltd.) and then purified by constant volume filtration to a filtering state. ratio of 5: 1. The filtrate was concentrated and lyophilized to give 50 g of powdered polysaccharide (formulation No. 7). Example V

600 ml of the medium pre-cultured with Bacillus polymyxa and pre-cultured for 24 hours was added to 12 liters of liquid containing 5% glucose, 0.3% yeast extract, 0.05% urea, 0.2% magnesium sulfate and 6 ppm manganese sulfate and a pH of 7.0. Cultivation was done at 28 ° C under aeration at 0.8 liters per liter of liquid per minute. After 120 hours, 40 liters of filtrate were obtained according to Example I and this was also generated according to Example 8102628 * w - 6, except that now methanol was used for the precipitation. This gave 40 g of powdered polysaccharide (formulation no. 8).

The analytical data of the last three preparations are shown in Table B.

Table B

Preparation Average mole Protein content Ash content no. Molecular weight (%) (%) 6 104 x 104 2.0 8.1 10 7 100 x 104 1.4 6.2 8 136 x 104 1.9 7.5

Example VI

600 ml of the medium inoculated with Bacillus polymyxa and pre-cultured for 24 hours was added to 12 liters of liquid containing 4% glucose, 0.5% peptone, 0.5% IG ^ PO ^, 0.1%

MgSO 2 3 0 6 ppm MnSO 2 SO 2 and 0.1% silicone against the foam, the pH of which was 7.0. Cultivation was done at 28 ° C under aeration at 0.8 liters per liter of liquid per minute.

After 96 hours, the viscosity of the liquid was 14,000 cP. This was diluted with 5 parts of hot water and worked up according to example III to give 53 g of powdered polysaccharide (preparation no. 9).

Example VII

40 ml of saturated NaCl solution was added to 40 liters of the filtrate of Example V, then 80 liters of ethanol. Thereafter, according to example III, it was worked up to give 55 g of powdered polysaccharide (preparation no. 10). Example VIII.

40 liters of filtrate obtained from the first m Example VI were filtered with dilution with no more than the same volume of water, to which was added 300 ml cation exchanger Amberlite 200 (H-form) and 600 ml anion exchanger Amberlite IRA-411 (OH-form) . After stirring at 40 ° C for 3 hours, the thus-treated liquid was further purified by ultrafiltration (UF-SDIM-85V-1, membrane-module Abcor SWM-85V) to a dilution ratio of 5: 1. This filtrate was concentrated and lyophilized to give 50 g of powdered polysaccharide (formulation No. 11).

81 0 2 6 2 8 0 \ - 7 -

Example IX

In accordance with Example VIII, part of the filtrate from Example VI was treated with an ion exchanger, to which was added 50 ml of 5% KCl solution. After standing overnight, work-up was continued according to Example VIII to give 51 g of powdered polysaccharide (formulation no. 12). Example X.

Example IX was repeated except that now 20 ml of 5% NaCl solution was added. After work-up, 51 g of powdered polysaccharide was obtained (formulation no. 13).

The analytical data of the last five preparations are shown in Table C.

Table C

15 Prep. Avg. protein as Cation content (ppm Total Mate no. mol and mg / g) - wt. %% Na K Mg Ca salt formation 20 9 114x104 1.46 7.4 5.400 18.000 3.400 <50 0.23 0.46 0.28 0.002 0.97 0.73 10 98 1.23 7.2 15,000 9,700 2,700 <50 0.65 0.25 0.22 0.002 1.12 0.84 11 118 0.74 3.0 590 120 270 <50 25 0.025 0.003 0.022 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 6,900 130 270 <50 0.30 0.003 0.022 0.002 0.33 0.25 30 ___

Example XI

600 ml of the medium pre-cultured with Bacillus polymyxa and pre-cultured for 24 hours was added to 12 liters of medium containing 5% glucose, 0.625% peptone, 0.5% Kl 2 PO 4, 0.02% 35 MgSO 4 .7H 2 O, 0.5 Z CaCO 3, 6 ppm MnSO 4-5H 2 O and 0.1% soybean oil, the pH of which was 7.0. Cultivation was done at 28 ° C under aeration at 0.8 liters per liter per minute. After 96 hours 8102628

* V

- δ - the viscosity of the liquid was 16,000 cP. 40 liters of the filtered culture liquid was worked up according to example III to give 58 g of powdered polysaccharide (preparation no. 14).

Example XII

40 liters of the filtrate obtained in Example XI were worked up according to Example VIII to give 62 g of a powdered polysaccharide (formulation No. 15).

Example XIII

40 liters of the filtrate obtained in Example XI were diluted with an equal volume of water and demineralized by passing through a column with 300 ml Amberlite 200 (H-form) and 600 ml Amberlite IRA-411 (OH-form). A mixture of 35 g K ^ PO ^ and 15 g MgSO ^ ^ E ^ O (amount calculated from the 15 originally present amount of salts) in a little water was added, then the mixture was neutralized with NaOH, and overnight standing, according to Example VIII, underwent ultrafiltration and work-up to give 60 g of powdered polysaccharide (formulation no. 16).

Example XIV

An ion-exchanged portion of the filtrate of Example XIII was ultrafiltered with no salts added. This resulted in 56 g of powdered polysaccharide (formulation no. 17).

Analytical data of the last four preparations are shown in Table D.

30 81 02 62 8 β

- 9 -Table D

Prep. Mol. protein as Cation content (ppm and Total Mate no. r * wt. mg / g) __r— Of salt 5 ___%% Ha K Mg Ca Form [14 104x10 ^ 2.0 8.1 5,470 18,800 7,850 4,250 - 0.24 0.48 0.65 0.21 1.58 1.18 15 100 1.4 8.9 6,670 15,600 4,420 4,120 - 10 0.29 0.40 0.36 0.21 1.26 0.95 16 89 0.9 6.1 5.610 16.350 6.700 920 - 0.24 0.42 0.55 0.0½ 1.26 0.95 17 91 0.9 1.2 670 70 310 3.19 (- 0.029 0.002 0.025 0, 16 0.216 0.16 15 _____

Example XV

600 ml of the liquid incubated with Bacillus polymyxa and incubated for 24 hours was added to 12 liters of medium containing 4% glucose, 0.5% peptone, 0.5% K ^ PO ^, 0.5% CaCO ^, 20 0.1 % MgSO ^ .71 ^ 0.6, MnSO ^ .5 ^ 0 and 0.1% silicone against the foam, the pH of which was 7.0. Incubation was done at 28 ° G under aeration at 0.8 liters per liter per minute.

After 72 hours, the viscosity of the liquid was 10,000 cP. Now it was diluted with 5 parts by volume of hot water and the whole was heated to 70-80 ° C. The bacteria cells were filtered through a filter paper on which a layer of diatomaceous earth was first applied.

40 liters of the filtrate were ultra-filtered (UF-SWM-85V-1, membrane module Abcor SWM-85V) keeping the volume above the filter constant until 200 liters of filtrate were obtained.

This was concentrated and lyophilized to give 50 g of powdered polysaccharide (formulation no. 18).

Example XVI

From the first filtrate obtained in Example XV, 6 samples were diluted with an equal amount of water and then demineralized by passing each through a 5 cm diameter column filled with 300 ml Amberlite 200 81 0 2 628 w - 10 - (H form) and 600 ml Amberlite IRA-411 (OH form). Nothing was added to one batch (no. 19) and to the other five 1 liter portions 10% solutions of NaCl, KCl, MgC 2 CaCl 2 and NH 2 Cl, respectively. Each mixture was left overnight and then ultrafiltered in accordance with Example I. This gave 40-45 g of polysaceharide salts (formulations No. 20 to 24).

Example XVII

1 liter of 10% FeCl 2 solution was added to a portion treated with an ion exchanger according to Example XVI. After standing overnight with occasional stirring, the resulting precipitate was centrifuged off, washed twice with demineralized water and dried under vacuum. This gave the pmps ferric salt (formulation no. 25).

Example XVIII

1 liter of 10% AlCl 2 solution was added to a preparation according to example XVI treated with ion exchanger. After a. overnight with occasional stirring, the precipitate was centrifuged according to example XVII, washed and dried. This gave the aluminum salt at 20 pmps.

The analytical data of the last nine preparations are shown in Table E.

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Animal testing 1

Seven groups of 5 rats, all 4 weeks old, were fed 4 days with a diet of the composition specified below. One group received only the standard-5 diet, a second group additionally received cholesterol, and the other five groups each received 5% of the preparations 1 to 5.

The diet compositions are shown in Table F. Water and food were available ad libitum.

Table F

10 Composition of the standard diet (in% by weight)

Standard Blank Trial _ Diet _ Diet_ Diet_

Casein 22 22 22 .....

Salt mixture ^ 4 4 4 (2) 15 Vitamin mixture 0.85 0.85 0.85 (water soluble) soybean oil ^ 1 1 1 (containing lipophilic vitamins) 20 choline 0.15 0.15 0.15 bacon 10 10 10 cholesterol 0 0.5 0.5 sodium cholate 0 0.25 0.25 pmps 0 0 5 (3) 25 sucrose 62 61.25 56.25 _ (58.25)

Total_100_100_100_ (1) Harper salt mixture, This contains CaCO2 29.29;

CaHPO4.2H20 0.43; KH2PO4 34.31; NaCl 25.06; 30 MgSO4.7H20 9.98 and others.

(2) Vitamin mix according to Harper.

(3) Containing 3000 IU of vitamin A, 300 IU of vitamin D and 100 mg of vitamin E per kg.

(4) The amount in brackets if 3% pmps was added.

8102628 - 13 -

After the experiment, the rats were anesthetized with ether, their hoods were cut open and blood was drained from the abdominal vein. The livers were cut out and weighed.

The blood samples were centrifuged and the serum separated.

Livers and serum samples were stored under freezer. The serum cholesterol level was measured directly using the Determiner TC from Kyowa Hakko Co., Ltd. The livers were saponified and the cholesterol content was determined in the unsaponifiable residues with the same Determiner TC. HDL cholesterol was determined using HDL sterozyme from Fuji Zoki Seiyaku Co., Ltd. The results are shown in Table G.

Table G

Test Cholesterol level (mg / dl) Atherogenic Total growth serum HDL liver (mg / g) * -n (* ex_, in ° deSlever standard 91 + 16 59.6 + 16.0 3.7 y + 1.2 0 .6 + O.4 - diet blank- 291 + 53 31.6 + 7.0 22.2 + 0.6 8.2 + 2.0 50.4 + 4.7 diet 20 pilot diet with 5% of prep. 1 116 + 16 39.5 + 4.8 7.6 + 1.1 1.9 + 0.3 14.2 + 1.7 prep. 2 129 + 40 38.9 + 8.4 10.2 +1.9 2.4 + 1.0 16.6 + 6.4 25 prep. 3 165 + 41 34.5 + 4.7 9.6 + 2.5 2.8 + 1.2 15.9+ 4.7 prep. 4 190 + 37 40.1 + 9.7 12.9 + 0.9 4.2 + 2.6 20.6 + 6.0 prep. 5 243 + 35 30.5 + 4.8 20.6 + 1.7 7.0 + 2.0 35.4 + 5.3

As seen in Table G, the 30 pmps with molecular weights above 200,000 (formulations 1 through 4) significantly reduce the cholesterol content of serum and liver.

The liver of the rats on the standard diet looked reddish brown, and that of the rats on the blank diet yellowish brown, apparently the color of fatty liver. Only 35 rat livers on diets at 5% pmps 1 to 4 had the • clfi healthy reddish brown color. The livers of rats from the 7 group (preparation no. 5) looked yellowish brown, just like those on the blank diet; they also had fatty livers.

8102628 "- 14 -

Animal testing 2

The experimental design was the same as in the first rat experiment, except now preparations 6, 7 and 8 were used and in two different dosages. The results-5 of this are shown in table H.

Table H

Test Cholesterol level (mg / dl) atherogenic Total group index cholesterol serum_HDL liver (mg / g) _ in the liver 10 standard 96 + 13 61.8j + 4.5 2.9 + 0.2 0.6 + 0 , 2 diet blank- 323 + 19 36.3 + 1.2 22.4 + 1.0 8.3 + 0.4 52.4 + 3.8 diet pilot diet 15 with 3% prep. 6 213 + 11 32.1 + 1.5 11.9 + 0.6 5.7 + 0.5 19.8 + 2.5 5% prep. 6 150 + 11 39.7 + 2.1 9.5 + 0.5 2.9 + 0.5 14.6 + 1.4 3% 20 prep. 7 138 + 11 34.9 + 1.2 7.9 + 0.5 3.0 + 0.3 9.9 + 1.1 5% prep. 7 90 ^ 3 45.0 ^ + 2.4 5, 4 + 0.6 1.0 + 0.1 3.8 y + 1.3 3% prep. 8 201 + 8 35.8 + 2.2 11.3 + 0.8 4.5 + 0.3 18. 8 + 2.0 25 5% prep. 8 155 + 14 37.1 + 1.9 9.6 + 0.3 3.0 + 0.5 14.7 + 1.5

As can be clearly seen from Table H, the pmps of the invention significantly reduce the elevated serum and liver cholesterol levels and atherogenic indices, both on 3% and 5% formulation in the feed. This is especially evident with formulation No. 7 which had undergone ultrafiltration. Preparation no. 8 with the highest molecular weight of 1,360,000 was slightly less good, but still did not lag much behind preparation no. 6 with a molecular weight of 1,040,000. The efficacy of these preparations does not seem to increase further if the molecular weight is above 1,000,000.

The livers of the rats in the test groups with the compositions of the invention had the reddish brown color, just like those on the standard diet.

8102628 - 15 -

Animal testing 3

In accordance with Runs 1 and 2, Formulations 9-13 were also tested, only at the 5% dose. The results are shown in Table I.

Table I

Test Cholesterol content (mg / dl) atherogenic Total group index cholesterol ____serum_HDL_ liver (mg / g) _ liver standard- 88 + 5 50.9 + 4.2 3.2 + 0.3 0.7 + 0, 1 - 10 diet blank- 251 + 26 28.9 + 2.7 20.6 + 0.5 8.2 + 1.8 49.8 + 2.2 diet pilot diet with 5% 15 prep. 9 106 + 6 49.9 + 6.0 7.7 + 1.1 I> 3 + 0.3 8.6 + 2.9 5% prep. 10 116 + 16 41.0 + 6.1 7.4 + 1.4 2.2 + 0.7 7.8 + 4.2 5% prep. 11 191 + 47 31.5 + 3.2 23, 4 + 0.5 8.4 + 1.3 51.4 + 1.8 20 5% prep. 12 105 + 11 32.0 + 4.2 8.3 + 1.6 2.3 + 0.7 7.5 + 3.1 5% prep. 13 154 + 21 28.1 + 2.7 9.8 + 1.6 4.8 + 1.1 12.9 + 4.0 25 As can be clearly seen from Table I, lowered preparations No. 9, 10, 12 and 13, wherein the acid is more than 0.2 salt (0.73, 0.84, 0.47, and 0.25, respectively) the cholesterol levels of blood serum and provides substantial. Diet with 5% formulation No. 11, of which the salt formation amounted to only 0.039, behaved much like the blank diet.

Compositions 12 and 13, to which metals had been added after demineralization, had quite a good effect, as did preparations 9 and 10. The presence of cations in these pmps appears to be essential for the cholesterol-lowering activity. The sheer presence of any cation does not appear to be sufficient for this, as shown in Table F, where 4% salt mixture did not elevate the control diet.

When examining the livers of the test animals, the animals were fed test diets with pmps according to 8102628: / V t t! The invention again reddish brown, and that of the blank group yellowish brown. The livers of the rats fed preparations containing pmps looked much better than those of the blank group, as in animal experiments 1 and 2.

5 Animal testing 4

In accordance with the previous tests, preparations 14 to 17 were now tested in two different dosages. The results of this are shown in table J.

Table J

10 Test Cholesterol content in (mg / dl) atherogenic Total group index cholesterol _serum HDL_Lever (mg / g) _ in the liver standard- 96 + j3 61.8 ^ 4.5 2.9 + 0.2 0.6 +0.2 diet 15 blank- 323 + 19 36.3 + 1.2 22.4 + 1.0 8.3 + 0.4 52.4 + 3.8 diet pilot diet with 3% prep. 14 213 + 11 32.1 + 1.5 11.9 + 0.6 5.7 + 0.5 19.8 + 2.5 20 5% prep. 14 150 + 11 39.7 + 2.1 9.5 + 0.5 2.9 + 0.5 14.6 + 1.4 3% prep. 15 138 + 11 34.9 + 1.2 7.9 + 0.5 3.0 + 0.3 9.9 + 1.1 5% 25 prep. 15 90 + 3 45.0 + 2.4 5.4 + 0.6 1.0 + 0.1 3.8 + 1.3 3% prep. 16 127 + 16 42.2 + 1.0 6.6 + 0.4 2.0 + 0.4 6.5 + 1.1 5% prep. 16 91 + 8 45.1 + 4.3 5.3 + 0.4 1.1 + 0.3 2.5 + 1.2 30 3% prep. 17 251 + 17 33.9 + 1.2 15.6 + 1.5 6.4 + ', 4 31.3 + 3.4 5% prep. 17 227 + 12 32.4 + 1.6 15.5 + 1.3 6.1 + 0.4 29.1 + 3.2 35 As can be clearly seen from Table J, diets in which preparations 14, 15 and 16 much better than with the blank group. Preparation No. 17, with a salt formation of only 0.16, had a somewhat weaker effect. Preparation no. 16, with a salt formation of 0.95, prepared by adding salts to de-40 mineralized polysaccharide and ultrafiltration, had almost as much effect as preparation no. 15, also with a salt formation of 0, 95, prepared by ultrafiltration without demineralization. The cholesterol-lowering effect is apparently completely restored by adding salts.

Again, the livers of rats fed on pmps diets of the invention looked much better than those of the blank groups, as in the previous animal experiments.

Animal Test 5 These again were entirely in accordance with the previous tests, now with preparations no. 18 to 25 inclusive, of which 4% were always added to the test diet. The results of this are shown in table K.

Table K.

15 Test Cholesterol content (mg / dl) atherogenic Total group index cholesterol _serum_HDL liver (mg / g) _ in the liver standard- 92j + I4 43.3 ^ 7.3 2.5jf0.3 1.2jK), 5 - diet 20 blank- 248 + 31 31.8 + 5.4 17.7 + 0.6 8.1 + 1.8 44.1 + 3.1 diet pilot diet with 4% prep. 18 114 + 16 39.1 + 13.9 7.3 + 1.1 1.9 + 0.3 12.2 + 2.4 25 prep. 19 (Na salt) 106 + 13 34.6 + 6.5 7.8 + 1.0 1.8 + 0.6 12.8 + 3.7 prep. 20 (K salt) 92 + 25 40.7 + 12.7 6.5 + 2.2 1.6 + 1.4 8.5 + 5.3 prep. 21 30 (Mg salt) 88 + 35 50.3 + 13.2 6.2 + 1.8 1.1 + 1.7 7.7 + 5.0 prep. 22 (Ca salt) 85 + 24 47.8 + 11.7 5.8 + 1.6 0.9 + 0.9 6.1 + 3.9 prep. 23 (NH4 salt) 72 + 17 46.9 + 4.9 4.6 + 1.8 0.5 + 0.2 3.9 + 5.0 35 prep. 24 (Fe salt) 102 + 17 37.0 + 6.1 7.7 + 1.3 1.8 + 0.7 12.1 + 3.3 prep. 25 (Al salt) 110 + 17 42.5 + 17.0 6.5 + 1.9 1.9 + 1.1 8.6 + 5.6 81 0 2 6 2 8 *. I v - 18 -

As can be seen in Table K, the salts of the pmps with combinations of cations significantly reduce the cholesterol content in serum and liver and also the atherogenic index when compared to the test diet. Perhaps combining 5 cations gives an extra effect. The ammonium, calcium and magnesium salts of the pmps in particular had remarkably high activities, which can be seen mainly from the atherogenic indices.

Also in this test series, the livers of rats fed the pmps salt diet according to the invention looked much better than those on the blank diet.

8102628

Claims (9)

1. Process for preparing a polysaccharide with biological activity, in which a bacterium is grown and the separated polysaccharide is recovered, characterized in that a polysaccharide with cholesterol-lowering activity is obtained by Bacillus polymyxa No. 271 in an aqueous medium containing suitable sources of assimilable carbon and nitrogen and salts. 2. A method according to claim 1, characterized in that incubation is longer than 20 hours. Process according to claim 1 or 2, characterized in that the obtained solution with polysaccharide is subjected to ultrafiltration before the polysaccharide is separated from it. Process according to any one of the preceding claims, characterized in that Bacillus polymyxa is grown in a medium containing at least 0.005 M cations and has a pH above 4.5. 5. A method according to any one of the preceding claims, characterized in that salts are added to the liquid after the cultivation of the bacterium but before the separation of the polysaccharide. 6. Process for preparing a salt polysaccharide according to any one of the preceding claims, characterized in that the polysaccharide cations are those of alkali metals, alkaline earth metals, transition metals, manganese, aluminum, zinc, copper and / or ammonium. 7. Process according to any one of the preceding claims, characterized in that the polysaccharide or salt is separated therefrom by adding alcohol to the aqueous solution. 8. Method mainly according to description and / or examples. 9. A cholesterol-lowering effect preparation obtained by the method according to any one of the preceding claims. 8102628 -V · ê ο— Improvement of errata in the description associated with patent application No. 81-02628 Ned. proposed by Applicant dd. b Ml Hg] Add in line 25 of Page. 2 behind "lies" in "Preferably, however, it is not above 1,000,000." Complete line 37 of page 14 with "is coming" * • JKr / RR 8102628 -2 / - Improvement of errata in the description pertaining to patent application no. 81.02628 Ned. proposed by applicant dd · 17 AUS. 1981______________________ In page 13, line 25 (preparation 3) in the column "Atherogenic index" change the number 2.8 + _ 1.2 to 3.8 _ + 1.2. In line 19 of page 5 (preparation 11) in the column "Serum" change the number 191 47 to 291 _ + 47. JKr / JvdB 8102S28